Julia Ring

Induction of autophagy by spermidine promotes longevity

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

A Stress-Responsive System for Mitochondrial Protein Degradation

We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive--Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function, and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular, and organismal viability.

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

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

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

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

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

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

Amyloid-β Peptide Induces Mitochondrial Dysfunction by Inhibition of Preprotein Maturation

Most mitochondrial proteins possess N-terminal presequences that are required for targeting and import into the organelle. Upon import, presequences are cleaved off by matrix processing peptidases and subsequently degraded by the peptidasome Cym1/PreP, which also degrades Amyloid-beta peptides (Aβ). Here we find that impaired turnover of presequence peptides results in feedback inhibition of presequence processing enzymes. Moreover, Aβ inhibits degradation of presequence peptides by PreP, resulting in accumulation of mitochondrial preproteins and processing intermediates. Dysfunctional preprotein maturation leads to rapid protein degradation and an imbalanced organellar proteome. Our findings reveal a general mechanism by which Aβ peptide can induce the multiple diverse mitochondrial dysfunctions accompanying Alzheimers disease.

Fatty acids trigger mitochondrion-dependent necrosis

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.

Loss of peroxisome function triggers necrosis

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.

Neurotoxic 43-kDa TAR DNA-binding Protein (TDP-43) Triggers Mitochondrion-dependent Programmed Cell Death in Yeast

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.

Endonuclease G mediates α‐synuclein cytotoxicity during Parkinson's disease

Malfunctioning of the protein α‐synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinsons 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.