Didac Carmona‐Gutiérrez

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Administration of spermidine, a polyamine whose concentration declines during ageing, extends lifespan in yeast, flies, worms and in human immune cells. Spermidine prevents early oxidative stress and necrotic cell death and increases the expression of autophagy genes by inhibiting histone acetyltransferases action on histone H3. 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.

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.

A cell's 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 baker's 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.

The increase in life expectancy has boosted the incidence of age-related pathologies beyond social and economic sustainability. Consequently, there is an urgent need for interventions that revert or at least prevent the pathogenic age-associated deterioration. The permanent or periodic reduction of calorie intake without malnutrition (caloric restriction and fasting) is the only strategy that reliably extends healthspan in mammals including non-human primates. However, the strict and life-long compliance with these regimens is difficult, which has promoted the emergence of caloric restriction mimetics (CRMs). We define CRMs as compounds that ignite the protective pathways of caloric restriction by promoting autophagy, a cytoplasmic recycling mechanism, via a reduction in protein acetylation. Here, we describe the current knowledge on molecular, cellular, and organismal effects of known and putative CRMs in mice and humans. We anticipate that CRMs will become part of the pharmacological armamentarium against aging and age-related cardiovascular, neurodegenerative, and malignant diseases.

Polyamines are polycations that interact with negatively charged molecules such as DNA, RNA and proteins.They play multiple roles in cell growth, survival and proliferation.Changes in polyamine levels have been associated with aging and diseases.Their levels decline continuously with age and polyamine (spermidine or high-polyamine diet) supplementation increases life span in model organisms.Polyamines have also been involved in stress resistance.On the other hand, polyamines are increased in cancer cells and are a target for potential chemotherapeutic agents.In this review, we bring together these various results and draw a picture of the state of our knowledge on the roles of polyamines in aging, stress and diseases.www.impactaging.com

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.

Endonuclease G (EndoG) is located in mitochondria yet translocates into the nucleus of apoptotic cells during human degenerative diseases. Nonetheless, a direct involvement of EndoG in cell-death execution remains equivocal, and the mechanism for mitochondrio-nuclear translocation is not known. Here, we show that the yeast homolog of EndoG (Nuc1p) can efficiently trigger apoptotic cell death when excluded from mitochondria. Nuc1p induces apoptosis in yeast independently of metacaspase or of apoptosis inducing factor. Instead, the permeability transition pore, karyopherin Kap123p, and histone H2B interact with Nuc1p and are required for cell death upon Nuc1p overexpression, suggesting a pathway in which mitochondrial pore opening, nuclear import, and chromatin association are successively involved in EndoG-mediated death. Deletion of NUC1 diminishes apoptotic death when mitochondrial respiration is increased but enhances necrotic death when oxidative phosphorylation is repressed, pointing to dual--lethal and vital--roles for EndoG.

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.

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.

Reduced supply of the amino acid methionine increases longevity across species through an as yet elusive mechanism. Here, we report that methionine restriction (MetR) extends yeast chronological lifespan in an autophagy-dependent manner. Single deletion of several genes essential for autophagy (ATG5, ATG7 or ATG8) fully abolished the longevity-enhancing capacity of MetR. While pharmacological or genetic inhibition of TOR1 increased lifespan in methionine-prototroph yeast, TOR1 suppression failed to extend the longevity of methionine-restricted yeast cells. Notably, vacuole-acidity was specifically enhanced by MetR, a phenotype that essentially required autophagy. Overexpression of vacuolar ATPase components (Vma1p or Vph2p) suffices to increase chronological lifespan of methionine-prototrophic yeast. In contrast, lifespan extension upon MetR was prevented by inhibition of vacuolar acidity upon disruption of the vacuolar ATPase. In conclusion, autophagy promotes lifespan extension upon MetR and requires the subsequent stimulation of vacuolar acidification, while it is epistatic to the equally autophagy-dependent anti-aging pathway triggered by TOR1 inhibition or deletion.

Lysosomes are the main catabolic organelles of a cell and play a pivotal role in a plethora of cellular processes, including responses to nutrient availability and composition, stress resistance, programmed cell death, plasma membrane repair, development, and cell differentiation. In line with this pleiotropic importance for cellular and organismal life and death, lysosomal dysfunction is associated with many age-related pathologies like Parkinson’s and Alzheimer’s disease, as well as with a decline in lifespan. Conversely, targeting lysosomal functional capacity is emerging as a means to promote longevity. Here, we analyze the current knowledge on the prominent influence of lysosomes on aging-related processes, such as their executory and regulatory roles during general and selective macroautophagy, or their storage capacity for amino acids and ions. In addition, we review and discuss the roles of lysosomes as active players in the mechanisms underlying known lifespan-extending interventions like, for example, spermidine or rapamycin administration. In conclusion, this review aims at critically examining the nature and pliability of the different layers, in which lysosomes are involved as a control hub for aging and longevity.

Autophagy is a cellular recycling program that retards ageing by efficiently eliminating damaged and potentially harmful organelles and intracellular protein aggregates. Here, we show that the abundance of phosphatidylethanolamine (PE) positively regulates autophagy. Reduction of intracellular PE levels by knocking out either of the two yeast phosphatidylserine decarboxylases (PSD) accelerated chronological ageing-associated production of reactive oxygen species and death. Conversely, the artificial increase of intracellular PE levels, by provision of its precursor ethanolamine or by overexpression of the PE-generating enzyme Psd1, significantly increased autophagic flux, both in yeast and in mammalian cell culture. Importantly administration of ethanolamine was sufficient to extend the lifespan of yeast (Saccharomyces cerevisiae), mammalian cells (U2OS, H4) and flies (Drosophila melanogaster). We thus postulate that the availability of PE may constitute a bottleneck for functional autophagy and that organismal life or healthspan could be positively influenced by the consumption of ethanolamine-rich food.

Many microbial communities live in highly competitive surroundings, in which the fight for resources determines their survival and genetic persistence.Humans live in a close relationship with microbial communities, which includes the health-and disease-determining interactions with our microbiome.Accordingly, the understanding of microbial competitive activities are essential at physiological and pathophysiological levels.Here we provide a brief overview on microbial competition and discuss some of its roles and consequences that directly affect humans.

Annually, over 150 million severe cases of fungal infections occur worldwide, resulting in approximately 1.7 million deaths per year. Alarmingly, these numbers are continuously on the rise with a number of social and medical developments during the past decades that have abetted the spread of fungal infections. Additionally, the long-term therapeutic application and prophylactic use of antifungal drugs in high-risk patients have promoted the emergence of (multi)drug-resistant fungi, including the extremely virulent strain Candida auris. Hence, fungal infections are already a global threat that is becoming increasingly severe. In this article, we underline the importance of more and effective research to counteract fungal infections and their consequences.

Cardiovascular diseases are the most prominent maladies in aging societies. Indeed, aging promotes the structural and functional declines of both the heart and the blood circulation system. In this review, we revise the contribution of known longevity pathways to cardiovascular health and delineate the possibilities to interfere with them. In particular, we evaluate autophagy, the intracellular catabolic recycling system associated with life- and health-span extension. We present genetic models, pharmacological interventions, and dietary strategies that block, reduce, or enhance autophagy upon age-related cardiovascular deterioration. Caloric restriction or caloric restriction mimetics like metformin, spermidine, and rapamycin (all of which trigger autophagy) are among the most promising cardioprotective interventions during aging. We conclude that autophagy is a fundamental process to ensure cardiac and vascular health during aging and outline its putative therapeutic importance.

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.

Decreased cognitive performance is a hallmark of brain aging, but the underlying mechanisms and potential therapeutic avenues remain poorly understood. Recent studies have revealed health-protective and lifespan-extending effects of dietary spermidine, a natural autophagy-promoting polyamine. Here, we show that dietary spermidine passes the blood-brain barrier in mice and increases hippocampal eIF5A hypusination and mitochondrial function. Spermidine feeding in aged mice affects behavior in homecage environment tasks, improves spatial learning, and increases hippocampal respiratory competence. In a Drosophila aging model, spermidine boosts mitochondrial respiratory capacity, an effect that requires the autophagy regulator Atg7 and the mitophagy mediators Parkin and Pink1. Neuron-specific Pink1 knockdown abolishes spermidine-induced improvement of olfactory associative learning. This suggests that the maintenance of mitochondrial and autophagic function is essential for enhanced cognition by spermidine feeding. Finally, we show large-scale prospective data linking higher dietary spermidine intake with a reduced risk for cognitive impairment in humans.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

Age-associated diseases are rising to pandemic proportions, exposing the need for efficient and low-cost methods to tackle these maladies at symptomatic, behavioral, metabolic, and physiological levels. While nutrition and health are closely intertwined, our limited understanding of how diet precisely influences disease often precludes the medical use of specific dietary interventions. Caloric restriction (CR) has approached clinical application as a powerful, yet simple, dietary modulation that extends both life- and healthspan in model organisms and ameliorates various diseases. However, due to psychological and social-behavioral limitations, CR may be challenging to implement into real life. Thus, CR-mimicking interventions have been developed, including intermittent fasting, time-restricted eating, and macronutrient modulation. Nonetheless, possible side effects of CR and alternatives thereof must be carefully considered. We summarize key concepts and differences in these dietary interventions in humans, discuss their molecular effects, and shed light on advantages and disadvantages.

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.

As our society ages, neurodegenerative disorders like Parkinson`s disease (PD) are increasing in pandemic proportions. While mechanistic understanding of PD is advancing, a treatment with well tolerable drugs is still elusive. Here, we show that administration of the naturally occurring polyamine spermidine, which declines continuously during aging in various species, alleviates a series of PD-related degenerative processes in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, two established model systems for PD pathology. In the fruit fly, simple feeding with spermidine inhibited loss of climbing activity and early organismal death upon heterologous expression of human α-synuclein, which is thought to be the principal toxic trigger of PD. In this line, administration of spermidine rescued α-synuclein-induced loss of dopaminergic neurons, a hallmark of PD, in nematodes. Alleviation of PD-related neurodegeneration by spermidine was accompanied by induction of autophagy, suggesting that this cytoprotective process may be responsible for the beneficial effects of spermidine administration.

Antimicrobial peptides, natural or synthetic, appear as promising molecules for antimicrobial therapy because of their both broad antimicrobial activity and mechanism of action. Herein, we determine the anti-Candida and antimycobacterial activities, mechanism of action on yeasts, and cytotoxicity on mammalian cells in the presence of the bioinspired peptide CaDef2.1G27-K44.CaDef2.1G27-K44 was designed to attain the following criteria: high positive net charge; low molecular weight (<3000 Da); Boman index ≤2.5; and total hydrophobic ratio ≥ 40%. The mechanism of action was studied by growth inhibition, plasma membrane permeabilization, ROS induction, mitochondrial functionality, and metacaspase activity assays. The cytotoxicity on macrophages, monocytes, and erythrocytes were also determined.CaDef2.1G27-K44 showed inhibitory activity against Candida spp. with MIC100 values ranging from 25 to 50 μM and the standard and clinical isolate of Mycobacterium tuberculosis with MIC50 of 33.2 and 55.4 μM, respectively. We demonstrate that CaDef2.1G27-K44 is active against yeasts at different salt concentrations, induced morphological alterations, caused membrane permeabilization, increased ROS, causes loss of mitochondrial functionality, and activation of metacaspases. CaDef2.1G27-K44 has low cytotoxicity against mammalian cells.The results obtained showed that CaDef2.1G27-K44 has great antimicrobial activity against Candida spp. and M. tuberculosis with low toxicity to host cells. For Candida spp., the treatment with CaDef2.1G27-K44 induces a process of regulated cell death with apoptosis-like features.We show a new AMP bioinspired with physicochemical characteristics important for selectivity and antimicrobial activity, which is a promising candidate for drug development, mainly to control Candida infections.

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-X(L) 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.

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 <i>YCA1</i>, the apoptosis-inducing factor <i>AIF1</i>, or the serine protease <i>OMI</i>) 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.

We show that the antifungal plant defensin Raphanus sativus antifungal protein 2 (RsAFP2) from radish induces apoptosis and concomitantly triggers activation of caspases or caspase-like proteases in the human pathogen Candida albicans. Furthermore, we demonstrate that deletion of C. albicans metacaspase 1, encoding the only reported (putative) caspase in C. albicans, significantly affects caspase activation by the apoptotic stimulus acetic acid, but not by RsAFP2. To our knowledge, this is the first report on the induction of apoptosis with concomitant caspase activation by a defensin in this pathogen. Moreover, our data point to the existence of at least two different types of caspases or caspase-like proteases in C. albicans.

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.

Spermidine is a natural polyamine that stimulates cytoprotective macroautophagy/autophagy. External supplementation of spermidine extends lifespan and health span across species, including in yeast, nematodes, flies and mice. In humans, spermidine levels decline with aging, and a possible connection between reduced endogenous spermidine concentrations and age-related deterioration has been suggested. Recent epidemiological data support this notion, showing that an increased uptake of this polyamine with spermidine-rich food diminishes overall mortality associated with cardiovascular diseases and cancer. Here, we discuss nutritional and other possible routes to counteract the age-mediated decline of spermidine levels.

Abstract Ageing constitutes the most important risk factor for all major chronic ailments, including malignant, cardiovascular and neurodegenerative diseases. However, behavioural and pharmacological interventions with feasible potential to promote health upon ageing remain rare. Here we report the identification of the flavonoid 4,4′-dimethoxychalcone (DMC) as a natural compound with anti-ageing properties. External DMC administration extends the lifespan of yeast, worms and flies, decelerates senescence of human cell cultures, and protects mice from prolonged myocardial ischaemia. Concomitantly, DMC induces autophagy, which is essential for its cytoprotective effects from yeast to mice. This pro-autophagic response induces a conserved systemic change in metabolism, operates independently of TORC1 signalling and depends on specific GATA transcription factors. Notably, we identify DMC in the plant Angelica keiskei koidzumi , to which longevity- and health-promoting effects are ascribed in Asian traditional medicine. In summary, we have identified and mechanistically characterised the conserved longevity-promoting effects of a natural anti-ageing drug.

In the search for interventions against aging and age-related diseases, biological screening platforms are indispensable tools to identify anti-aging compounds among large substance libraries. The budding yeast, Saccharomyces cerevisiae, has emerged as a powerful chemical and genetic screening platform, as it combines a rapid workflow with experimental amenability and the availability of a wide range of genetic mutant libraries. Given the amount of conserved genes and aging mechanisms between yeast and human, testing candidate anti-aging substances in yeast gene-deletion or overexpression collections, or de novo derived mutants, has proven highly successful in finding potential molecular targets. Yeast-based studies, for example, have led to the discovery of the polyphenol resveratrol and the natural polyamine spermidine as potential anti-aging agents. Here, we present strategies for pharmacological anti-aging screens in yeast, discuss common pitfalls and summarize studies that have used yeast for drug discovery and target identification.

Autophagy is a catabolic pathway with multifaceted roles in cellular homeostasis. This process is also involved in the antiviral response at multiple levels, including the direct elimination of intruding viruses (virophagy), the presentation of viral antigens, the fitness of immune cells, and the inhibition of excessive inflammatory reactions. In line with its central role in immunity, viruses have evolved mechanisms to interfere with or to evade the autophagic process, and in some cases, even to harness autophagy or constituents of the autophagic machinery for their replication. Given the devastating consequences of the current COVID-19 pandemic, the question arises whether manipulating autophagy might be an expedient approach to fight the novel coronavirus SARS-CoV-2. In this piece, we provide a short overview of the evidence linking autophagy to coronaviruses and discuss whether such links may provide actionable targets for therapeutic interventions.

Natural polyamines (spermidine and spermine) are small, positively charged molecules that are ubiquitously found within organisms and cells. They exert numerous (intra)cellular functions and have been implicated to protect against several age-related diseases. Although polyamine levels decline in a complex age-dependent, tissue-, and cell type–specific manner, they are maintained in healthy nonagenarians and centenarians. Increased polyamine levels, including through enhanced dietary intake, have been consistently linked to improved health and reduced overall mortality. In preclinical models, dietary supplementation with spermidine prolongs life span and health span. In this review, we highlight salient aspects of nutritional polyamine intake and summarize the current knowledge of organismal and cellular uptake and distribution of dietary (and gastrointestinal) polyamines and their impact on human health. We further summarize clinical and epidemiological studies of dietary polyamines.

Otto Warburg observed that cancer cells are often characterized by intense glycolysis in the presence of oxygen and a concomitant decrease in mitochondrial respiration. Research has mainly focused on a possible connection between increased glycolysis and tumor development whereas decreased respiration has largely been left unattended. Therefore, a causal relation between decreased respiration and tumorigenesis has not been demonstrated.For this purpose, colonies of Saccharomyces cerevisiae, which is suitable for manipulation of mitochondrial respiration and shows mitochondria-mediated cell death, were used as a model. Repression of respiration as well as ROS-scavenging via glutathione inhibited apoptosis and conferred a survival advantage during seeding and early development of this fast proliferating solid cell population. In contrast, enhancement of respiration triggered cell death.Thus, the Warburg effect might directly contribute to the initiation of cancer formation--not only by enhanced glycolysis--but also via decreased respiration in the presence of oxygen, which suppresses apoptosis.

Plant defensins are active against plant and human pathogenic fungi (such as Candida albicans) and baker's yeast. However, they are non-toxic to human cells, providing a possible source for treatment of fungal infections. In this study, we characterized the mode of action of the antifungal plant defensin HsAFP1 from coral bells by screening the Saccharomyces cerevisiae deletion mutant library for mutants with altered HsAFP1 sensitivity and verified the obtained genetic data by biochemical assays in S. cerevisiae and C. albicans. We identified 84 genes, which when deleted conferred at least fourfold hypersensitivity or resistance to HsAFP1. A considerable part of these genes were found to be implicated in mitochondrial functionality. In line, sodium azide, which blocks the respiratory electron transport chain, antagonized HsAFP1 antifungal activity, suggesting that a functional respiratory chain is indispensable for HsAFP1 antifungal action. Since mitochondria are the main source of cellular reactive oxygen species (ROS), we investigated the ROS-inducing nature of HsAFP1. We showed that HsAFP1 treatment of C. albicans resulted in ROS accumulation. As ROS accumulation is one of the phenotypic markers of apoptosis in yeast, we could further demonstrate that HsAFP1 induced apoptosis in C. albicans. These data provide novel mechanistic insights in the mode of action of a plant defensin.

Pathological neuronal inclusions of the 43-kDa TAR DNA-binding protein (TDP-43) are implicated in dementia and motor neuron disorders; however, the molecular mechanisms of the underlying cell loss remain poorly understood. Here we used a yeast model to elucidate cell death mechanisms upon expression of human TDP-43. TDP-43-expressing cells displayed markedly increased markers of oxidative stress, apoptosis, and necrosis. Cytotoxicity was dose- and age-dependent and was potentiated upon expression of disease-associated variants. TDP-43 was localized in perimitochondrial aggregate-like foci, which correlated with cytotoxicity. Although the deleterious effects of TDP-43 were significantly decreased in cells lacking functional mitochondria, cell death depended neither on the mitochondrial cell death proteins apoptosis-inducing factor, endonuclease G, and cytochrome c nor on the activity of cell death proteases like the yeast caspase 1. In contrast, impairment of the respiratory chain attenuated the lethality upon TDP-43 expression with a stringent correlation between cytotoxicity and the degree of respiratory capacity or mitochondrial DNA stability. Consistently, an increase in the respiratory capacity of yeast resulted in enhanced TDP-43-triggered cytotoxicity, oxidative stress, and cell death markers. These data demonstrate that mitochondria and oxidative stress are important to TDP-43-triggered cell death in yeast and may suggest a similar role in human TDP-43 pathologies.

The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.

Yeast mother cell-specific aging constitutes a model of replicative aging as it occurs in stem cell populations of higher eukaryotes. Here, we present a new long-lived yeast deletion mutation,afo1 (for aging factor one), that confers a 60% increase in replicative lifespan. AFO1/MRPL25 codes for a protein that is contained in the large subunit of the mitochondrial ribosome. Double mutant experiments indicate that the longevity-increasing action of the afo1 mutation is independent of mitochondrial translation, yet involves the cytoplasmic Tor1p as well as the growth-controlling transcription factor Sfp1p. In their final cell cycle, the long-lived mutant cells do show the phenotypes of yeast apoptosis indicating that the longevity of the mutant is not caused by an inability to undergo programmed cell death. Furthermore, the afo1 mutation displays high resistance against oxidants. Despite the respiratory deficiency the mutant has paradoxical increase in growth rate compared to generic petite mutants. A comparison of the single and double mutant strains for afo1 and fob1 shows that the longevity phenotype of afo1 is independent of the formation of ERCs (ribosomal DNA minicircles). AFO1/MRPL25 function establishes a new connection between mitochondria, metabolism and aging.

Caspases are cysteine proteases that cleave their substrates after an aspartate residue. Besides their multiple vital roles ranging from immune regulation to spermatogenesis, they are crucial in most cell death pathways, representing a sort of executing sword in the hands of apoptosis. In 2000, a psi-Blast in-silico approach led Uren et al. to identify two novel caspase-related families: metacaspases and paracaspases. Paracaspases are involved in the development of MALT lymphoma, but not in cell death execution, and are found both in eukaryotes owning caspases (animals), as well as in organisms lacking caspases. Metacaspases, on the other hand, are found only in eukaryotes that are devoid of caspases, for example, plants, protists and fungi. Similar to caspases, they contain a caspase-specific catalytic diad of histidine and cysteine, as well as a caspase-like secondary structure. Our lab was among the first to perform experiments on metacaspases showing that the sole metacaspase encoded by the genome of Saccharomyces cerevisiae (which we termed YCA1) is involved in oxidative stress-induced cell death. Overexpression of YCA1 caused a type of cell death that was accompanied by apoptotic markers, while deletion of YCA1 protected against apoptosis caused by reactive oxygen species or chronological aging. We thus had revealed that one metacaspase, YCA1, was involved in the same process as caspases, namely programmed cell death (PCD). Indeed, many groups subsequently unfolded the crucial contribution of metacaspases to cell death execution upon various stresses in yeast and other fungi, as well as in plants. Bozhkov, Zhivotovsky and colleagues could even demonstrate that the plant metacaspase mcII-Pa shapes the embryo of Norway spruce (Picea abies) during development, presumably through its implication in developmental cell death. Together with the unequivocal fact of a common evolutionary origin, these results made us conclude that, in functional terms, metacaspases behave like caspases, thus unchaining a stormy scientific debate. Driven by the fact that caspases occur in animals but metacaspases are present in all kingdoms except animals, many authors deduced that, although cell death is a universal and fundamental process, the implication of caspases in lethal processes is not necessarily conserved. The discovery that metacaspases have a different cleavage specificity than caspases – they hydrolyze proteins after arginine or lysine (basic residues), not after aspartate (an acidic residue) 7,9 – added further fuel to the discussion. In fact, the exclusion of metacaspases from the caspase family and their regrouping into a separate family in the CD clan of cysteine peptidases was demanded. In other words, it was implicated that metacaspases are not caspases. Nevertheless, we strongly felt that Yca1p, a protein that possesses sequence homology to human caspases, and the knockout of which rescues roughly 40% of all cell death scenarios tested in yeast, should retain the name ‘metacaspase’. Nomenclature reaches beyond particular points of divergence (such as localization, structural features or, as it is for metacaspases, the amino acid specificity) when it refers to functional groups. For instance, we use the expression ‘nucleic acids’ for DNA or RNA from prokaryotes that by definition lack nuclei. Similarly, we talk about ‘mitochondrial DNA’ even though this pool of DNA is located in the mitochondria, not in the nucleus. Admittedly, the question whether metacaspases are caspases (or not) can only be answered by enzymology rather than by the vague comparison of their implication in various lethal signaling pathways. Do caspases and metacaspases cleave similar death-related substrates? We reasoned that, although a major divergence of the amino acid specificity of caspases and metacaspases may have occurred during evolution, their target proteins should fall into similar functional groups if the role of caspases and metacaspases was conserved. Hence, do the degradomes of caspases and metacaspases overlap? In a recent paper published in Nature Cell Biology, Peter Bozhkov and colleagues identified the first metacaspase substrate, which they showed to be a functional substrate of mammalian caspase-3 as well. In their seminal study, they demonstrated that both caspases and metacaspases cleave the phylogenetically conserved protein TSN (Tudor staphylococcal nuclease). The authors revealed that TSN from P. abies (PaTSN) is cleaved by its metacaspase (mcII-Pa) at four different sites. This process was blocked either by adding a metacaspase inhibitor or by mutation of a catalytic cysteine. Importantly, PaTSN was shown to be a component of the degradome during both stress-induced and developmental PCD. During embryogenesis, metacaspase activity correlated with proteolysis of endogenous PaTSN in the different embryo stages (high in early and low in mature embryos). Consistently, knockdown of mcII-Pa via

Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.

Mitochondrial dysfunction is a common feature of many neurodegenerative diseases, including proteinopathies such as Alzheimer's or Parkinson's disease, which are characterized by the deposition of aggregated proteins in the form of insoluble fibrils or plaques. The distinct molecular processes that eventually result in mitochondrial dysfunction during neurodegeneration are well studied but still not fully understood. However, defects in mitochondrial fission and fusion, mitophagy, oxidative phosphorylation and mitochondrial bioenergetics have been linked to cellular demise. These processes are influenced by the lipid environment within mitochondrial membranes as, besides membrane structure and curvature, recruitment and activity of different proteins also largely depend on the respective lipid composition. Hence, the interaction of neurotoxic proteins with certain lipids and the modification of lipid composition in different cell compartments, in particular mitochondria, decisively impact cell death associated with neurodegeneration. Here, we discuss the relevance of mitochondrial lipids in the pathological alterations that result in neuronal demise, focussing on proteinopathies.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons, which arises from a yet elusive concurrence between genetic and environmental factors. The protein α-synuclein (αSyn), the principle toxic effector in PD, has been shown to interfere with neuronal Ca(2+) fluxes, arguing for an involvement of deregulated Ca(2+) homeostasis in this neuronal demise. Here, we identify the Golgi-resident Ca(2+)/Mn(2+) ATPase PMR1 (plasma membrane-related Ca(2+)-ATPase 1) as a phylogenetically conserved mediator of αSyn-driven changes in Ca(2+) homeostasis and cytotoxicity. Expression of αSyn in yeast resulted in elevated cytosolic Ca(2+) levels and increased cell death, both of which could be inhibited by deletion of PMR1. Accordingly, absence of PMR1 prevented αSyn-induced loss of dopaminergic neurons in nematodes and flies. In addition, αSyn failed to compromise locomotion and survival of flies when PMR1 was absent. In conclusion, the αSyn-driven rise of cytosolic Ca(2+) levels is pivotal for its cytotoxicity and requires PMR1.

The aging process drives the progressive deterioration of an organism and is thus subject to a complex interplay of regulatory and executing mechanisms. Our understanding of this process eventually aims at the delay and/or prevention of age-related pathologies, among them the age-dependent decrease in cognitive performance (e.g., learning and memory). Using the fruit fly Drosophila melanogaster, which combines a generally high mechanistic conservation with an efficient experimental access regarding aging and memory studies, we have recently unveiled a protective function of polyamines (including spermidine) against age-induced memory impairment (AMI). The flies' age-dependent decline of aversive olfactory memory, an established model for AMI, can be rescued by both pharmacological treatment with spermidine and genetic modulation that increases endogenous polyamine levels. Notably, we find that this effect strictly depends on autophagy, which is remarkable in light of the fact that autophagy is considered a key regulator of aging in other contexts. Given that polyamines in general and spermidine in particular are endogenous metabolites, our findings place them as candidate target substances for AMI treatment.

Loss of cardiac macroautophagy/autophagy impairs heart function, and evidence accumulates that an increased autophagic flux may protect against cardiovascular disease. We therefore tested the protective capacity of the natural autophagy inducer spermidine in animal models of aging and hypertension, which both represent major risk factors for the development of cardiovascular disease. Dietary spermidine elicits cardioprotective effects in aged mice through enhancing cardiac autophagy and mitophagy. In salt-sensitive rats, spermidine supplementation also delays the development of hypertensive heart disease, coinciding with reduced arterial blood pressure. The high blood pressure-lowering effect likely results from improved global arginine bioavailability and protection from hypertension-associated renal damage. The polyamine spermidine is naturally present in human diets, though to a varying amount depending on food type and preparation. In humans, high dietary spermidine intake correlates with reduced blood pressure and decreased risk of cardiovascular disease and related death. Altogether, spermidine represents a cardio- and vascular-protective autophagy inducer that can be readily integrated in common diets.

Caloric restriction mimetics (CRMs) are natural or synthetic compounds that mimic the health-promoting and longevity-extending effects of caloric restriction. CRMs provoke the deacetylation of cellular proteins coupled to an increase in autophagic flux in the absence of toxicity. Here, we report the identification of a novel candidate CRM, namely 3,4-dimethoxychalcone (3,4-DC), among a library of polyphenols. When added to several different human cell lines, 3,4-DC induced the deacetylation of cytoplasmic proteins and stimulated autophagic flux. At difference with other well-characterized CRMs, 3,4-DC, however, required transcription factor EB (TFEB)- and E3 (TFE3)-dependent gene transcription and mRNA translation to trigger autophagy. 3,4-DC stimulated the translocation of TFEB and TFE3 into nuclei both in vitro and in vivo, in hepatocytes and cardiomyocytes. 3,4-DC induced autophagy in vitro and in mouse organs, mediated autophagy-dependent cardioprotective effects, and improved the efficacy of anticancer chemotherapy in vivo. Altogether, our results suggest that 3,4-DC is a novel CRM with a previously unrecognized mode of action.

Obesity is characterized by lipid accumulation in non-adipose tissues, leading to organ degeneration and a wide range of diseases, including diabetes, heart attack, and liver cirrhosis. Free fatty acids (FFA) are believed to be the principal toxic triggers mediating the adverse cellular effects of lipids. Here, we show that various cooking oils used in human nutrition cause cell death in yeast in the presence of a triacylglycerol lipase, mimicking the physiological microenvironment of the small intestine. Combining genetic and cell death assays, we demonstrate that elevated FFA concentrations lead to necrotic cell death, as evidenced by loss of membrane integrity and release of nuclear HMGB1. FFA-mediated necrosis depends on functional mitochondria and leads to the accumulation of reactive oxygen species. We conclude that lipotoxicity is executed via a mitochondrial necrotic pathway, challenging the dogma that the adverse effects of lipid stress are exclusively apoptotic.

We previously isolated a Saccharomyces cerevisiae mutant (HsTnII), which displays 40% reduced chronological lifespan as compared to the wild type (WT). In this study, we found HsTnII cultures to be characterized by fragmented and dysfunctional mitochondria, and by increased initiation of apoptosis during chronological aging as compared to WT. Expression of genes encoding subunits of mitochondrial electron transport chain and ATP synthase is significantly downregulated in HsTnII, and as a consequence, HsTnII is not able to respire ethanol. All these data confirm the importance of functional mitochondria and respiration in determining yeast chronological lifespan and apoptosis.

The lysosomal endoprotease cathepsin D (CatD) is an essential player in general protein turnover and specific peptide processing. CatD-deficiency is associated with neurodegenerative diseases, whereas elevated CatD levels correlate with tumor malignancy and cancer cell survival. Here, we show that the CatD ortholog of the budding yeast Saccharomyces cerevisiae (Pep4p) harbors a dual cytoprotective function, composed of an anti-apoptotic part, conferred by its proteolytic capacity, and an anti-necrotic part, which resides in the protein's proteolytically inactive propeptide. Thus, deletion of PEP4 resulted in both apoptotic and necrotic cell death during chronological aging. Conversely, prolonged overexpression of Pep4p extended chronological lifespan specifically through the protein's anti-necrotic function. This function, which triggered histone hypoacetylation, was dependent on polyamine biosynthesis and was exerted via enhanced intracellular levels of putrescine, spermidine and its precursor S-adenosyl-methionine. Altogether, these data discriminate two pro-survival functions of yeast CatD and provide first insight into the physiological regulation of programmed necrosis in yeast.

As the major lysosomal degradation pathway, autophagy represents the guardian of cellular homeostasis, removing damaged and potentially harmful material and replenishing energy reserves in conditions of starvation. Given its vast physiological importance, autophagy is crucially involved in the process of aging and associated pathologies. Although the regulation of autophagy strongly depends on nutrient availability, specific metabolites that modulate autophagic responses are poorly described. Recently, we revealed nucleo-cytosolic acetyl-coenzyme A (AcCoA) as a phylogenetically conserved inhibitor of starvation-induced and age-associated autophagy. AcCoA is the sole acetyl-group donor for protein acetylation, explaining why pharmacological or genetic manipulations that modify the concentrations of nucleo-cytosolic AcCoA directly affect the levels of protein acetylation. The acetylation of histones and cytosolic proteins inversely correlates with the rate of autophagy in yeast and mammalian cells, respectively, despite the fact that the routes of de novo AcCoA synthesis differ across phyla. Thus, we propose nucleo-cytosolic AcCoA to act as a conserved metabolic rheostat, linking the cellular metabolic state to the regulation of autophagy via effects on protein acetylation.

Autophagy, a membrane-dependent catabolic process, ensures survival of aging cells and depends on the cellular energetic status. Acetyl-CoA carboxylase 1 (Acc1) connects central energy metabolism to lipid biosynthesis and is rate-limiting for the <i>de novo</i> synthesis of lipids. However, it is unclear how <i>de novo</i> lipogenesis and its metabolic consequences affect autophagic activity. Here, we show that in aging yeast, autophagy levels highly depend on the activity of Acc1. Constitutively active Acc1 (<i>acc1^S/A</i>) or a deletion of the Acc1 negative regulator, Snf1 (yeast AMPK), shows elevated autophagy levels, which can be reversed by the Acc1 inhibitor soraphen A. Vice versa, pharmacological inhibition of Acc1 drastically reduces cell survival and results in the accumulation of Atg8-positive structures at the vacuolar membrane, suggesting late defects in the autophagic cascade. As expected, <i>acc1^S/A</i> cells exhibit a reduction in acetate/acetyl-CoA availability along with elevated cellular lipid content. However, concomitant administration of acetate fails to fully revert the increase in autophagy exerted by <i>acc1^S/A</i>. Instead, administration of oleate, while mimicking constitutively active Acc1 in WT cells, alleviates the vacuolar fusion defects induced by Acc1 inhibition. Our results argue for a largely lipid-dependent process of autophagy regulation downstream of Acc1. We present a versatile genetic model to investigate the complex relationship between acetate metabolism, lipid homeostasis, and autophagy and propose Acc1-dependent lipogenesis as a fundamental metabolic path downstream of Snf1 to maintain autophagy and survival during cellular aging.

Disturbance of peroxisome function can lead to various degenerative diseases during ageing. Here, we show that in yeast deletion of PEX6, encoding a protein involved in a key step of peroxisomal protein import, results in an increased accumulation of reactive oxygen species and an enhanced loss of viability upon acetic acid treatment and during early stationary phase. Cell death of ageing-like yeast cells lacking PEX6 does not depend on the apoptotic key players Yca1p and Aif1p, but instead shows markers of necrosis. Thus, we conclude that loss of peroxisomal function leads to a form of necrotic cell death.

Summary To identify new genetic regulators of cellular aging and senescence, we performed genome‐wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress‐induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.

Spermidine is a natural polyamine involved in many important cellular functions, whose supplementation in food or water increases life span and stress resistance in several model organisms. In this work, we expand spermidine's range of age-related beneficial effects by demonstrating that it is also able to improve locomotor performance in aged flies. Spermidine's mechanism of action on aging has been primarily related to general protein hypoacetylation that subsequently induces autophagy. Here, we suggest that the molecular targets of spermidine also include lipid metabolism: Spermidine-fed flies contain more triglycerides and show altered fatty acid and phospholipid profiles. We further determine that most of these metabolic changes are regulated through autophagy. Collectively, our data suggests an additional and novel lipid-mediated mechanism of action for spermidine-induced autophagy.

We evaluated the effects of dihydroxyacetone (DHA) on Trypanosoma brucei bloodstream forms. DHA is considered an energy source for many different cell types. T. brucei takes up DHA readily due to the presence of aquaglyceroporins. However, the parasite is unable to use it as a carbon source because of the absence of DHA kinase (DHAK). We could not find a homolog of the relevant gene in the genomic database of T. brucei and have been unable to detect DHAK activity in cell lysates of the parasite, and the parasite died quickly if DHA was the sole energy source in the medium. In addition, during trypanosome cultivation, DHA induced growth inhibition with a 50% inhibitory concentration of about 1 mM, a concentration that is completely innocuous to mammals. DHA caused cell cycle arrest in the G(2)/M phase of up to 70% at a concentration of 2 mM. Also, DHA-treated parasites showed profound ultrastructural alterations, including an increase of vesicular structures within the cytosol and the presence of multivesicular bodies, myelin-like structures, and autophagy-like vacuoles, as well as a marked disorder of the characteristic mitochondrion structure. Based on the toxicity of DHA for trypanosomes compared with mammals, we consider DHA a starting point for a rational design of new trypanocidal drugs.

The activation of ceramide-generating enzymes, the blockade of ceramide degradation, or the addition of ceramide analogues can trigger apoptosis or necrosis in human cancer cells. Moreover, endogenous ceramide plays a decisive role in the killing of neoplastic cells by conventional anticancer chemotherapeutics. Here, we explored the possibility that membrane-permeable C2-ceramide might kill budding yeast (Saccharomyces cerevisiae) cells under fermentative conditions, where they exhibit rapid proliferation and a Warburg-like metabolism that is reminiscent of cancer cells. C2-ceramide efficiently induced the generation of reactive oxygen species (ROS), as well as apoptotic and necrotic cell death, and this effect was not influenced by deletion of the sole yeast metacaspase. However, C2-ceramide largely failed to cause ROS hypergeneration and cell death upon deletion of the mitochondrial genome. Thus, mitochondrial function is strictly required for C2-ceramide-induced yeast lethality. Accordingly, mitochondria from C2-ceramide-treated yeast cells exhibited major morphological alterations including organelle fragmentation and aggregation. Altogether, our results point to a pivotal role of mitochondria in ceramide-induced yeast cell death.

For this study, we performed a genetic screen of S. cerevisiae's deletion library for mutants sensitive to dehydration stress, with which we aimed to discover cell dehydration-tolerant genes.We used a yeast gene deletion set (YGDS) of 4850 viable mutant haploid strains to perform a genome-wide screen for the identification of desiccation stress modifiers. SIP18 is among the genes identified as essential for cells surviving to drying/rehydration process. Deletion of SIP18 promotes the accumulation of reactive oxygen species and enhances apoptotic and necrotic cell death in response to dehydration/rehydration process.SIP18p acts as an inhibitor of apoptosis in yeast under dehydration stress, as suggested by its antioxidative capacity through the ROS accumulation reduction after an H(2) O(2) attack.To our knowledge, this is the first systematic screen for the identification of putative genes essential to overcoming cell dehydration process. A broad range of identified genes could help to understand why some strains of high biotechnological interest cannot cope with the drying and rehydration treatments. Dehydration sensitivity makes these strains not suitable to be commercialized by yeast manufactures.

Abstract Resveratrol is a polyphenol suggested to play a protective role against ageing and age‐related diseases. We demonstrate that administering low‐doses of resveratrol causes ROS accumulation and transcriptional changes in yeast cells and human adipocytes. These changes in gene expression depend on the oxidative transcription factor Yap1p. In particular, resveratrol induces expression of Yap1p gene targets, such as TRX2 , TRR1 or AHP1 , in a Yap1p‐dependent mode. Under resveratrol treatment, Yap1p is phosphorylated and accumulated in the nucleus. Yap1p knockout causes resveratrol sensitivity, which totally depends on the presence of the C‐terminal region of Yap1p. Thus, resveratrol may enhance cellular lifespan by hormetic ROS accumulation, which leads to strengthening the cells' antioxidant capacity. Copyright © 2012 John Wiley &amp; Sons, Ltd.

All living organisms need to adapt to ever changing adverse conditions in order to survive.The phenomenon termed hormesis describes an evolutionarily conserved process by which a cell or an entire organism can be preconditioned, meaning that previous exposure to low doses of an insult protects against a higher, normally harmful or lethal dose of the same stressor.Growing evidence suggests that hormesis is directly linked to an organism's (or cell's) capability to cope with pathological conditions such as aging and age-related diseases.Here, we condense the conceptual and potentially therapeutic importance of hormesis by providing a short overview of current evidence in favor of the cytoprotective impact of hormesis, as well as of its underlying molecular mechanisms.

A recent prospective epidemiological study suggested that an increase in the nutritional uptake of the natural polyamine spermidine is associated with reduced overall and cancer-specific mortality. Here, we speculate through which mechanisms spermidine might exert such oncopreventive effects.Abbreviations: ACLY, ATP citrate lyase; ATG, autophagy-related gene; CoA, coenzyme A; NSCLC, non-small cell lung cancer

Neither the molecular mechanisms whereby cancer cells intrinsically are or become resistant to the DNA-damaging agent cisplatin nor the signaling pathways that account for cisplatin cytotoxicity have thus far been characterized in detail. In an attempt to gain further insights into the molecular cascades elicited by cisplatin (leading to resistance or underpinning its antineoplastic properties), we comparatively investigated the ability of cisplatin, C2-ceramide and cadmium dichloride, alone or in the presence of an array of mitochondrion-protective agents, to trigger the permeabilization of purified mitochondria. In addition, we compared the transcriptional response triggered by cisplatin, C2-ceramide and cadmium dichloride in non-small cell lung carcinoma A549 cells. Finally, we assessed the capacity of cisplatin, C2-ceramide and cadmium dichloride to reduce the clonogenic potential of a battery of yeast strains lacking proteins involved in the regulation of cell death, DNA damage signaling and stress management. This multipronged experimental approach revealed that cisplatin elicits signaling pathways that are for the most part "private," i.e., that manifest limited overlap with the molecular cascades ignited by other inducers of mitochondrial apoptosis, and triggers apoptosis mainly in a transcription-independent fashion. Indeed, bona fide cisplatin-response modifiers that we have recently identified by a functional genome-wide siRNA screen are either not transcriptionally regulated during cisplatin-induced cell death or their transcriptional modulation reflects the activation of an adaptive response promoting cisplatin resistance.

The identification and characterization of the molecular determinants governing ageing represents the key to counteracting age-related diseases and eventually prolonging our health span. A large number of fundamental insights into the ageing process have been provided by research into the budding yeast Saccharomyces cerevisiae, which couples a wide array of technical advantages with a high degree of genetic, proteomic and mechanistic conservation. Indeed, this unicellular organism harbours regulatory pathways, such as those related to programmed cell death or nutrient signalling, that are crucial for ageing control and are reminiscent of other eukaryotes, including mammals. Here, we summarize and discuss three different paradigms of yeast ageing: replicative, chronological and colony ageing. We address their physiological relevance as well as the specific and common characteristics and regulators involved, providing an overview of the network underlying ageing in one of the most important eukaryotic model organisms.

The loss of lipid homeostasis can lead to lipid overload and is associated with a variety of disease states. However, little is known as to how the disruption of lipid regulation or lipid overload affects cell survival. In this study we investigated how excess diacylglycerol (DG), a cardinal metabolite suspected to mediate lipotoxicity, compromises the survival of yeast cells. We reveal that increased DG achieved by either genetic manipulation or pharmacological administration of 1,2-dioctanoyl-sn-glycerol (DOG) triggers necrotic cell death. The toxic effects of DG are linked to glucose metabolism and require a functional Rim101 signaling cascade involving the Rim21-dependent sensing complex and the activation of a calpain-like protease. The Rim101 cascade is an established pathway that triggers a transcriptional response to alkaline or lipid stress. We propose that the Rim101 pathway senses DG-induced lipid perturbation and conducts a signaling response that either facilitates cellular adaptation or triggers lipotoxic cell death. Using established models of lipotoxicity, i.e., high-fat diet in Drosophila and palmitic acid administration in cultured human endothelial cells, we present evidence that the core mechanism underlying this calpain-dependent lipotoxic cell death pathway is phylogenetically conserved.

Huntington's disease (HD) is a neurodegenerative disorder that leads to progressive neuronal loss, provoking impaired motor control, cognitive decline, and dementia. So far, HD remains incurable, and available drugs are effective only for symptomatic management. HD is caused by a mutant form of the huntingtin protein, which harbors an elongated polyglutamine domain and is highly prone to aggregation. However, many aspects underlying the cytotoxicity of mutant huntingtin (mHTT) remain elusive, hindering the efficient development of applicable interventions to counteract HD. An important strategy to obtain molecular insights into human disorders in general is the use of eukaryotic model organisms, which are easy to genetically manipulate and display a high degree of conservation regarding disease-relevant cellular processes. The budding yeast Saccharomyces cerevisiae has a long-standing and successful history in modeling a plethora of human maladies and has recently emerged as an effective tool to study neurodegenerative disorders, including HD. Here, we summarize some of the most important contributions of yeast to HD research, specifically concerning the elucidation of mechanistic features of mHTT cytotoxicity and the potential of yeast as a platform to screen for pharmacological agents against HD.

The age-related decline in organismal fitness results in vulnerability to pathologies and eventual lethal decay. One way to counteract cellular aging and to delay and/or prevent the onset of age-related maladies is the reduction of calorie intake or the institution of fasting regimens. Caloric restriction mimetics (CRMs) have the ability to imitate the health-promoting and lifespan-extending effects of caloric restriction without the need for dietary restriction. CRMs induce an increase in autophagic flux in response to the deacetylation of cellular proteins in the absence of cytotoxicity. Here we report the development of a high-throughput discovery platform for novel CRMs that uses systems biology approaches, in vitro validation and functional tests employing in vivo disease models. This workflow led to the identification of 3,4-dimethoxychalcone (3,4-DC) as a novel CRM that stimulated TFEB (transcription factor EB)- and TFE3 (transcription factor E3)-dependent macroautophagy/autophagy. 3,4-DC showed cardioprotective effects and stimulated anticancer immunosurveillance in the context of immunogenic chemotherapy.

Candida auris is a multidrug resistant (MDR) fungal pathogen with a crude mortality rate of 30-60%. First identified in 2009, C. auris has been rapidly emerging to become a global risk in clinical settings and was declared an urgent health threat by the Centers for Disease Control and Prevention (CDC). A concerted global action is thus needed to successfully tackle the challenges created by this emerging fungal pathogen. In this brief article, we underline the importance of unique virulence traits,including its easy transformation, its persistence outside the host and its resilience against multiple cellular stresses, as well as of environmental factors that have mainly contributed to the rise of this superbug.

Cell death in yeast is oftenaccompanied by diagnostic apoptotic markers such asexternalization of phosphatidylserine to the outer leaflet ofthe plasma membrane, DNA degradation, chromatin con-densation, and the generation of reactive oxygen species, allof which can be measured both at a qualitative (microscopic)and at a quantitative (e.g. flow cytometric) level.

A cell's reaction to any change in the endogenous or exogenous conditions often involves a complex response that eventually either leads to cell adaptation and survival or to the initiation and execution of (programmed) cell death. The molecular decision whether to live or die, while depending on a cell's genome, is fundamentally influenced by its actual metabolic status. Thus, the collection of all metabolites present in a biological system at a certain time point (the so-called metabolome) defines its physiological, developmental and pathological state and determines its fate during changing and stressful conditions. The budding yeast Saccharomyces cerevisiae is a unicellular organism that allows to easily modify and monitor conditions affecting the cell's metabolome, for instance through a simple change of the nutrition source. Such changes can be used to mimic and study (patho)physiological scenarios, including caloric restriction and longevity, the Warburg effect in cancer cells or changes in mitochondrial mass affecting cell death. In addition, disruption of single genes or generation of respiratory deficiency (via abrogation of mitochondrial DNA) assists in revealing connections between metabolism and apoptosis. In this minireview, we discuss recent studies using the potential of the yeast model to provide new insights into the processes of stress defense, cell death and longevity.

Spermidine is a naturally occurring polyamine involved in multiple biological processes, including DNA metabolism, autophagy and aging. Like other polyamines, spermidine is also indispensable for successful reproduction at several stages. However, a direct influence on the actual fertilization process, i.e., the fusion of an oocyte with a spermatocyte, remains uncertain. To explore this possibility, we established the mating process in the yeast Saccharomyces cerevisiae as a model for fertilization in higher eukaryotes. During human fertilization, the sperm capacitates and the acrosome reaction is necessary for penetration of the oocyte. Similarly, sexually active yeasts form a protrusion called "shmoo" as a prerequisite for mating. In this study, we demonstrate that pheromone-induced shmoo formation requires spermidine. In addition, we show that spermidine is essential for mating in yeast as well as for egg fertilization in the nematode Caenorhabditis elegans. In both cases, this occurs independently from autophagy. In synthesis, we identify spermidine as an important mating component in unicellular and multicellular model organisms, supporting an unprecedented evolutionary conservation of the mechanisms governing fertilization-related cellular fusion.

Following microbial pathogen invasion, the human immune system of activated phagocytes generates and releases the potent oxidant hypochlorous acid (HOCl), which contributes to the killing of menacing microorganisms. Though tightly controlled, HOCl generation by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils/monocytes may occur in excess and lead to tissue damage. It is thus of marked importance to delineate the molecular pathways underlying HOCl cytotoxicity in both microbial and human cells. Here, we show that HOCl induces the generation of reactive oxygen species (ROS), apoptotic cell death and the formation of specific HOCl-modified epitopes in the budding yeast Saccharomyces cerevisiae. Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect. The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification. By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.

Supported by the Austrian Science Fund FWF (grants LIPOTOX, I1000‐B20, P23490‐B12, and P24381‐B20 to F.M.). We gratefully acknowledge support from NAWI Graz and BioTechMed Graz. Also supported by the Austrian Research Association and the Faculty of Natural Sciences, University of Graz (to A.Z.). Potential conflict of interest: Nothing to report. See Article on Page 1896 Macroautophagy (hereafter termed “autophagy”) is a process of cellular self‐digestion that targets superfluous or damaged cell components for lysosomal degradation. Thereby, cytosolic portions containing the target molecules/organelles are enveloped by double‐membrane vesicles (autophagosomes), which then fuse with lysosomes. There, the cargo is degraded and the resulting compounds (e.g., amino acids, fatty acids) are released into the cytoplasm for recycling and/or energy production. Accordingly, lipophagy defines the autophagic turnover of cytosolic lipid droplets (LDs), the main neutral lipid stores in the cell: LDs are engulfed by autophagosomes and, after lysosomal fusion, degraded by acid lipases. Together with lipolysis, which involves the direct docking of cytoplasmic lipases to the LD surface for hydrolysis, lipophagy is key for the regulation of LD catabolism. Imbalanced LD homeostasis, as may result from genetic factors, infection, or a particular lifestyle (e.g., high‐fat diet, excessive alcohol consumption), may lead to multiple afflictions. Obesity, for example, can cause the so‐called metabolic syndrome, a cluster of metabolic disorders that increases the risk of cardiovascular diseases, type 2 diabetes, and liver pathologies. In fact, the liver, the central transfer site for lipid trafficking, is particularly susceptible to such deregulation, which may result in hepatic disorders such as nonalcoholic fatty liver disease and alcohol‐induced hepatic steatosis. Interestingly, autophagy has been associated with several pathophysiological scenarios in the liver. For instance, inhibition of autophagy at both the cellular and the organismal level promotes hepatocellular steatosis.1 Still, many mechanistic details underlying the regulation of autophagic LD turnover in the liver remain unknown. The study by Schroeder et al. in this issue of hepatology2 gives detailed insight into how LDs interact with the autophagic pathway, determining the Rab7 guanosine triphosphatase as a key regulator in this process. It is known that Rab7 is associated with LD membranes and a regulator of transport and maturation of the late endocytic and autophagic compartments. Using nutrient starvation conditions, under which autophagy is strongly induced, the authors demonstrate increased Rab7 activation and recruitment to LDs of hepatoma cells. This is a prerequisite specifically for LD breakdown because genetic and pharmacological inhibition of Rab7 impairs the ability to degrade LDs (but does not alter LD formation).2 Thus, it seems as if active Rab7 would “prime” LDs for autophagic degradation. Likewise, nutrient deprivation also activates Rab7 at diverse degradative compartments of the autophagic pathway, including autophagosomes. This mediates their recruitment through a physical interaction with Rab7‐primed LDs. Activation of Rab7 also occurs at multivesicular bodies and is necessary for lipophagy.2 Multivesicular bodies usually form an intermediate compartment (amphisome) with autophagosomes before fusion with the lysosome. Whether the formation of an amphisome is required or this association represents a molecular platform to ease an interaction with lysosomes remains to be explored. In addition, the authors show a direct interaction between autophagosomal LDs and lysosomes.2 Using fluorescent microscopy analyses on fixed and live cells, they show a kiss‐and‐run mechanism: (1) recruitment of lysosomes to Rab7‐primed autophagosomal LDs, (2) docking for a short period of time, and (3) departing of the lysosome. They interpret that LD breakdown might be facilitated through such a sequential and repeated “nibbling off” by different lysosomes. This regulatory capacity of Rab7 depends on the ability to associate to membranes because the expression of a prenylation‐defective Rab7 mutant in Rab7 knockdown cells is not able to restore the lipophagy defect.2 In fact, this mutation precludes Rab7 from associating with LDs as well as actual guanosine triphosphatase activity. Interestingly, a further Rab7 mutant unable to bind to Rab7‐interacting lysosomal protein (RILP), a downstream effector of Rab7 involved in endolysosomal trafficking, remains associated to LDs and maintains its guanosine triphosphatase activity but still fails to restore lipophagy when Rab7 is knocked down.2 Importantly, Rab7 is known to regulate microtubule minus end–directed transport through simultaneous binding of RILP and oxysterol‐binding protein–related protein 1L in order to recruit the motor protein complex dynein/dynactin.3 At the same time, FYVE and coiled‐coil domain containing 1 (FYCO1), another Rab7‐interacting protein, regulates plus end–directed transport of autophagosomes, probably through interaction with LC3 (Atg8), phosphoinositol‐3‐phosphate, and the kinesin motor.4 Thus, Rab7 seems to directly control the recruitment and docking events between primed LDs and degradative compartments through microtubule‐assisted regulation. Further analysis of other Rab7 downstream effectors, like the homotypic fusion and protein‐sorting (HOPS) complex, which is required for autophagosome–lysosome fusion, may shed further light on lipophagic execution. Interestingly, the HOPS complex has been recently shown to directly interact with Pleckstrin homology domain containing protein family member 1, which contains an LC3‐interacting region that mediates its binding to autophagosomal membranes.5 Besides such downstream effectors, it will also be interesting to explore how prolipophagic upstream signals feed into Rab7 activation and how the Rab7 network might integrate into the regulation of different autophagy types. Altogether, the article by Schroeder et al. provides exciting mechanistic insights into Rab7 as the executory and regulatory factor during lipophagy. The significance of unveiling the regulatory network governing the lipophagic process in general and hepatic LD turnover in particular is evident given the medical implications of LD imbalance. This importance is further underscored by the increasing indications of the pleiotropic nature of autophagy for lipid degradation in different tissues.6 In the liver, lipophagy induction may prevent adverse conditions resulting from LD accumulation. For this purpose, different strategies that could counteract the aging phenomenon through autophagy induction may be applied, among them lifestyle (e.g., fasting, exercise) and pharmacological strategies (e.g., rapamycin, spermidine).7 Of note, polyphenols from coffee have recently been identified as proautophagic compounds.8 Epidemiologic studies further support the idea that drinking coffee reduces the risk for diverse afflictions in nonalcoholic fatty liver disease patients. In addition, caffeine protects against fatty liver by coordinating the induction of lipophagy and mitochondrial β‐oxidation.9 Nevertheless, gross autophagy activation in the liver might not be appropriate under specific conditions. For instance, in hepatocarcinogenesis, autophagic induction suppresses early tumorigenesis through its intracellular cleaning function. However, in established tumors, this cytoprotective effect may contribute to chemotherapy resistance and counteract the adversity of the hypoxic environment around the tumor. A recent study using mice with hepatocyte‐specific knockout of the autophagy‐essential Atg5 also suggests that after hepatocarcinogenesis initiation, autophagy might restrain the expression of tumor suppressors and thus fuel the progression of hepatocellular carcinoma.10 Similarly, autophagy might have an ambivalent impact on liver fibrosis, a typical wound‐healing reaction to apoptosis and necrosis‐mediated chronic liver injury that is associated with inflammation. Among other effects, this leads to the transdifferentiation of hepatic stellate cells to hepatic myofibroblasts that drive the fibrogenic process. In this context, autophagy exerts antifibrogenic effects through its cytoprotective and anti‐inflammatory properties but at the same time has profibrogenic properties due to its direct contribution to the process of hepatic stellate cell activation.11 These examples show that cell‐specific activation of autophagy is crucial to effectively prevent and/or counteract at least some hepatic pathologies. In sum, a deeper understanding of the molecular mechanisms of hepatic autophagy in general and lipophagy in particular is necessary for pathophysiological and therapeutical reasons. With the characterization of Rab7 as the lipophagic network orchestrator, a big step has been accomplished toward revealing the complexity of and allowing an intervention in how the liver controls its lipid balance. Author names in bold designate shared co‐first authorship.

Autophagy is a catabolic process that is crucial for cellular homeostasis and adaptive response to changing environments. Importantly, autophagy has been shown to be induced in many longevity-associated scenarios and to be required to maintain lifespan extension. Notably, autophagy is a highly conserved cellular process among eukaryotes, and the yeast Saccharomyces cerevisiae has become a universal model system for unraveling the molecular machinery underlying autophagic mechanisms. Here, we discuss different protocols to monitor survival and autophagy of yeast cells upon chronological aging. These include the use of propidium iodide to assess the loss of cell membrane integrity, as well as clonogenic assays to directly determine survival rates. Additionally, we describe methods to quantify autophagic flux, including the alkaline phosphatase activity or the GFP liberation assays, which measure the delivery of autophagosomal cargo to the vacuole. In sum, we have recapped established protocols used to evaluate a link between lifespan extension and autophagy in yeast.

The recent announcement of the 2016 Nobel Prize in Physiology or Medicine, awarded to Yoshinori Ohsumi for the discoveries of mechanisms governing autophagy, underscores the importance of intracellular degradation and recycling. At the same time, it further cements yeast, in which this field decisively developed, as a prolific model organism. Here we provide a quick historical overview that mirrors both the importance of autophagy as a conserved and essential process for cellular life and death as well as the crucial role of yeast in its mechanistic characterization.

The degeneration of dopaminergic neurons during Parkinson’s disease is intimately linked to malfunction of α-synuclein, the main component of the proteinaceous intracellular inclusions characteristic for this pathology. The cytotoxicity of α-synuclein has been attributed to disturbances in several biological processes conserved from yeast to humans, including Ca2+ homeostasis, general lysosomal function, and autophagy. However, the precise sequence of events that eventually results in cell death remains unclear. Here, we establish a connection between the major lysosomal protease cathepsin D and the Ca2+/calmodulin-dependent phosphatase calcineurin. In a yeast model for Parkinson’s disease, high levels of human α-synuclein triggered cytosolic acidification and reduced vacuolar hydrolytic capacity, finally leading to cell death. This could be counteracted by overexpression of yeast cathepsin D (Pep4), which re-installed pH homeostasis and vacuolar proteolytic function, decreased α-synuclein oligomers and aggregates, and provided cytoprotection. Interestingly, these beneficial effects of Pep4 were independent of autophagy. Instead, they required functional calcineurin signaling, since deletion of calcineurin strongly reduced both the proteolytic activity of endogenous Pep4 and the cytoprotective capacity of overexpressed Pep4. Calcineurin contributed to proper endosomal targeting of Pep4 to the vacuole and the recycling of the Pep4 sorting receptor Pep1 from prevacuolar compartments back to the trans-Golgi network. Altogether, we demonstrate that stimulation of this novel calcineurin-Pep4 axis reduces α-synuclein cytotoxicity.

Abstract A number of natural plant products have a long-standing history in both traditional and modern medical applications. Some secondary metabolites induce autophagy and mediate autophagy-dependent healthspan- and lifespan-extending effects in suitable mouse models. Here, we identified isobacachalcone (ISO) as a non-toxic inducer of autophagic flux that acts on human and mouse cells in vitro, as well as mouse organs in vivo. Mechanistically, ISO inhibits AKT as well as, downstream of AKT, the mechanistic target of rapamycin complex 1 (mTORC1), coupled to the activation of the pro-autophagic transcription factors EB (TFEB) and E3 (TFE3). Cells equipped with a constitutively active AKT mutant failed to activate autophagy. ISO also stimulated the AKT-repressible activation of all three arms of the unfolded stress response (UPR), including the PERK-dependent phosphorylation of eukaryotic initiation factor 2α (eIF2α). Knockout of TFEB and/or TFE3 blunted the UPR, while knockout of PERK or replacement of eIF2α by a non-phosphorylable mutant reduced TFEB/TFE3 activation and autophagy induced by ISO. This points to crosstalk between the UPR and autophagy. Of note, the administration of ISO to mice improved the efficacy of immunogenic anticancer chemotherapy. This effect relied on an improved T lymphocyte-dependent anticancer immune response and was lost upon constitutive AKT activation in, or deletion of the essential autophagy gene Atg5 from, the malignant cells. In conclusion, ISO is a bioavailable autophagy inducer that warrants further preclinical characterization.

Endonuclease G is a mitochondrio-nuclear located nuclease with dual- vital and lethal- functions. Besides its role in apoptosis execution, we have recently shown that depletion of endonuclease G leads to necrotic cell death in yeast. Here, we present further mechanistic elucidation of endonuclease G's vital functions. The deletion of the yeast Endonuclease G gene causes the complete elimination of tetraploid cells during exponential growth. Consistently, conditional knockdown of mammalian endonuclease G selectively kills tetraploid but not diploid clones of the human HCT116 colon carcinoma cell line. We conclude that endonuclease G is important for the viability of polyploid mammalian and yeast cells.

As time goes by, a postmitotic cell ages following a degeneration process ultimately ending in cell death. This phenomenon is evolutionary conserved and present in unicellular eukaryotes as well, making the yeast chronological aging system an appreciated model. Here, single cells die in a programmed fashion (both by apoptosis and necrosis) for the benefit of the whole population. Besides its meaning for aging and cell death research, age-induced programmed cell death represents the first experimental proof for the so-called group selection theory: Apoptotic genes became selected during evolution because of the benefits they might render to the whole cell culture and not to the individual cell. Many anti‐aging stimuli have been discovered in the yeast chronological aging system and have afterwards been confirmed in higher cells or organisms. New work from the Burhans group (this issue) now demonstrates that glucose signaling has a progeriatric effect on chronologically aged yeast cells: Glucose administration results in a diminished efficacy of cells to enter quiescence, finally causing superoxide‐mediated replication stress and apoptosis.

The multifaceted process of aging inevitably leads to disturbances in cellular metabolism and protein homeostasis. To meet this challenge, cells make use of autophagy, which is probably one of the most important pathways preserving cellular protection under stressful conditions. Thus, efficient autophagic flux is required for healthy aging in many if not all eukaryotic organisms. The regulation of autophagy itself is affected by changing metabolic conditions, but the precise metabolic circuitries are poorly understood. Recently, we found that the nucleocytosolic pool of acetyl-coenzyme A (AcCoA) functions as a major and dominant suppressor of cytoprotective autophagy during aging. Here, we propose an epigenetic mechanism for AcCoA-mediated autophagy suppression that causally involves the regulation of histone acetylation and changes in the autophagy-relevant transcriptome.

Alcoholic fermentation probably represents the oldest biotechnological utilization of a microorganism, even beyond history recording. Over time, yeast – especially the budding yeast Saccharomyces cerevisiae – has gained a unique role of cultural, social and industrial importance that ranges from its use in the millenary art of brewing beer and making wine to its modern application in the production of ethanol-based biofuel. Massive production of ethanol as an eco-friendly liquid fuel might help counteract the alarming repercussions of climate change, as well as the persisting insecurity of petroleum markets, yet relies on increasing fermentation performance to maintain its economic feasibility. The same applies to the alcoholic beverage industry, where stuck or sluggish fermentation accounts for substantial economic losses. For this reason, resolving a basic yeast cell biology problem like fermentative limitation may have a major impact on the biotechnology industry. This limitation is mainly caused by stress situations during the fermentative process, such as high osmotic pressure, large pH changes or accumulating fermentation products that can compromise yeast cell growth and survival. Thus, improving tolerance of yeast cells to different stresses can be expected to amend fermentative efficiency.

Metabolites in aging and autophagy – INTRODUCTION Autophagy, the main lysosomal degradative machinery, plays a major role in maintaining cellular homeostasis and thus a healthy state in an organism. This process recycles unnecessary or damaged material, therefore, not only providing nutrients to maintain vital cellular functions in times of starvation but also eliminating potentially harmful cellular material . Importantly, the autophagic rate (...)

In this issue of Molecular Cell, Dwyer et al. (2012) characterize a RecA-dependent and ClpXP-regulated pathway that controls the acquisition of several apoptotic markers upon bactericidal treatment of prokaryotes, placing the hypothetical origin of apoptosis further downstream in evolution.

Autophagy extends lifespan via vacuolar acidification – In view of the fact that macroautophagy (hereafter described as autophagy) constitutes (one of) the major anti-aging pathway(s), we evaluated the hypothesis that methionine could act as an important and potent autophagic regulator. Indeed, we discovered that MetR induces a rapid and long-lasting increase in autophagic flux. Importantly, deletions of genes that are essential for (...)

We demonstrated that a yeast deletion mutant in IPT1 and SKN1, encoding proteins involved in the biosynthesis of mannosyldiinositolphosphoryl ceramides, is characterized by increased autophagy and DNA fragmentation upon nitrogen (N) starvation as compared with the single deletion mutants or wild type (WT). Apoptotic features were not significantly different between single and double deletion mutants upon N starvation, pointing to increased autophagy in the double Deltaipt1 Deltaskn1 deletion mutant independent of apoptosis. We observed increased basal levels of phytosphingosine in membranes of the double Deltaipt1 Deltaskn1 deletion mutant as compared with the single deletion mutants or WT. These data point to a negative regulation of autophagy by both Ipt1 and Skn1 in yeast, with a putative involvement of phytosphingosine in this process.

Hereditary spastic paraplegias, a group of neurodegenerative disorders, can be caused by loss-of-function mutations in the protein spartin. However, the physiological role of spartin remains largely elusive. Here we show that heterologous expression of human or Drosophila spartin extends chronological lifespan of yeast, reducing age-associated ROS production, apoptosis, and necrosis. We demonstrate that spartin localizes to the proximity of mitochondria and physically interacts with proteins related to mitochondrial and respiratory metabolism. Interestingly, Nde1, the mitochondrial external NADH dehydrogenase, and Pda1, the core enzyme of the pyruvate dehydrogenase complex, are required for spartin-mediated cytoprotection. Furthermore, spartin interacts with the glycolysis enhancer phospo-fructo-kinase-2,6 (Pfk26) and is sufficient to complement for PFK26-deficiency at least in early aging. We conclude that mitochondria-related energy metabolism is crucial for spartin's vital function during aging and uncover a network of specific interactors required for this function.

The steroid hormone progesterone is not only a crucial sex hormone, but also serves as a neurosteroid, thus playing an important role in brain function.Epidemiological data suggest that progesterone improves the recovery of patients after traumatic brain injury.Brain injuries are often connected to elevated calcium spikes, reactive oxygen species (ROS) and programmed cell death affecting neurons.Here, we establish a yeast model to study progesterone-mediated cytoprotection.External supply of progesterone protected yeast cells from apoptosis-inducing stress stimuli and resulted in elevated mitochondrial oxygen uptake accompanied by a drop in ROS generation and ATP levels during chronological aging.In addition, cellular Ca 2+ concentrations were reduced upon progesterone treatment, and this effect occurred independently of known Ca 2+ transporters and mitochondrial respiration.All effects were also independent of Dap1, the yeast orthologue of the progesterone receptor.Altogether, our observations provide new insights into the cytoprotective effects of progesterone.

The age-induced deterioration of the organism results in detrimental and ultimately lethal pathologies. The process of aging itself involves a plethora of different mechanisms that should be subverted concurrently to delay and/or prevent age-related maladies. We have identified a natural compound, 4,4'-dimethoxychalcone (DMC), which promotes longevity in yeast, worms and flies, and protects mice from heart injury and liver toxicity. Interestingly, both the DMC-mediated lifespan extension and the cardioprotection depend on macroautophagy/autophagy whereas hepatoprotection does not. DMC induces autophagy by inhibiting specific GATA transcription factors (TFs), independently of the TORC1 kinase pathway. The autophagy-independent beneficial effects of DMC might involve its antioxidative properties. DMC treatment results in a phylogenetically conserved, systemic impact on the metabolome, which is most prominently characterized by changes in cellular amino acid composition. Altogether, DMC exerts multiple, geroprotective effects by igniting distinct pathways, and thus represents a potential pharmacological agent that delays aging through multipronged effects.

Comment on: Palermo V, et al. Cell Cycle 2010; 9:3991-6.

An important paper by David Allis and colleagues (Ahn et al., 2006 [this issue of Molecular Cell]) describes a specific deacetylation/phosphorylation crosstalk at the histone H2B tail required for apoptosis induction in yeast, thus giving first insights into the poorly understood epigenetic regulation of cell death.

GATA transcription factors (TFs) are a conserved family of zinc-finger TFs that fulfill diverse functions across eukaryotes. Accumulating evidence suggests that GATA TFs also play a role in lifespan regulation. In a recent study, we have identified a natural compound, 4,4' dimethoxychalcone (DMC) that extends lifespan depending on reduced activity of distinct GATA TFs. Prolonged lifespan by DMC treatment depends on autophagy, a protective cellular self-cleaning mechanism. In yeast, DMC reduces the activity of the GATA TF Gln3 and, at the same time, deletion of GLN3 increases autophagy levels during cellular aging per se. Here, we examine current data on the involvement of GATA TFs in the regulation of both autophagy and lifespan in different organisms and explore, if GATA TFs are suitable targets for anti-aging interventions.

The polyamine spermidine is essential for protein translation in eukaryotes, both as a substrate for the hypusination of the translation initiation factor eIF5A as well as general translational fidelity. Dwindling spermidine levels during aging have been implicated in reduced immune cell function through insufficient eIF5A hypusination, which can be restored by external supplementation. Recent findings characterize a group of novel Mendelian disorders linked to EIF5A missense and nonsense variants that cause protein translation defects. In model organisms that recapitulate these mutations, spermidine supplementation was able to alleviate at least some of the concomitant protein translation defects. Here, we discuss the role of spermidine in protein translation and possible therapeutic avenues for translation-associated disorders.

Viral, bacterial, fungal and protozoal biology is of cardi-nal importance for the evolutionary history of life, ecol-ogy, biotechnology and infectious diseases. Various mi-crobiological model systems have fundamentally con-tributed to the understanding of molecular and cellular processes, including the cell cycle, cell death, mitochon-drial biogenesis, vesicular fusion and autophagy, among many others. Microbial interactions within the envi-ronment have profound effects on many fields of biolo-gy, from ecological diversity to the highly complex and multifaceted impact of the microbiome on human health. Also, biotechnological innovation and corre-sponding industrial operations strongly depend on mi-crobial engineering. With this wide range of impact in mind, the peer-reviewed and open access journal Mi-crobial Cell was founded in 2014 and celebrates its 100th issue this month. Here, we briefly summarize how the vast diversity of microbiological subjects influences our personal and societal lives and shortly review the mile-stones achieved by Microbial Cell during the last years.

Ethanolamine (Etn) is a naturally occurring aminoalcohol necessary for synthesis of the phospholipid phosphatidylethanolamine (PE), a major component of biological membranes. We recently reported that Etn treatment increases cellular PE levels, thereby inducing cytoprotective autophagy and protecting against aging across species.

The modulation of cell death is a complex process. Its deregulation can lead to several types of diseases ranging from cancer to neurodegenerative disorders in humans. The finding that Saccharomyces cerevisiae can also undergo apoptosis has opened doors to investigate programmed cell death in a clear-cut model organism that unifies both technical advantages and a eukaryotic “cell room.” So far, cell death in yeast has been described under multiple conditions, including exposure to different drugs, failure in several cellular processes, or heterologous expression of human proapoptotic genes. Yeast apoptosis has also been shown to occur in physiological scenarios such as aging or failed mating, thus suggesting a teleological explanation for the death of a unicellular organism. Finally, several yeast orthologues of crucial metazoan apoptotic regulators have been identified and characterized, including a caspase and the apoptosis-inducing factor. Uncovering apoptosis in yeast as well as in other fungi and unicellular parasites is of great medical interest. Moreover, it is helping to decipher the molecular mechanisms of cell death in higher organisms.

One cell, one love: a journal for microbial research – INTRODUCTION With their broad utility for biotechnology, their continuous menace as infectious pathogens, and as an integral part of our bodies (intestinal flora), unicellular organisms remain in the focus of global research. This interest has been further stimulated by the challenge to counteract the emergence of multi-resistant microbes, as well as by the recent advances in (...)

Chronological age represents the time that passes between birth and a given date. To understand the complex network of factors contributing to chronological lifespan, a variety of model organisms have been implemented. One of the best studied organisms is the yeast Saccharomyces cerevisiae, which has greatly contributed toward identifying conserved biological mechanisms that act on longevity. Here, we discuss high- und low-throughput protocols to monitor and characterize chronological lifespan and chronological aging-associated cell death in S. cerevisiae. Included are propidium iodide staining with the possibility to quantitatively assess aging-associated cell death via flow cytometry or qualitative assessments via microscopy, cell viability assessment through plating and cell counting and cell death characterization via propidium iodide/AnnexinV staining and subsequent flow cytometric analysis or microscopy. Importantly, all of these methods combined give a clear picture of the chronological lifespan under different conditions or genetic backgrounds and represent a starting point for pharmacological or genetic interventions.

This spring, more than a hundred scientists from around the world gathered in Canterbury, the historic city in the county of Kent in South East England, to attend the eighth International Meeting on Yeast Apoptosis (IMYA). As with every IMYA conference since its inception in 2002, the feeling of being part of a community that is almost a family was evident. In addition, this year's meeting has shown that the field of yeast programmed cell death (PCD) is growing, not only in numbers, but also in its thematic scope.

Unicellular organisms like yeast can undergo controlled demise in a manner that is partly reminiscent of mammalian cell death. This is true at the levels of both mechanistic and functional conservation. Yeast offers the combination of unparalleled genetic amenability and a comparatively simple biology to understand both the regulation and evolution of cell death. In this minireview, we address the capacity of the nucleus as a regulatory hub during yeast regulated cell death (RCD), which is becoming an increasingly central question in yeast RCD research. In particular, we explore and critically discuss the available data on stressors and signals that specifically impinge on the nucleus. Moreover, we also analyze the current knowledge on nuclear factors as well as on transcriptional control and epigenetic events that orchestrate yeast RCD. Altogether we conclude that the functional significance of the nucleus for yeast RCD in undisputable, but that further exploration beyond correlative work is necessary to disentangle the role of nuclear events in the regulatory network.

It was a unique atmosphere when 60 birds of paradise from all over the world gathered in Kutna Hora, an old silver-digger town near Prague. No justifications, no explanations, no excuses regarding the decision to work on such an exotic topic were offered. Instead, the community, which is almost a family since a core group of people and a growing number of others meet regularly to offer advice and help with plasmids and strains, suddenly realized that a turning point in the field had been reached. Prior to this meeting, most publications had demonstrated that yeast apoptosis (Madeo, 1997) is comparable to mammalian apoptosis. Here, for the first time, the majority of groups presented results that provided new insights into apoptosis research as a whole and that could give new directions to research in other model organisms or cell-culture systems. Chronological ageing of yeast, defined as the lifespan of a postdiauxic but still metabolic active culture, has been used as a valuable model with which to study oxidative damage and molecularly conserved ageing pathways of postmitotic tissues in higher organisms. Replicative ageing, defined by the number of daughters born from a single mother, represents a model for asymmetrically dividing stem cells. Both replicative and chronological ageing of yeast cells have been demonstrated to culminate in cell death with an apoptotic phenotype (Laun, 2001; Fabrizio, 2004; Herker, 2004). It was Ian Dawes (Sydney, Australia) who opened the meeting with an introduction about how reactive oxygen species (ROS) contribute to ageing by damaging nucleic acids, proteins and lipids. In addition, he gave details of a study aiming to evaluate the influence of diverse antioxidants on replicative lifespan and discussed the correlation between oxidative stress and replicative ageing (Laun, 2001). On a more methodological level, David Goldfarb (Rochester, USA) presented …

Autophagy is an intracellular recycling program that is ubiquitously present in eukaryotes, where it is crucial for the maintenance of cellular homeostasis. Thereby, superfluous or damaged proteins, organelles and cytoplasmic portions are targeted either in bulk or selectively for delivery to the lysosome (or vacuole in yeast). There, they are subsequently digested to provide substrates and building blocks. The core components as well as the regulatory circuits governing this well-orchestrated catabolic pathway are highly conserved among eukaryotes. The baker's yeast Saccharomyces cerevisiae has contributed fundamentally to the understanding of the molecular principles and the discovery of major regulators of autophagy, and remains a favorable model organism for the study of different aspects of this pathway. Here, we provide detailed protocols for two of the main established assays used to monitor autophagic activity in yeast, the green fluorescent protein (GFP) liberation assay and the alkaline phosphatase (ALP) activity assay. Notably, both assays follow the delivery of autophagosomal cargo into the yeast vacuole and thus provide a valuable tool for the measurement of actual autophagic flux. We further recapitulate experimental setups for both assays and provide information on how they can be used to study non-selective and selective forms of autophagy in yeast.