New genes tied to endocrine, metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans genomic RNAi screen

Most of our knowledge about the regulation of aging comes from mutants originally isolated for other phenotypes. To ask whether our current view of aging has been affected by selection bias, and to deepen our understanding of known longevity pathways, we screened a genomic Caenorhabditis elegans RNAi library for clones that extend lifespan. We identified 23 new longevity genes affecting signal transduction, the stress response, gene expression, and metabolism and assigned these genes to specific longevity pathways. Our most important findings are (i) that dietary restriction extends C. elegans' lifespan by down-regulating expression of key genes, including a gene required for methylation of many macromolecules, (ii) that integrin signaling is likely to play a general, evolutionarily conserved role in lifespan regulation, and (iii) that specific lipophilic hormones may influence lifespan in a DAF-16/FOXO-dependent fashion. Surprisingly, of the new genes that have conserved sequence domains, only one could not be associated with a known longevity pathway. Thus, our current view of the genetics of aging has probably not been distorted substantially by selection bias.     

The genetics of aging

Once thought to be an extremely complex conundrum of weak genetic and environmental effects, exceptional longevity is beginning to yield genetic findings. Numerous lower organism and mammalian models demonstrate genetic mutations that increase life-span markedly. These variations, some of them evolutionarily conserved, inform us about biochemical pathways that significantly impact upon longevity. Centenarian studies have also proven useful as they are a cohort that, relative to younger age groups, lacks genotypes linked to age-related lethal diseases and premature mortality. Pedigree studies have demonstrated a significant familial component to the ability to survive to extreme old age and a recent study demonstrates a locus on chromosome 4 linked to exceptional longevity indicating the likely existence of at least one longevity enabling gene in humans. Thus, a number of laboratories are making substantial and exciting strides in the understanding of the genetics of aging and longevity which should lead to the discovery of genes and ultimately drugs that slow down the aging process and facilitate people's ability to delay and perhaps escape age-associated diseases.     

Shortest-Path Network Analysis Is a Useful Approach toward Identifying Genetic Determinants of Longevity

Background

Identification of genes that modulate longevity is a major focus of aging-related research and an area of intense public interest. In addition to facilitating an improved understanding of the basic mechanisms of aging, such genes represent potential targets for therapeutic intervention in multiple age-associated diseases, including cancer, heart disease, diabetes, and neurodegenerative disorders. To date, however, targeted efforts at identifying longevity-associated genes have been limited by a lack of predictive power, and useful algorithms for candidate gene-identification have also been lacking.

Methodology/Principal Findings

We have utilized a shortest-path network analysis to identify novel genes that modulate longevity in Saccharomyces cerevisiae. Based on a set of previously reported genes associated with increased life span, we applied a shortest-path network algorithm to a pre-existing protein–protein interaction dataset in order to construct a shortest-path longevity network. To validate this network, the replicative aging potential of 88 single-gene deletion strains corresponding to predicted components of the shortest-path longevity network was determined. Here we report that the single-gene deletion strains identified by our shortest-path longevity analysis are significantly enriched for mutations conferring either increased or decreased replicative life span, relative to a randomly selected set of 564 single-gene deletion strains or to the current data set available for the entire haploid deletion collection. Further, we report the identification of previously unknown longevity genes, several of which function in a conserved longevity pathway believed to mediate life span extension in response to dietary restriction.

Conclusions/Significance

This work demonstrates that shortest-path network analysis is a useful approach toward identifying genetic determinants of longevity and represents the first application of network analysis of aging to be extensively validated in a biological system. The novel longevity genes identified in this study are likely to yield further insight into the molecular mechanisms of aging and age-associated disease.     

Evolutionary conservation of regulated longevity assurance mechanisms

Background

To what extent are the determinants of aging in animal species universal? Insulin/insulin-like growth factor (IGF)-1 signaling (IIS) is an evolutionarily conserved (public) regulator of longevity; yet it remains unclear whether the genes and biochemical processes through which IIS acts on aging are public or private (that is, lineage specific). To address this, we have applied a novel, multi-level cross-species comparative analysis to compare gene expression changes accompanying increased longevity in mutant nematodes, fruitflies and mice with reduced IIS.

Results

Surprisingly, there is little evolutionary conservation at the level of individual, orthologous genes or paralogous genes under IIS regulation. However, a number of gene categories are significantly enriched for genes whose expression changes in long-lived animals of all three species. Down-regulated categories include protein biosynthesis-associated genes. Up-regulated categories include sugar catabolism, energy generation, glutathione-S-transferases (GSTs) and several other categories linked to cellular detoxification (that is, phase 1 and phase 2 metabolism of xenobiotic and endobiotic toxins). Protein biosynthesis and GST activity have recently been linked to aging and longevity assurance, respectively.

Conclusion

These processes represent candidate, regulated mechanisms of longevity-control that are conserved across animal species. The longevity assurance mechanisms via which IIS acts appear to be lineage-specific at the gene level (private), but conserved at the process level (or semi-public). In the case of GSTs, and cellular detoxification generally, this suggests that the mechanisms of aging against which longevity assurance mechanisms act are, to some extent, lineage specific.     

Tetz’s theory and law of longevity

Here, we present new theory and law of longevity intended to evaluate fundamental factors that control lifespan. This theory is based on the fact that genes affecting host organism longevity are represented by subpopulations: genes of host eukaryotic cells, commensal microbiota, and non-living genetic elements. Based on Tetz’s theory of longevity, we propose that lifespan and aging are defined by the accumulation of alterations over all genes of macroorganism and microbiome and the non-living genetic elements associated with them. Tetz’s law of longevity states that longevity is limited by the accumulation of alterations to the limiting value that is not compatible with life. Based on theory and law, we also propose a novel model to calculate several parameters, including the rate of aging and the remaining lifespan of individuals. We suggest that this theory and model have explanatory and predictive potential to eukaryotic organisms, allowing the influence of diseases, medication, and medical procedures to be re-examined in relation to longevity. Such estimates also provide a framework to evaluate new fundamental aspects that control aging and lifespan.     

Longevity regulation in Saccharomyces cerevisiae: linking metabolism, genome stability, and heterochromatin.

When it was first proposed that the budding yeast Saccharomyces cerevisiae might serve as a model for human aging in 1959, the suggestion was met with considerable skepticism. Although yeast had proved a valuable model for understanding basic cellular processes in humans, it was difficult to accept that such a simple unicellular organism could provide information about human aging, one of the most complex of biological phenomena. While it is true that causes of aging are likely to be multifarious, there is a growing realization that all eukaryotes possess surprisingly conserved longevity pathways that govern the pace of aging. This realization has come, in part, from studies of S. cerevisiae, which has emerged as a highly informative and respected model for the study of life span regulation. Genomic instability has been identified as a major cause of aging, and over a dozen longevity genes have now been identified that suppress it. Here we present the key discoveries in the yeast-aging field, regarding both the replicative and chronological measures of life span in this organism. We discuss the implications of these findings not only for mammalian longevity but also for other key aspects of cell biology, including cell survival, the relationship between chromatin structure and genome stability, and the effect of internal and external environments on cellular defense pathways. We focus on the regulation of replicative life span, since recent findings have shed considerable light on the mechanisms controlling this process. We also present the specific methods used to study aging and longevity regulation in S. cerevisiae.     

Predicting aging/longevity-related genes in the nematode Caenorhabditis elegans

We present a novel mathematical/computational strategy for predicting genes/proteins associated with aging/longevity. The novelty of our method arises from the topological analysis of an organismal longevity gene/protein network (LGPN), which extends the existing cellular networks. The LGPN nodes represent both genes and corresponding proteins. Links stand for all known interactions between the nodes. The LGPN of C. elegans incorporated 362 genes/proteins, 160 connecting and 202 age-related ones, from a list of 321 with known impact on aging/longevity. A 'longevity core' of 129 directly interacting genes or proteins was identified. This core may shed light on the large-scale mechanisms of aging. Predictions were made, based upon the finding that LGPN hubs and centrally located nodes have higher likelihoods of being associated with aging/longevity than do randomly selected nodes. Analysis singled-out 15 potential aging/longevity-related genes for further examination: mpk-1, gei-4, csp-1, pal-1, mkk-4, 4O210, sem-5, gei-16, 1O814, 5M722, ife-3, ced-10, cdc-42, 1O776Co, and 1O690.     

Longevity Regulation in Saccharomyces cerevisiae: Linking Metabolism, Genome Stability, and Heterochromatin

When it was first proposed that the budding yeast Saccharomyces cerevisiae might serve as a model for human aging in 1959, the suggestion was met with considerable skepticism. Although yeast had proved a valuable model for understanding basic cellular processes in humans, it was difficult to accept that such a simple unicellular organism could provide information about human aging, one of the most complex of biological phenomena. While it is true that causes of aging are likely to be multifarious, there is a growing realization that all eukaryotes possess surprisingly conserved longevity pathways that govern the pace of aging. This realization has come, in part, from studies of S. cerevisiae, which has emerged as a highly informative and respected model for the study of life span regulation. Genomic instability has been identified as a major cause of aging, and over a dozen longevity genes have now been identified that suppress it. Here we present the key discoveries in the yeast-aging field, regarding both the replicative and chronological measures of life span in this organism. We discuss the implications of these findings not only for mammalian longevity but also for other key aspects of cell biology, including cell survival, the relationship between chromatin structure and genome stability, and the effect of internal and external environments on cellular defense pathways. We focus on the regulation of replicative life span, since recent findings have shed considerable light on the mechanisms controlling this process. We also present the specific methods used to study aging and longevity regulation in S. cerevisiae.     

Long-lived worms and aging

Several investigators have generated long-lived nematode worms (Caenorhabditis elegans) in the past decade by mutation of genes in the organism in order to study the genetics of aging and longevity. Dozens of longevity assurance genes (LAG) that dramatically increase the longevity of this organism have been identified. All long-lived mutants of C. elegans are also resistant to environmental stress, such as high temperature, reactive oxygen species (ROS), and ultraviolet irradiation. Double mutations of some LAGs further extended life span up to 400%, providing more insight into cellular mechanisms that put limits on the life span of organisms. With the availability of the LAG mutants and the combined DNA microarray and RNAi technology, the understanding of actual biochemical processes that determine life span is within reach: the downstream signal transduction pathway may regulate life span by up-regulating pro-longevity genes such as those that encode antioxidant enzymes and/or stress-response proteins, and down-regulating specific life-shortening genes. Furthermore, longevity could be modified through chemical manipulation. Results from these studies further support the free radical theory of aging, suggest that the molecular mechanism of aging process may be shared in all organisms, and provide insight for therapeutic intervention in age-related diseases.     

Human longevity: Genetics or Lifestyle? It takes two to tango

Healthy aging and longevity in humans are modulated by a lucky combination of genetic and non-genetic factors. Family studies demonstrated that about 25 % of the variation in human longevity is due to genetic factors. The search for genetic and molecular basis of aging has led to the identification of genes correlated with the maintenance of the cell and of its basic metabolism as the main genetic factors affecting the individual variation of the aging phenotype. In addition, studies on calorie restriction and on the variability of genes associated with nutrient-sensing signaling, have shown that ipocaloric diet and/or a genetically efficient metabolism of nutrients, can modulate lifespan by promoting an efficient maintenance of the cell and of the organism. Recently, epigenetic studies have shown that epigenetic modifications, modulated by both genetic background and lifestyle, are very sensitive to the aging process and can either be a biomarker of the quality of aging or influence the rate and the quality of aging.On the whole, current studies are showing that interventions modulating the interaction between genetic background and environment is essential to determine the individual chance to attain longevity.     

Genetics of healthy aging and longevity

Longevity and healthy aging are among the most complex phenotypes studied to date. The heritability of age at death in adulthood is approximately 25 %. Studies of exceptionally long-lived individuals show that heritability is greatest at the oldest ages. Linkage studies of exceptionally long-lived families now support a longevity locus on chromosome 3; other putative longevity loci differ between studies. Candidate gene studies have identified variants at APOE and FOXO3A associated with longevity; other genes show inconsistent results. Genome-wide association scans (GWAS) of centenarians vs. younger controls reveal only APOE as achieving genome-wide significance (GWS); however, analyses of combinations of SNPs or genes represented among associations that do not reach GWS have identified pathways and signatures that converge upon genes and biological processes related to aging. The impact of these SNPs, which may exert joint effects, may be obscured by gene-environment interactions or inter-ethnic differences. GWAS and whole genome sequencing data both show that the risk alleles defined by GWAS of common complex diseases are, perhaps surprisingly, found in long-lived individuals, who may tolerate them by means of protective genetic factors. Such protective factors may ‘buffer’ the effects of specific risk alleles. Rare alleles are also likely to contribute to healthy aging and longevity. Epigenetics is quickly emerging as a critical aspect of aging and longevity. Centenarians delay age-related methylation changes, and they can pass this methylation preservation ability on to their offspring. Non-genetic factors, particularly lifestyle, clearly affect the development of age-related diseases and affect health and lifespan in the general population. To fully understand the desirable phenotypes of healthy aging and longevity, it will be necessary to examine whole genome data from large numbers of healthy long-lived individuals to look simultaneously at both common and rare alleles, with impeccable control for population stratification and consideration of non-genetic factors such as environment.     

Aging and longevity genes

The genetics of aging has made substantial strides in the past decade. This progress has been confined primarily to model organisms, such as filamentous fungi, yeast, nematodes, fruit flies, and mice, in which some thirty-five genes that determine life span have been cloned. These genes encode a wide array of cellular functions, indicating that there must be multiple mechanisms of aging. Nevertheless, some generalizations are already beginning to emerge. It is now clear that there are at least four broad physiological processes that play a role in aging: metabolic control, resistance to stress, gene dysregulation, and genetic stability. The first two of these at least are common themes that connect aging in yeast, nematodes, and fruit flies, and this convergence extends to caloric restriction, which postpones senescence and increases life span in rodents. Many of the human homologs of the longevity genes found in model organisms have been identified. This will lead to their use as candidate human longevity genes in population genetic studies. The urgency for such studies is great: The population is graying, and this research holds the promise of improvement in the quality of the later years of life.     

Why genes extending lifespan in model organisms have not been consistently associated with human longevity and what it means to translation research

A recent paper by Deelen et al. (2014) in Human Molecular Genetics reports the largest genome-wide association study of human longevity to date. While impressive, there is a remarkable lack of association of genes known to considerably extend lifespan in rodents with human longevity, both in this latest study and in genetic association studies in general. Here, I discuss several possible explanations, such as intrinsic limitations in longevity association studies and the complex genetic architecture of longevity. Yet one hypothesis is that the lack of correlation between longevity-associated genes in model organisms and genes associated with human longevity is, at least partly, due to intrinsic limitations and biases in animal studies. In particular, most studies in model organisms are conducted in strains of limited genetic diversity which are then not applicable to human populations. This has important implications and, together with other recent results demonstrating strain-specific longevity effects in rodents due to caloric restriction, it questions our capacity to translate the exciting findings from the genetics of aging to human therapies.     

An intervention resembling caloric restriction prolongs life span and retards aging in yeast.

The yeast Saccharomyces cerevisiae has a finite life span that is measured by the number of daughter cells an individual produces. The 20 genes known to determine yeast life span appear to function in more than one pathway, implicating a variety of physiological processes in yeast longevity. Less attention has been focused on environmental effects on yeast aging. We have examined the role that nutritional status plays in determining yeast life span. Reduction of the glucose concentration in the medium led to an increase in life span and to a delay in appearance of an aging phenotype. The increase in life span was the more extensive the lower the glucose levels. Life extension was also elicited by decreasing the amino acids content of the medium. This suggests that it is the decline in calories and not a particular nutrient that is responsible, in striking similarity to the effect on aging of caloric restriction in mammals. The caloric restriction effect did not require the induction of the retrograde response pathway, which signals the functional status of the mitochondrion and determines longevity. Furthermore, deletion of RTG3, a downstream mediator in this pathway, and caloric restriction had an additive effect, resulting in the largest increase (123%) in longevity described thus far in yeast. Thus, retrograde response and caloric restriction operate along distinct pathways in determining yeast longevity. These pathways may be exclusive, at least in part. This provides evidence for multiple mechanisms of metabolic control in yeast aging. Inasmuch as caloric restriction lowers blood glucose levels, this study raises the possibility that reduced glucose alters aging at the cellular level in mammals.     

A genomic analysis of chronological longevity factors in budding yeast

Chronological life span (CLS) has been studied as an aging paradigm in yeast. A few conserved aging genes have been identified that modulate both chronological and replicative longevity in yeast as well as longevity in the nematode Caenorhabditis elegans; however, a comprehensive analysis of the relationship between genetic control of chronological longevity and aging in other model systems has yet to be reported. To address this question, we performed a functional genomic analysis of chronological longevity for 550 single-gene deletion strains, which accounts for approximately 12% of the viable homozygous diploid deletion strains in the yeast ORF deletion collection. This study identified 33 previously unknown determinants of CLS. We found no significant enrichment for enhanced CLS among deletions corresponding to yeast orthologs of worm aging genes or among replicatively long-lived deletion strains, although a trend toward overlap was noted. In contrast, a subset of gene deletions identified from a screen for reduced acidification of culture media during growth to stationary phase was enriched for increased CLS. These results suggest that genetic control of CLS under the most commonly utilized assay conditions does not strongly overlap with longevity determinants in C. elegans, with the existing confined to a small number of genetic pathways. These data also further support the model that acidification of the culture medium plays an important role in survival during chronological aging in synthetic medium, and suggest that chronological aging studies using alternate medium conditions may be more informative with regard to aging of multicellular eukaryotes.     

Age-specific changes in epistatic effects on mortality rate in Drosophila melanogaster

Models for the evolution of senescence assume that genes with age-specific effects act independently of one another. Although recent empirical data show that longevity is influenced in part by interactions between genes, there are currently few data on whether epistasis influences age-specific components of mortality. To gauge if and how interactions affect age-specific traits, we incorporated the Drosophila visible marker mutations ebony, forked, and purple into seven wild-caught strains of D. melanogaster to examine gene x genetic background interactions. We found significant natural genetic variation for longevity and baseline mortality rates. Gene x genetic background interactions were prevalent not only for longevity but also for baseline mortality rates and age-specific mortality rates. We conclude that gene x genetic background epistasis is prevalent for aging-related traits and could play a significant role in the evolution of aging. These results suggest that future genetic models for the evolution of aging should incorporate the effects of epistasis.     

A more efficient search strategy for aging genes based on connectivity

MOTIVATION: Many aging genes have been found from unbiased screens in model organisms. Genetic interventions promoting longevity are usually quantitative, while in many other biological fields (e.g. development) null mutations alone have been very informative. Therefore, in the case of aging the task is larger and the need for a more efficient genetic search strategy is especially strong. RESULTS: The topology of genetic and metabolic networks is organized according to a scale-free distribution, in which hubs with large numbers of links are present. We have developed a computational model of aging genes as the hubs of biological networks. The computational model shows that, after generalized damage, the function of a network with scale-free topology can be significantly restored by a limited intervention on the hubs. Analyses of data on aging genes and biological networks support the applicability of the model to biological aging. The model also might explain several of the properties of aging genes, including the high degree of conservation across different species. The model suggests that aging genes tend to have a higher number of connections and therefore supports a strategy, based on connectivity, for prioritizing what might otherwise be a random search for aging genes.     

Mitochondrial DNA involvement in human longevity

The main message of this review can be summarized as follows: aging and longevity, as complex traits having a significant genetic component, likely depend on a number of nuclear gene variants interacting with mtDNA variability both inherited and somatic. We reviewed the data available in the literature with particular attention to human longevity, and argued that what we hypothesize for aging and longevity could have a more general relevance and be extended to other age-related complex traits such as Alzheimer's and Parkinson's diseases. The genetics which emerges for complex traits, including aging and longevity, is thus even more complicated than previously thought, as epistatic interactions between nuclear gene polymorphisms and mtDNA variability (both somatic and inherited) as well as between mtDNA somatic mutations (tissue specific) and mtDNA inherited variants (haplogroups and sub-haplogroups) must be considered as additional players capable of explaining a part of the aging and longevity phenotype. To test this hypothesis is one of the main challenge in the genetics of aging and longevity in the next future.     

A genomic analysis of chronological longevity factors in budding yeast

Chronological life span (CLS) has been studied as an aging paradigm in yeast. A few conserved aging genes have been identified that modulate both chronological and replicative longevity in yeast as well as longevity in the nematode Caenorhabditis elegans; however, a comprehensive analysis of the relationship between genetic control of chronological longevity and aging in other model systems has yet to be reported. To address this question, we performed a functional genomic analysis of chronological longevity for 550 single-gene deletion strains, which accounts for approximately 12% of the viable homozygous diploid deletion strains in the yeast ORF deletion collection. This study identified 33 previously unknown determinants of CLS. We found no significant enrichment for enhanced CLS among deletions corresponding to yeast orthologs of worm aging genes or among replicatively long-lived deletion strains, although a trend toward overlap was noted. In contrast, a subset of gene deletions identified from a screen for reduced acidification of culture media during growth to stationary phase was enriched for increased CLS. These results suggest that genetic control of CLS under the most commonly utilized assay conditions does not strongly overlap with longevity determinants in C. elegans, with the existing confined to a small number of genetic pathways. These data also further support the model that acidification of the culture medium plays an important role in survival during chronological aging in synthetic medium, and suggest that chronological aging studies using alternate medium conditions may be more informative with regard to aging of multicellular eukaryotes.Key words: aging, genomic, screen, lifespan, yeast, C. elegans, pH, chronological, replicative