SIN-3 transcriptional coregulator maintains mitochondrial homeostasis and polyamine flux.

Mitochondrial function relies on the coordinated transcription of mitochondrial and nuclear genomes to assemble respiratory chain complexes. Across species, the SIN3 coregulator influences mitochondrial functions, but how its loss impacts mitochondrial homeostasis and metabolism in the context of a whole organism is unknown. Exploring this link is important because SIN3 haploinsufficiency causes intellectual disability/autism syndromes and SIN3 plays a role in tumor biology. Here we show that loss of C. elegans SIN-3 results in transcriptional deregulation of mitochondrial- and nuclear-encoded mitochondrial genes, potentially leading to mito-nuclear imbalance. Consistent with impaired mitochondrial function, sin-3 mutants show extensive mitochondrial fragmentation by transmission electron microscopy (TEM) and in vivo imaging, and altered oxygen consumption. Metabolomic analysis of sin-3 mutant animals revealed a mitochondria stress signature and deregulation of methionine flux, resulting in decreased S-adenosyl methionine (SAM) and increased polyamine levels. Our results identify SIN3 as a key regulator of mitochondrial dynamics and metabolic flux, with important implications for human pathologies.

Considering Caenorhabditis elegans Aging on a Temporal and Tissue Scale: The Case of Insulin/IGF-1 Signaling.

The aging process is inherently complex, involving multiple mechanisms that interact at different biological scales. The nematode Caenorhabditis elegans is a simple model organism that has played a pivotal role in aging research following the discovery of mutations extending lifespan. Longevity pathways identified in C. elegans were subsequently found to be conserved and regulate lifespan in multiple species. These pathways intersect with fundamental hallmarks of aging that include nutrient sensing, epigenetic alterations, proteostasis loss, and mitochondrial dysfunction. Here we summarize recent data obtained in C. elegans highlighting the importance of studying aging at both the tissue and temporal scale. We then focus on the neuromuscular system to illustrate the kinetics of changes that take place with age. We describe recently developed tools that enabled the dissection of the contribution of the insulin/IGF-1 receptor ortholog DAF-2 to the regulation of worm mobility in specific tissues and at different ages. We also discuss guidelines and potential pitfalls in the use of these new tools. We further highlight the opportunities that they present, especially when combined with recent transcriptomic data, to address and resolve the inherent complexity of aging. Understanding how different aging processes interact within and between tissues at different life stages could ultimately suggest potential intervention points for age-related diseases.

Loss of SET1/COMPASS methyltransferase activity reduces lifespan and fertility in Caenorhabditis elegans.

Changes in histone post-translational modifications are associated with aging through poorly defined mechanisms. Histone 3 lysine 4 (H3K4) methylation at promoters is deposited by SET1 family methyltransferases acting within conserved multiprotein complexes known as COMPASS. Previous work yielded conflicting results about the requirement for H3K4 methylation during aging. Here, we reassessed the role of SET1/COMPASS-dependent H3K4 methylation in Caenorhabditis elegans lifespan and fertility by generating set-2(syb2085) mutant animals that express a catalytically inactive form of SET-2, the C. elegans SET1 homolog. We show that set-2(syb2085) animals retain the ability to form COMPASS, but have a marked global loss of H3K4 di- and trimethylation (H3K4me2/3). Reduced H3K4 methylation was accompanied by loss of fertility, as expected; however, in contrast to earlier studies, set-2(syb2085) mutants displayed a significantly shortened, not extended, lifespan and had normal intestinal fat stores. Other commonly used set-2 mutants were also short-lived, as was a cfp-1 mutant that lacks the SET1/COMPASS chromatin-targeting component. These results challenge previously held views and establish that WT H3K4me2/3 levels are essential for normal lifespan in C. elegans.

Auxin confers protection against ER stress in Caenorhabditis elegans.

Auxins are plant growth regulators that influence most aspects of plant development through complex mechanisms. The development of an auxin-inducible degradation (AID) system has enabled rapid, conditional protein depletion in yeast and cultured cells. More recently, the system was successfully adapted to Caenorhabditiselegans to achieve auxin-dependent degradation of targets in all tissues and developmental stages. Whether auxin treatment alone has an impact on nematode physiology is an open question. Here we show that indole-3-acetic acid (IAA), the auxin most commonly used to trigger AID in worms, functions through the conserved IRE-1/XBP-1 branch of the Unfolded Protein Response (UPR) to promote resistance to endoplasmic reticulum (ER) stress. Because the UPR not only plays a central role in restoring ER homeostasis, but also promotes lipid biosynthesis and regulates lifespan, we suggest that extreme caution should be exercised when using the AID system to study these and related processes.

Histone Methylation and Memory of Environmental Stress.

Cellular adaptation to environmental stress relies on a wide range of tightly controlled regulatory mechanisms, including transcription. Changes in chromatin structure and organization accompany the transcriptional response to stress, and in some cases, can impart memory of stress exposure to subsequent generations through mechanisms of epigenetic inheritance. In the budding yeast Saccharomyces cerevisiae, histone post-translational modifications, and in particular histone methylation, have been shown to confer transcriptional memory of exposure to environmental stress conditions through mitotic divisions. Recent evidence from Caenorhabditis elegans also implicates histone methylation in transgenerational inheritance of stress responses, suggesting a more widely conserved role in epigenetic memory.

HIF-1-dependent regulation of lifespan in Caenorhabditis elegans by the acyl-CoA-binding protein MAA-1.

In yeast, the broadly conserved acyl-CoA-binding protein (ACBP) is a negative regulator of stress resistance and longevity. Here, we have turned to the nematode C. elegans as a model organism in which to determine whether ACBPs play similar roles in multicellular organisms. We systematically inactivated each of the seven C. elegans ACBP paralogs and found that one of them, maa-1 (which encodes membrane-associated ACBP 1), is indeed involved in the regulation of longevity. In fact, loss of maa-1 promotes lifespan extension and resistance to different types of stress. Through genetic and gene expression studies we have demonstrated that HIF-1, a master transcriptional regulator of adaptation to hypoxia, plays a central role in orchestrating the anti-aging response induced by MAA-1 deficiency. This response relies on the activation of molecular chaperones known to contribute to maintenance of the proteome. Our work extends to C. elegans the role of ACBP in aging, implicates HIF-1 in the increase of lifespan of maa-1-deficient worms, and sheds light on the anti-aging function of HIF-1. Given that both ACBP and HIF-1 are highly conserved, our results suggest the possible involvement of these proteins in the age-associated decline in proteostasis in mammals.

The Role of Dafachronic Acid Signaling in Development and Longevity in Caenorhabditis elegans: Digging Deeper Using Cutting-Edge Analytical Chemistry.

Steroid hormones regulate physiological processes in species ranging from plants to humans. A wide range of steroid hormones exist, and their contributions to processes, such as growth, reproduction, development, and aging, is almost always complex. Understanding the biosynthetic pathways that generate steroid hormones and the signaling pathways that mediate their effects is thus of fundamental importance. In this work, we review recent advances in (i) the biological role of steroid hormones in the roundworm Caenorhabditis elegans and (ii) the development of novel methods to facilitate the detection and identification of these molecules. Our current understanding of steroid signaling in this simple organism serves to illustrate the challenges we face moving forward. First, it seems clear that we have not yet identified all of the enzymes responsible for steroid biosynthesis and/or degradation. Second, perturbation of steroid signaling affects a wide range of phenotypes, and subtly different steroid molecules can have distinct effects. Finally, steroid hormone levels are critically important, and minute variations in quantity can profoundly impact a phenotype. Thus, it is imperative that we develop innovative analytical tools and combine them with cutting-edge approaches including comprehensive and highly selective liquid chromatography coupled to mass spectrometry based on new methods such as supercritical fluid chromatography coupled to mass spectrometry (SFC-MS) if we are to obtain a better understanding of the biological functions of steroid signaling.

Serine- and threonine/valine-dependent activation of PDK and Tor orthologs converge on Sch9 to promote aging.

Dietary restriction extends longevity in organisms ranging from bacteria to mice and protects primates from a variety of diseases, but the contribution of each dietary component to aging is poorly understood. Here we demonstrate that glucose and specific amino acids promote stress sensitization and aging through the differential activation of the Ras/cAMP/PKA, PKH1/2 and Tor/S6K pathways. Whereas glucose sensitized cells through a Ras-dependent mechanism, threonine and valine promoted cellular sensitization and aging primarily by activating the Tor/S6K pathway and serine promoted sensitization via PDK1 orthologs Pkh1/2. Serine, threonine and valine activated a signaling network in which Sch9 integrates TORC1 and Pkh signaling via phosphorylation of threonines 570 and 737 and promoted intracellular relocalization and transcriptional inhibition of the stress resistance protein kinase Rim15. Because of the conserved pro-aging role of nutrient and growth signaling pathways in higher eukaryotes, these results raise the possibility that similar mechanisms contribute to aging in mammals.

Chronological aging in Saccharomyces cerevisiae.

The two paradigms to study aging in Saccharomyces cerevisiae are the chronological life span (CLS) and the replicative life span (RLS). The chronological life span is a measure of the mean and maximum survival time of non-dividing yeast populations while the replicative life span is based on the mean and maximum number of daughter cells generated by an individual mother cell before cell division stops irreversibly. Here we review the principal discoveries associated with yeast chronological aging and how they are contributing to the understanding of the aging process and of the molecular mechanisms that may lead to healthy aging in mammals. We will focus on the mechanisms of life span regulation by the Tor/Sch9 and the Ras/adenylate Ras/adenylate cyclase/PKA pathways with particular emphasis on those implicating age-dependent oxidative oxidative stress stress and DNA damage/repair.

Endosomal protein sorting and autophagy genes contribute to the regulation of yeast life span.

Accumulating evidence from various organisms points to a role for autophagy in the regulation of life span. By performing a genome-wide screen to identify novel life span determinants in Saccharomyces cerevisiae, we have obtained further insights into the autophagy-related and -unrelated degradation processes that may be important for preventing cellular senescence. The generation of multivesicular bodies and their fusion with the vacuole in the endosomal pathway emerged as novel cell functions involved in yeast chronological survival and longevity extension.

Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation.

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

Comparative analyses of time-course gene expression profiles of the long-lived sch9Delta mutant.

In an attempt to elucidate the underlying longevity-promoting mechanisms of mutants lacking SCH9, which live three times as long as wild type chronologically, we measured their time-course gene expression profiles. We interpreted their expression time differences by statistical inferences based on prior biological knowledge, and identified the following significant changes: (i) between 12 and 24 h, stress response genes were up-regulated by larger fold changes and ribosomal RNA (rRNA) processing genes were down-regulated more dramatically; (ii) mitochondrial ribosomal protein genes were not up-regulated between 12 and 60 h as wild type were; (iii) electron transport, oxidative phosphorylation and TCA genes were down-regulated early; (iv) the up-regulation of TCA and electron transport was accompanied by deep down-regulation of rRNA processing over time; and (v) rRNA processing genes were more volatile over time, and three associated cis-regulatory elements [rRNA processing element (rRPE), polymerase A and C (PAC) and glucose response element (GRE)] were identified. Deletion of AZF1, which encodes the transcriptional factor that binds to the GRE element, reversed the lifespan extension of sch9Delta. The significant alterations in these time-dependent expression profiles imply that the lack of SCH9 turns on the longevity programme that extends the lifespan through changes in metabolic pathways and protection mechanisms, particularly, the regulation of aerobic respiration and rRNA processing.

Tor1/Sch9-regulated carbon source substitution is as effective as calorie restriction in life span extension.

The effect of calorie restriction (CR) on life span extension, demonstrated in organisms ranging from yeast to mice, may involve the down-regulation of pathways, including Tor, Akt, and Ras. Here, we present data suggesting that yeast Tor1 and Sch9 (a homolog of the mammalian kinases Akt and S6K) is a central component of a network that controls a common set of genes implicated in a metabolic switch from the TCA cycle and respiration to glycolysis and glycerol biosynthesis. During chronological survival, mutants lacking SCH9 depleted extracellular ethanol and reduced stored lipids, but synthesized and released glycerol. Deletion of the glycerol biosynthesis genes GPD1, GPD2, or RHR2, among the most up-regulated in long-lived sch9Delta, tor1Delta, and ras2Delta mutants, was sufficient to reverse chronological life span extension in sch9Delta mutants, suggesting that glycerol production, in addition to the regulation of stress resistance systems, optimizes life span extension. Glycerol, unlike glucose or ethanol, did not adversely affect the life span extension induced by calorie restriction or starvation, suggesting that carbon source substitution may represent an alternative to calorie restriction as a strategy to delay aging.

Chronological aging-induced apoptosis in yeast.

Saccharomyces cerevisiae is the simplest among the major eukaryotic model organisms for aging and diseases. Longevity in the chronological life span paradigm is measured as the mean and maximum survival period of populations of non-dividing yeast. This paradigm has been used successfully to identify several life-regulatory genes and three evolutionary conserved pro-aging pathways. More recently, Schizosaccharomyces pombe has been shown to age chronologically in a manner that resembles that of S. cerevisiae and that depends on the activity of the homologues of two pro-aging proteins previously identified in the budding yeast. Both yeast show features of apoptotic death during chronological aging. Here, we review some fundamental aspects of the genetics of chronological aging and the overlap between yeast aging and apoptotic processes with particular emphasis on the identification of an aging/death program that favors the dedifferentiation and regrowth of a few better adapted mutants generated within populations of aging S. cerevisiae. We also describe the use of a genome-wide screening technique to gain further insights into the mechanisms of programmed death in populations of chronologically aging S. cerevisiae.

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

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

Life span extension by calorie restriction depends on Rim15 and transcription factors downstream of Ras/PKA, Tor, and Sch9.

Calorie restriction (CR), the only non-genetic intervention known to slow aging and extend life span in organisms ranging from yeast to mice, has been linked to the down-regulation of Tor, Akt, and Ras signaling. In this study, we demonstrate that the serine/threonine kinase Rim15 is required for yeast chronological life span extension caused by deficiencies in Ras2, Tor1, and Sch9, and by calorie restriction. Deletion of stress resistance transcription factors Gis1 and Msn2/4, which are positively regulated by Rim15, also caused a major although not complete reversion of the effect of calorie restriction on life span. The deletion of both RAS2 and the Akt and S6 kinase homolog SCH9 in combination with calorie restriction caused a remarkable 10-fold life span extension, which, surprisingly, was only partially reversed by the lack of Rim15. These results indicate that the Ras/cAMP/PKA/Rim15/Msn2/4 and the Tor/Sch9/Rim15/Gis1 pathways are major mediators of the calorie restriction-dependent stress resistance and life span extension, although additional mediators are involved. Notably, the anti-aging effect caused by the inactivation of both pathways is much more potent than that caused by CR.

Significant and systematic expression differentiation in long-lived yeast strains.

BACKGROUND: Recent studies suggest that the regulation of longevity may be partially conserved in many eukaryotes ranging from yeast to mammals. The three yeast mutants sch9Delta, ras2Delta, tor1Delta show extended chronological life span up to three folds. Our aim is to dissect the mechanisms that lead to the yeast life span extension. METHODOLOGY/PRINCIPAL FINDINGS: We obtain gene expression profiles of sch9Delta, ras2Delta, tor1Delta as well as that for a wild type at day 2.5 in SDC medium using Affymetrix Yeast2.0 arrays. To accurately estimate the expression differentiation between the wild type and the long-lived mutants, we use sub-array normalization followed by a variant of the median-polishing summarization. The results are validated by the probe sets of S. pombe on the same chips. To translate the differentiation into changes of biological activities, we make statistical inference by integrating the expression profiles with biological gene subsets defined by Gene Ontology, KEGG pathways, and cellular localization of proteins. Other than subset-versus-other comparisons, we also make local comparisons between two directly-related gene subsets such as cytosolic and mitochondrial ribosomes. Our consensus is obtained by cross-examination of these inferences. The significant and systematic differentiation in the three long-lived strains includes: lower transcriptional activities; down-regulation of TCA cycle and oxidative phosphorylation versus up-regulation of the KEGG pathway Glycolysis/Gluconeogenesis; the overall reduction of mitochondrial activities. We also report some different expression patterns such as reduction of the activities relating to mitosis in ras2Delta. CONCLUSIONS/SIGNIFICANCE: The modification of energy pathways and modification of compartment activities such as down-regulation of mitochondrial ribosome proteins versus up-regulation of cytosolic ribosome proteins are directly associated with the life span extension in yeast. The results provide a new and systematic S. cerevisiae version of the free radical theory from the perspective of functional genomics.

Inference of transcription modification in long-live yeast strains from their expression profiles.

BACKGROUND: Three kinases: Sch9, PKA and TOR, are suggested to be involved in both the replicative and chronological ageing in yeast. They function in pathways whose down-regulation leads to life span extension. Several stress response proteins, including two transcription factors Msn2 and Msn4, mediate the longevity extension phenotype associated with decreased activity of either Sch9, PKA, or TOR. However, the mechanisms of longevity, especially the underlying transcription program have not been fully understood. RESULTS: We measured the gene expression profiles in wild type yeast and three long-lived mutants: sch9Delta, ras2Delta, and tor1Delta. To elucidate the transcription program that may account for the longevity extension, we identified the transcription factors that are systematically and significantly associated with the expression differentiation in these mutants with respect to wild type by integrating microarray expression data with motif and ChIP-chip data, respectively. Our analysis suggests that three stress response transcription factors, Msn2, Msn4 and Gis1, are activated in all the three mutants. We also identify some other transcription factors such as Fhl1 and Hsf1, which may also be involved in the transcriptional modification in the long-lived mutants. CONCLUSION: Combining microarray expression data with other data sources such as motif and ChIP-chip data provides biological insights into the transcription modification that leads to life span extension. In the chronologically long-lived mutant: sch9Delta, ras2Delta, and tor1Delta, several common stress response transcription factors are activated compared with the wild type according to our systematic transcription inference.

The chronological life span of Saccharomyces cerevisiae.

The chronological life span of yeast, which is measured as the survival time of populations of nondividing cells, has been used successfully for the identification of key pathways responsible for the regulation of aging. These pathways have remarkable similarities with those that regulate the life span in higher eukaryotes, suggesting that longevity depends on the activity of genes and signaling pathways that share a common evolutionary origin. Thus, the unicellular Saccharomyces cerevisiae is a simple model system that can provide significant insights into the human genetics and molecular biology of aging. Here, we describe the standard procedures to measure the chronological life span, including both the normal and calorie restriction paradigms.