Publications from researchers at the Buck Institute for Research on Aging http://www.buckinstitute.org/kennedyLab © 2011 Buck Institute, All Rights Reserved In the past several decades the budding yeast Saccharomyces cerevisiae has emerged as a prominent model for aging research. The creation of a singlegene deletion collection covering the majority of open reading frames in the yeast genome and advances in genomic technologies have opened yeast research to genomescale screens for a variety of phenotypes. A number of screens have been performed looking for genes that modify secondary ageassociated phenotypes such as stress resistance or growth rate. More recently, moderatethroughput methods for measuring replicative life spanreplicative life span and highthroughput methods for measuring chronological life spanchronological life span have allowed for the first unbiased screens aimed at directly identifying genes involved in determining yeast longevity. In this chapter we discuss largescale life span studies performed in yeast and their implications for research related to the basic biology of aging. http://www.ncbi.nlm.nih.gov/pubmed/22094426 Sat, 31 Dec 2011 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/22214665 Sat, 31 Dec 2011 00:00:00 -0800 In Saccharomyces cerevisiae, 59 of the 78 ribosomal proteins are encoded by duplicated genes that, in most cases, encode identical or very similar protein products. However, different sets of ribosomal protein genes have been identified in screens for various phenotypes, including lifespan, budding pattern and drug sensitivities. Due to potential suppressors of growth rate defects among this set of strains in the ORF deletion collection, we regenerated the entire set of haploid ribosomal protein gene deletion strains in a clean genetic background. The new strains were used to create double deletions lacking both paralogs, allowing us to define a set of 14 nonessential ribosomal proteins. Replicative lifespan analysis of new strains corresponding to ORF deletion collection strains that likely carried suppressors of growth defects identified 12 new yeast replicative aging genes. Treatment of the collection of ribosomal protein gene deletion strains with tunicamycin revealed a significant correlation between slow growth and resistance to ER stress that was recapitulated by reducing translation of wildtype yeast with cycloheximide. Interestingly, enhanced tunicamycin resistance in ribosomal protein gene deletion mutants was independent of the unfolded protein response transcription factor Hac1. These data support a model in which reduced translation is protective against ER stress by a mechanism distinct from the canonical ER stress response pathway and further add to the diverse yet specific phenotypes associated with ribosomal protein gene deletions. http://www.ncbi.nlm.nih.gov/pubmed/22377630 Sat, 31 Dec 2011 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/22390715 Sat, 31 Dec 2011 00:00:00 -0800 Laminopathies, caused by mutations in Atype nuclear lamins, encompass a range of diseases, including forms of progeria and muscular dystrophy. In this issue, Chen etal. provide evidence that elevated expression of the nuclear inner membrane protein SUN1 drives pathology in multiple laminopathies. http://www.ncbi.nlm.nih.gov/pubmed/22541423 Sat, 31 Dec 2011 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/22508266 Sat, 31 Dec 2011 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/22461595 Sat, 31 Dec 2011 00:00:00 -0800 Yeast mother cells pay a sacrifice during budding: they keep the extrachromosomal rDNA circles (ERCs) so that their buds have a full lifespan ahead. Two new studies indicate that retention of ERCs in mother cells occurs not by tethering to the nuclear periphery but rather by the simple rules of diffusion in a timelimited and complex landscape. http://www.ncbi.nlm.nih.gov/pubmed/21334292 Mon, 31 Jan 2011 00:00:00 -0800 Studies in invertebrate model organisms have led to a wealth of knowledge concerning the ageing process. But which of these discoveries will apply to ageing in humans Recently, an assessment of the degree of conservation of ageing pathways between two of the leading invertebrate model organisms, Saccharomyces cerevisiae and Caenorhabditis elegans, was completed. The results (i) quantitatively indicated that pathways were conserved between evolutionarily disparate invertebrate species and (ii) emphasized the importance of the TOR kinase pathway in ageing. With recent findings that deletion of the mTOR substrate S6K1 or exposure of mice to the mTOR inhibitor rapamycin result in lifespan extension, mTOR signalling has become a major focus of ageing research. Here, we address downstream targets of mTOR signalling and their possible links to ageing. We also briefly cover other ageing genes identified by comparing worms and yeast, addressing the likelihood that their mammalian counterparts will affect longevity. http://www.ncbi.nlm.nih.gov/pubmed/21115526 Fri, 31 Dec 2010 00:00:00 -0800 Reduced fecundity has been associated with some alleles that enhance longevity in invertebrate and mammalian models. This observation has been suggested to support the antagonistic pleiotropy theory of aging, which predicts that alleles of some genes promoting fitness early in life have detrimental effects later in life that limit survival. In only a few cases, however, has the relative fitness of longlived mutants been quantified through direct competition with the wild type genotype. Here we report the first comprehensive analysis of longevity/fitness tradeoffs by measuring the relative fitness of 49 longlived yeast variants in a direct competition assay with wild type cells. We find that 32 (65) of these variants show a significant defect in fitness in this competition assay. In 26 (81) of these cases, this reduction in fitness can be partially accounted for by reduced maximal growth rate during early life, usually resulting from a G0/G1specific cell cycle defect. A majority of the less fit longevityenhancing variants are associated with reduced mRNA translation. These findings are therefore consistent with the idea that enhanced longevity often comes with a fitness cost and suggest that this cost is often associated with variation in a subset of longevity factors, such as those regulating mRNA translation, growth, and reproduction. http://www.ncbi.nlm.nih.gov/pubmed/21191185 Fri, 31 Dec 2010 00:00:00 -0800 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 singlegene 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 longlived 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. http://www.ncbi.nlm.nih.gov/pubmed/21447998 Fri, 31 Dec 2010 00:00:00 -0800 Although recent publications have linked the molecular events driving facioscapulohumeral muscular dystrophy (FSHD) to expression of the double homeobox transcription factor DUX4, overexpression of FRG1 has been proposed as one alternative causal agent as mice overexpressing FRG1 present with muscular dystrophy. Here, we characterize proliferative defects in two independent myoblast lines overexpressing FRG1. Myoblasts isolated from thigh muscle of FRG1 transgenic mice, an affected dystrophic muscle, exhibit delayed proliferation as measured by decreased clone size, whereas myoblasts isolated from the unaffected diaphragm muscle proliferated normally. To confirm the observation that overexpression of FRG1 could impair myoblast proliferation, we examined C2C12 myoblasts with inducible overexpression of FRG1, finding increased doubling time and G1phase cells in mass culture after induction of FRG1 and decreased levels of pRb phosphorylation. We propose that depressed myoblast proliferation may contribute to the pathology of mice overexpressing FRG1 and may play a part in FSHD. http://www.ncbi.nlm.nih.gov/pubmed/21603621 Fri, 31 Dec 2010 00:00:00 -0800 The "Trinations Aging Symposium" was held on the campus of Guangdong Medical College in Dongguan, China from April 28 to 30, 2011. The goal was to promote interaction, collaboration, and exchange of ideas between scientists in the field of aging research from Japan, South Korea, and China. Aging research is on the rise in Asia. This represents an important development, since Korea and Japan are the two longestlived countries in the world, and life expectancy is increasing rapidly in China and other Asian countries. The world will see a greater percentage of people over age 65 in coming years than any period in human history. Developing therapeutic approaches to increase healthspan has the potential not only to enhance quality of life, but would also help stem the looming economic crisis associated with a high percentage of elderly. The focus of the Trinations Aging Symposium was on the basic biology of aging, and topics discussed included genome maintenance, metabolism and aging, longevity genes and interventions, and new therapies for agerelated diseases. The meeting finished with a commitment for another symposium next year that will include additional Asian countries and the formation of a new scientific organization, the Asian Association for Aging Research. http://www.ncbi.nlm.nih.gov/pubmed/21771608 Fri, 31 Dec 2010 00:00:00 -0800 Defining the molecular events that precipitate multisystem decline is an important component of aging research. In this issue, Jin etal. (2011) show that increased expression of the histone demethylase, utx1, causes genomewide decreases in histone H3K27 trimethylation, which includes the insulin/IGF1 signaling (IIS) pathway that promotes aging. http://www.ncbi.nlm.nih.gov/pubmed/21803283 Fri, 31 Dec 2010 00:00:00 -0800 Activation of Sir2orthologs is proposed to increase lifespan downstream of dietary restriction (DR). Here we describe an examination of the effect of 32 different lifespanextending mutations and four methods of dietary restriction on replicative lifespan (RLS) in the shortlived sir2 yeast strain. In every case, deletion of SIR2 prevented RLS extension however, RLS extension was restored when both SIR2 and FOB1 were deleted in several cases, demonstrating that SIR2 is not directly required for RLS extension. These findings indicate that suppression of the sir2 lifespan defect is a rare phenotype among longevity interventions and suggest that sir2 cells senesce rapidly by a mechanism distinct from that of wildtype cells. They also demonstrate that failure to observe life span extension in a shortlived background, such as cells or animals lacking sirtuins, should be interpreted with caution. 2011 The Authors Aging Cell 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland. http://www.ncbi.nlm.nih.gov/pubmed/21902802 Fri, 31 Dec 2010 00:00:00 -0800 Aging is characterized by the accumulation of damaged cellular macromolecules caused by declining repair and elimination pathways. An integral component employed by cells to counter toxic protein aggregates is the conserved ubiquitin/proteasome system (UPS). Previous studies have described an agedependent decline of proteasomal function and increased longevity correlates with sustained proteasome capacity in centenarians and in naked mole rats, a longlived rodent. Proof for a direct impact of enhanced proteasome function on longevity, however, is still lacking. To determine the importance of proteasome function in yeast aging, we established a method to modulate UPS capacity by manipulating levels of the UPSrelated transcription factor Rpn4. While cells lacking RPN4 exhibit a decreased nonadaptable proteasome pool, loss of UBR2, an ubiquitin ligase that regulates Rpn4 turnover, results in elevated Rpn4 levels, which upregulates UPS components. Increased UPS capacity significantly enhances replicative lifespan (RLS) and resistance to proteotoxic stress, while reduced UPS capacity has opposing consequences. Despite tight transcriptional coregulation of the UPS and oxidative detoxification systems, the impact of proteasome capacity on lifespan is independent of the latter, since elimination of Yap1, a key regulator of the oxidative stress response, does not affect lifespan extension of cells with higher proteasome capacity. Moreover, since elevated proteasome capacity results in improved clearance of toxic huntingtin fragments in a yeast model for neurodegenerative diseases, we speculate that the observed lifespan extension originates from prolonged elimination of damaged proteins in old mother cells. Epistasis analyses indicate that proteasomemediated modulation of lifespan is at least partially distinct from dietary restriction, Tor1, and Sir2. These findings demonstrate that UPS capacity determines yeast RLS by a mechanism that is distinct from known longevity pathways and raise the possibility that interventions to promote enhanced proteasome function will have beneficial effects on longevity and agerelated disease in humans. http://www.ncbi.nlm.nih.gov/pubmed/21931558 Fri, 31 Dec 2010 00:00:00 -0800 http://www.ncbi.nlm.nih.gov/pubmed/22157188 Fri, 31 Dec 2010 00:00:00 -0800 In this, the fourth installment of our annual Hot Topics review on mRNA translation and aging, we have decided to expand our scope to include recent findings related to the role of TOR signaling in aging. As new data emerge, it is clear that TOR signaling acts upstream of mRNA translation, as well as a variety of other cellular processes, to modulate longevity and healthspan in evolutionarily diverse species. This Hot Topics review will cover important new findings in this area that have occurred over the past year. These include the demonstration that the TOR substrate ribosomal S6 kinase modulates longevity in mammals, the potential for TOR inhibitors as therapeutic treatments for Alzheimer's disease, and further studies emphasizing the importance of differential translation of specific mRNAs for healthy aging and enhanced longevity. http://www.ncbi.nlm.nih.gov/pubmed/21176090 Tue, 30 Nov 2010 00:00:00 -0800 Signaling by target of rapamycin (mTOR in mammals) has been shown to modulate lifespan in several model organisms ranging from yeast to mice. In mice, reduced mTOR signaling by chronic rapamycin treatment leads to life span extension, raising the possibility that rapamycin and its analogs may benefit the aging brain and serve as effective treatments of agerelated neurodegenerative diseases. Here, we review mTOR signaling and how neurons utilize mTOR to regulate brain function, including regulation of feeding, synaptic plasticity and memory formation. Additionally, we discuss recent findings that evaluate the mechanisms by which reduced mTOR activity might benefit the aging brain in normal and pathological states. We will focus on recent studies investigating mTOR and Alzheimer's disease, Parkinson's disease, and polyglutamine expansion syndromes such as Huntington's disease. http://www.ncbi.nlm.nih.gov/pubmed/20849946 Tue, 31 Aug 2010 00:00:00 -0700 In this issue of Molecular Cell, Feser etal. (2010) show that aging yeast lose chromatinassociated histones and, furthermore, that correcting this deficiency robustly enhances replicative life span, indicating that loss of normal chromatin packing contributes to the aging process. http://www.ncbi.nlm.nih.gov/pubmed/20832716 Tue, 31 Aug 2010 00:00:00 -0700 Although AIMP3/p18 is normally associated with the macromolecular tRNA synthetase complex, recent reports have revealed a new role of AIMP3 in tumor suppression. In this study, we generated a transgenic mouse that overexpresses AIMP3 and characterized the associated phenotype in vivo and in vitro. Surprisingly, the AIMP3 transgenic mouse exhibited a progeroid phenotype, and the cells that overexpressed AIMP3 showed accelerated senescence and defects in nuclear morphology. We found that overexpression of AIMP3 resulted in proteasomedependent degradation of mature lamin A, but not of lamin C, prelamin A, or progerin. The resulting imbalance in the protein levels of lamin A isoforms, namely altered stoichiometry of prelamin A and progerin to lamin A, appeared to be responsible for a phenotype that resembled progeria. An increase in the level of endogenous AIMP3 has been observed in aged human tissues and cells. The findings in this report suggest that AIMP3 is a specific regulator of mature lamin A and imply that enhanced expression of AIMP3 might be a factor driving cellular and/or organismal aging. http://www.ncbi.nlm.nih.gov/pubmed/20726853 Tue, 31 Aug 2010 00:00:00 -0700 One of the many debated topics in ageing research is whether progeroid syndromes are really accelerated forms of human ageing. The answer requires a better understanding of the normal ageing process and the molecular pathology underlying these rare diseases. Exciting recent findings regarding a severe human progeria, HutchinsonGilford progeria syndrome, have implicated molecular changes that are also linked to normal ageing, such as genome instability, telomere attrition, premature senescence and defective stem cell homeostasis in disease development. These observations, coupled with genetic studies of longevity, lead to a hypothesis whereby progeria syndromes accelerate a subset of the pathological changes that together drive the normal ageing process. http://www.ncbi.nlm.nih.gov/pubmed/20651707 Wed, 30 Jun 2010 00:00:00 -0700 BACKGROUND: Mutations in the LMNA gene, which encodes all Atype lamins, result in a variety of human diseases termed laminopathies. Lmna(/) mice appear normal at birth but become runted as early as 2 weeks of age and develop multiple tissue defects that mimic some aspects of human laminopathies. Lmna(/) mice also display smaller spleens and thymuses. In this study, we investigated whether altered lymphoid organ sizes are correlated with specific defects in lymphocyte development. PRINCIPAL FINDINGS: Lmna(/) mice displayed severe agedependent defects in T and B cell development which coincided with runting. Lmna(/) bone marrow reconstituted normal T and B cell development in irradiated wildtype recipients, driving generation of functional and selfMHC restricted CD4() and CD8() T cells. Transplantation of Lmna(/) neonatal thymus lobes into syngeneic wildtype recipients resulted in good engraftment of thymic tissue and normal thymocyte development. CONCLUSIONS: Collectively, these data demonstrate that the severe defects in lymphocyte development that characterize Lmna(/) mice do not result directly from the loss of Atype lamin function in lymphocytes or thymic stroma. Instead, the immune defects in Lmna(/) mice likely reflect indirect damage, perhaps resulting from prolonged stress due to the striated muscle dystrophies that occur in these mice. http://www.ncbi.nlm.nih.gov/pubmed/20405040 Wed, 31 Mar 2010 00:00:00 -0700 The cyclic adenosine monophosphatedependent protein kinase A (PKA) pathway helps regulate both cell growth and division, and triglyceride storage and metabolism in response to nutrient status. Studies in yeast show that disruption of this pathway promotes longevity in a manner similar to caloric restriction. Because PKA is highly conserved, it can be studied in mammalian systems. This report describes the metabolic phenotype of mice lacking the PKA catalytic subunit Cbeta. We confirmed that Cbeta has high levels of expression in the brain but also showed moderate levels in liver. Cbetanull animals had reduced basal PKA activity while appearing overtly normal when fed standard rodent chow. However, the absence of Cbeta protected mice from dietinduced obesity, steatosis, dyslipoproteinemia, and insulin resistance, without any differences in caloric intake or locomotor activity. These findings have relevant pharmacological implications because aging in mammals is characterized by metabolic decline associated with obesity, altered body fat distribution, and insulin resistance. http://www.ncbi.nlm.nih.gov/pubmed/19776218 Sat, 31 Oct 2009 00:00:00 -0700 In the last few years, links between regulation of mRNA translation and aging have been firmly established in invertebrate model organisms. This year, a possible relationship between mRNA translation and aging in mammals has been established with the report that rapamycin increases lifespan in mice. Other significant findings have connected translation control with other known longevity pathways and provided fodder for mechanistic hypotheses. Here, we summarize advances in this emerging field and raise questions for future studies. http://www.ncbi.nlm.nih.gov/pubmed/19747234 Sat, 31 Oct 2009 00:00:00 -0700 Several studies indicate that reduced TOR signaling underlies life span extension by dietary restriction. Recently, Zid et al. (Zid et al., 2009) linked the benefits of dietary restriction in flies to increased levels of the downstream TOR target 4EBP1 and corresponding changes in the relative translation rates of classes of mRNAs. http://www.ncbi.nlm.nih.gov/pubmed/19808017 Wed, 30 Sep 2009 00:00:00 -0700 Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron degenerative disease, and the inherited form, familial ALS (fALS), has been linked to over 100 different point mutations scattered throughout the CuZn superoxide dismutase protein (SOD1). The disease is likely due to a toxic gain of function caused by the misfolding, oligomerization, and eventual aggregation of mutant SOD1, but it is not yet understood how the structurally diverse mutations result in a common disease phenotype. The behavior of the apomonomer fALSassociated mutant protein A4V was explored using moleculardynamics simulations to elucidate characteristic structural changes to the protein that may allow the mutant form to improperly associate with other monomer subunits. Simulations showed that the mutant protein is less stable than the WT protein overall, with shifts in residueresidue contacts that lead to destabilization of the dimer and metalbinding sites, and stabilization of nonnative contacts that leads to a misfolded state. These findings provide a unifying explanation for disparate experimental observations, allow a better understanding of alterations of residue contacts that accompany loss of SOD1 structural integrity, and suggest sites where compensatory changes may stabilize the mutant structure. http://www.ncbi.nlm.nih.gov/pubmed/19751676 Mon, 31 Aug 2009 00:00:00 -0700 Studies in a variety of model organisms indicate that nutrient signaling is tightly coupled to longevity. In nutrient replete conditions, organisms develop, grow, and age quickly. When nutrients become sparse as with dietary restriction, growth and development decline, stress response pathways become induced and organisms live longer. Considerable effort has been devoted to understanding the molecular events mediating lifespan extension by dietary restriction. One central focus has been on nutrientresponsive signal transduction pathways including insulin/IGF1, AMP kinase, protein kinase A and the TOR pathway. Here we describe the increasingly prominent links between TOR signaling and aging in invertebrates. Longevity studies in mammals are not published to date. Instead, we highlight studies in mouse models, which indicate that dampening the TOR pathway leads to widespread protection from an array of agerelated diseases. http://www.ncbi.nlm.nih.gov/pubmed/19539012 Mon, 31 Aug 2009 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/19606132 Tue, 30 Jun 2009 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/19587684 Tue, 30 Jun 2009 00:00:00 -0700 Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice. Due to the lack of easily quantified ageassociated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage. Chronological life span refers to the length of time that a mother cell can survive in a nondividing, quiescencelike state, and is proposed to serve as a model for aging of postmitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiberoptic needle. Typical laboratory yeast strains produce 2030 daughter cells per mother and one life span experiment requires 23 weeks. http://www.ncbi.nlm.nih.gov/pubmed/19556967 Sun, 31 May 2009 00:00:00 -0700 Cells undergoing developmental processes are characterized by persistent nongenetic alterations in chromatin, termed epigenetic changes, represented by distinct patterns of DNA methylation and histone posttranslational modifications. Sirtuins, a group of conserved NAD()dependent deacetylases or ADPribosyltransferases, promote longevity in diverse organisms however, their molecular mechanisms in ageing regulation remain poorly understood. Yeast Sir2, the first member of the family to be found, establishes and maintains chromatin silencing by removing histone H4 lysine 16 acetylation and bringing in other silencing proteins. Here we report an ageassociated decrease in Sir2 protein abundance accompanied by an increase in H4 lysine 16 acetylation and loss of histones at specific subtelomeric regions in replicatively old yeast cells, which results in compromised transcriptional silencing at these loci. Antagonizing activities of Sir2 and Sas2, a histone acetyltransferase, regulate the replicative lifespan through histone H4 lysine 16 at subtelomeric regions. This pathway, distinct from existing ageing models for yeast, may represent an evolutionarily conserved function of sirtuins in regulation of replicative ageing by maintenance of intact telomeric chromatin. http://www.ncbi.nlm.nih.gov/pubmed/19516333 Sun, 31 May 2009 00:00:00 -0700 The molecular mechanisms that cause organismal aging are a topic of intense scrutiny and debate. Dietary restriction extends the life span of many organisms, including yeast, and efforts are underway to understand the biochemical and genetic pathways that regulate this life span extension in model organisms. Here we describe the mechanism by which dietary restriction extends yeast chronological life span, defined as the length of time stationary yeast cells remain viable in a quiescent state. We find that aging under standard culture conditions is the result of a cellextrinsic component that is linked to the pH of the culture medium. We identify acetic acid as a cellextrinsic mediator of cell death during chronological aging, and demonstrate that dietary restriction, growth in a nonfermentable carbon source, or transferring cells to water increases chronological life span by reducing or eliminating extracellular acetic acid. Other life span extending environmental and genetic interventions, such as growth in high osmolarity media, deletion of SCH9 or RAS2, increase cellular resistance to acetic acid. We conclude that acetic acid induced mortality is the primary mechanism of chronological aging in yeast under standard conditions. http://www.ncbi.nlm.nih.gov/pubmed/19305133 Tue, 31 Mar 2009 00:00:00 -0700 Regions of heterochromatin are often found at the periphery of the mammalian nucleus, juxtaposed to the nuclear lamina. Genes in these regions are likely maintained in a transcriptionally silent state, although other locations at the nuclear periphery associated with nuclear pores are sites of active transcription. As primary components of the nuclear lamina, A and Btype nuclear lamins are intermediate filament proteins that interact with DNA, histones and known transcriptional repressors, leading to speculation that they may promote establishment of repressive domains. However, no direct evidence of a role for nuclear lamins in transcriptional repression has been reported. Here we find that human lamin A, when expressed in yeast and cultured human cells as a fusion protein to the Gal4 DNAbinding domain (DBD), can mediate robust transcriptional repression of promoters with Gal4 binding sites. Full repression by lamin A requires both the coiledcoil rod domain and the Cterminal tail domain. In human cells, other intermediate filament proteins such as lamin B and vimentin are unable to confer robust repression as Gal4DBD fusions, indicating that this property is specific to Atype nuclear lamins. These findings indicate that Atype lamins can promote transcriptional repression when in proximity of a promoter. http://www.ncbi.nlm.nih.gov/pubmed/19272320 Sat, 28 Feb 2009 00:00:00 -0800 HutchinsonGilford progeria syndrome (HGPS) is a rare, debilitating disease with early mortality and rapid onset of agingassociated pathologies. It is linked to mutations in LMNA, which encodes Atype nuclear lamins. The most frequent HGPSassociated LMNA mutation results in a protein, termed progerin, with an internal 50 amino acid deletion and, unlike normal Atype lamins, stable farnesylation. The cellular consequences of progerin expression underlying the HGPS phenotype remain poorly understood. Here, we stably expressed lamin A mutants, including progerin, in otherwise identical primary human fibroblasts to compare the effects of different mutants on nuclear morphology and cell proliferation. We find that expression of progerin leads to inhibition of proliferation in a high percentage of cells and slightly premature senescence in the population. Expression of a stably farnesylated mutant of lamin A phenocopied the immediate proliferative defects but did not result in premature senescence. Either p53 inhibition or, more surprisingly, expression of the catalytic subunit of telomerase (hTERT) suppressed the early proliferative defects associated with progerin expression. These findings lead us to propose that progerin may interfere with telomere structure or metabolism in a manner suppressible by increased telomerase levels and possibly link mechanisms leading to progeroid phenotypes to those of cell immortalization. http://www.ncbi.nlm.nih.gov/pubmed/18843043 Sun, 30 Nov 2008 00:00:00 -0800 BACKGROUND: Identification of genes that modulate longevity is a major focus of agingrelated 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 ageassociated diseases, including cancer, heart disease, diabetes, and neurodegenerative disorders. To date, however, targeted efforts at identifying longevityassociated genes have been limited by a lack of predictive power, and useful algorithms for candidate geneidentification have also been lacking. METHODOLOGY/PRINCIPAL FINDINGS: We have utilized a shortestpath 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 shortestpath network algorithm to a preexisting proteinprotein interaction dataset in order to construct a shortestpath longevity network. To validate this network, the replicative aging potential of 88 singlegene deletion strains corresponding to predicted components of the shortestpath longevity network was determined. Here we report that the singlegene deletion strains identified by our shortestpath longevity analysis are significantly enriched for mutations conferring either increased or decreased replicative life span, relative to a randomly selected set of 564 singlegene 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 shortestpath 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 ageassociated disease. http://www.ncbi.nlm.nih.gov/pubmed/19030232 Fri, 31 Oct 2008 00:00:00 -0700 The important role that regulation of protein translation plays in determining longevity in invertebrate organisms became widely appreciated in 2007, with the publication of several papers discussed in last year's review. During 2008, several studies have further strengthened the idea that regulation of translation is one component of a highly evolutionarily conserved pathway that modifies longevity. Importantly, studies published this year also began to provide insights into specific mechanisms by which altered mRNA translation does (and in some cases does not) slow aging in invertebrate model organisms. http://www.ncbi.nlm.nih.gov/pubmed/18782345 Fri, 31 Oct 2008 00:00:00 -0700 BACKGROUND: Dietary restriction (DR) increases life span and delays ageassociated disease in many organisms. The mechanism by which DR enhances longevity is not well understood. RESULTS: Using bacterial food deprivation as a means of DR in C. elegans, we show that transient DR confers longterm benefits including stress resistance and increased longevity. Consistent with studies in the fruit fly and in mice, we demonstrate that DR also enhances survival when initiated late in life. DR by bacterial food deprivation significantly increases life span in worms when initiated as late as 24 days of adulthood, an age at which greater than 50 of the cohort have died. These survival benefits are, at least partially, independent of food consumption, as control fed animals are no longer consuming bacterial food at this advanced age. Animals separated from the bacterial lawn by a barrier of solid agar have a life span intermediate between control fed and food restricted animals. Thus, we find that life span extension from bacterial deprivation can be partially suppressed by a diffusible component of the bacterial food source, suggesting a calorieindependent mechanism for life span extension by dietary restriction. CONCLUSION: Based on these findings, we propose that dietary restriction by bacterial deprivation increases longevity in C. elegans by a combination of reduced food consumption and decreased food sensing. http://www.ncbi.nlm.nih.gov/pubmed/18457595 Sat, 31 May 2008 00:00:00 -0700 Dietary restriction increases lifespan and slows the onset of ageassociated disease in organisms from yeast to mammals. In humans, several agerelated diseases are associated with aberrant protein folding or aggregation, including neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases. We report here that dietary restriction dramatically suppresses ageassociated paralysis in three nematode models of proteotoxicity. Similar to its longevityenhancing properties, dietary restriction protects against proteotoxicity by a mechanism distinct from reduced insulin/IGF1like signaling. Instead, the heat shock transcription factor, hsf1, is required for enhanced thermotolerance, suppression of proteotoxicity, and lifespan extension by dietary restriction. These findings demonstrate that dietary restriction confers a general protective effect against proteotoxicity and promotes longevity by a mechanism involving hsf1. http://www.ncbi.nlm.nih.gov/pubmed/18331616 Wed, 30 Apr 2008 00:00:00 -0700 In nearly every organism studied, reduced caloric intake extends life span. In yeast, span extension from dietary restriction is thought to be mediated by the highly conserved, nutrientresponsive target of rapamycin (TOR), protein kinase A (PKA), and Sch9 kinases. These kinases coordinately regulate various cellular processes including stress responses, protein turnover, cell growth, and ribosome biogenesis. Here we show that a specific reduction of 60S ribosomal subunit levels slows aging in yeast. Deletion of genes encoding 60S subunit proteins or processing factors or treatment with a small molecule, which all inhibit 60S subunit biogenesis, are each sufficient to significantly increase replicative life span. One mechanism by which reduced 60S subunit levels leads to life span extension is through induction of Gcn4, a nutrientresponsive transcription factor. Genetic epistasis analyses suggest that dietary restriction, reduced 60S subunit abundance, and Gcn4 activation extend yeast life span by similar mechanisms. http://www.ncbi.nlm.nih.gov/pubmed/18423200 Mon, 31 Mar 2008 00:00:00 -0700 Studies in invertebrate model organisms have been a driving force in aging research, leading to the identification of many genes that influence life span. Few of these genes have been examined in the context of mammalian aging, however, and it remains an open question as to whether and to what extent the pathways that modulate longevity are conserved across different eukaryotic species. Using a comparative functional genomics approach, we have performed the first quantitative analysis of the degree to which longevity genes are conserved between two highly divergent eukaryotic species, the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans. Here, we report the replicative life span phenotypes for singlegene deletions of the yeast orthologs of worm aging genes. We find that 15 of these yeast deletions are longlived. In contrast, only 3.4 of a random set of deletion mutants are longliveda statistically significant difference. These data suggest that genes that modulate aging have been conserved not only in sequence, but also in function, over a billion years of evolution. Among the longevity determining ortholog pairs, we note a substantial enrichment for genes involved in an evolutionarily conserved pathway linking nutrient sensing and protein translation. In addition, we have identified several conserved aging genes that may represent novel longevity pathways. Together, these findings indicate that the genetic component of life span determination is significantly conserved between divergent eukaryotic species, and suggest pathways that are likely to play a similar role in mammalian aging. http://www.ncbi.nlm.nih.gov/pubmed/18340043 Mon, 31 Mar 2008 00:00:00 -0700 Chronological aging in yeast has been studied by maintaining cells in a quiescentlike stationary phase culture and monitoring cell survival over time. The composition of the growth medium can have a profound influence on chronological aging. For example, dietary restriction accomplished by lowering the glucose concentration of the medium significantly increases life span. Here we report a novel highthroughput method for measuring yeast chronological life span by monitoring outgrowth of aging cells using a Bioscreen C MBR machine. We show that this method provides survival data comparable to traditional methods, but with decreased variability. In addition to reducing the glucose concentration, we find that elevated amino acid levels or increased osmolarity of the growth medium is sufficient to increase chronological life span. We also report that lifespan extension from dietary restriction does not require any of the five yeast sirtuins (Sir2, Hst1, Hst2, Hst3, or Hst4) either alone or in combination. http://www.ncbi.nlm.nih.gov/pubmed/18314444 Fri, 29 Feb 2008 00:00:00 -0800 Using the Saccharomyces cerevisiae MATa/MATalpha ORF deletion collection, homozygous deletion strains were identified that undergo mating with MATa or MATalpha haploids. Seven homozygous deletions were identified that confer enhanced mating. Three of these, lacking CTF8, CTF18, and DCC1, mate at a low frequency with either MATa or MATalpha haploids. The products of these genes form a complex involved in sister chromatid cohesion. Each of these strains also exhibits increased chromosome loss rates, and mating likely occurs due to loss of one copy of chromosome III, which bears the MAT locus. Three other homozygous diploid deletion strains, ylr193cDelta/ylr193cDelta, yor305wDelta/yor305wDelta, and ypr170cDelta/ypr170cDelta, mate at very low frequencies with haploids of either or both mating types. However, an ist3Delta/ist3Delta strain mates only with MATa haploids. It is shown that IST3, previously linked to splicing, is required for efficient processing of the MATa1 message, particularly the first intron. As a result, the ist3Delta/ist3Delta strain expresses unbalanced ratios of Matalpha to Mata proteins and therefore mates with MATa haploids. Accordingly, mating in this diploid can be repressed by introduction of a MATa1 cDNA. In summary, this study underscores and elaborates upon predicted pathways by which mutations restore mating function to yeast diploids and identifies new mutants warranting further study. http://www.ncbi.nlm.nih.gov/pubmed/17995956 Thu, 31 Jan 2008 00:00:00 -0800 Translation of RNA to protein is essential for life. It should perhaps not be surprising, therefore, that appropriate regulation of translation plays a key role in determining longevity. This Hot Topic article discusses papers published in the last year related to the importance of translation and its regulation by signaling through the target of rapamycin kinase, in modulating aging and ageassociated diseases. http://www.ncbi.nlm.nih.gov/pubmed/17941970 Wed, 31 Oct 2007 00:00:00 -0700 BACKGROUND: Atype lamins, predominantly lamins A and C, are nuclear intermediate filaments believed to act as scaffolds for assembly of transcription factors. Lamin A/C is necessary for the retinoblastoma protein (pRB) stabilization through unknown mechanism(s). Two oncoproteins, gankyrin and MDM2, are known to promote pRB degradation in other contexts. Consequently, we tested the hypothesis that gankyrin and/or MDM2 are required for enhanced pRB degradation in Lmna/ fibroblasts. Principal Findings. To determine if gankyrin promotes pRB destabilization in the absence of lamin A/C, we first analyzed its protein levels in Lmna/ fibroblasts. Both gankyrin mRNA levels and protein levels are increased in these cells, leading us to further investigate its role in pRB degradation. Consistent with prior reports, overexpression of gankyrin in Lmna/ cells destabilizes pRB. This decrease is functionally significant, since gankyrin overexpressing cells are resistant to p16(ink4a)mediated cell cycle arrest. These findings suggest that lamin Amediated degradation of pRB would be gankyrindependent. However, effective RNAienforced reduction of gankyrin expression in Lmna/ cells was insufficient to restore pRB stability. To test the importance of MDM2, we disrupted the MDM2pRB interaction by transfecting Lmna/ cells with p14(arf). p14(arf) expression was also insufficient to stabilize pRB or confer cell cycle arrest, suggesting that MDM2 also does not mediate pRB degradation in Lmna/ cells. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that pRB degradation in Lmna/ cells occurs by gankyrin and MDM2independent mechanisms, leading us to propose the existence of a third proteasomedependent pathway for pRB degradation. Two findings from this study also increase the likelihood that lamin A/C functions as a tumor suppressor. First, protein levels of the oncoprotein gankyrin are elevated in Lmna/ fibroblasts. Second, Lmna/ cells are refractory to p14(arf)mediated cell cycle arrest, as was previously shown with p16(ink4a). Potential roles of lamin A/C in the suppression of tumorigenesis are discussed. http://www.ncbi.nlm.nih.gov/pubmed/17896003 Fri, 31 Aug 2007 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/17635418 Tue, 31 Jul 2007 00:00:00 -0700 In the last decade, research into the molecular determinants of aging has progressed rapidly and much of this progress can be attributed to studies in invertebrate eukaryotic model organisms. Of these, singlecelled yeast is the least complicated and most amenable to genetic and molecular manipulations. Supporting the use of this organism for aging research, increasing evidence has accumulated that a subset of pathways influencing longevity in yeast are conserved in other eukaryotes, including mammals. Here we briefly outline aging in yeast and describe recent findings that continue to keep this "simple" eukaryote at the forefront of aging research. http://www.ncbi.nlm.nih.gov/pubmed/17530929 Mon, 30 Apr 2007 00:00:00 -0700 Progeroid syndromes have been the focus of intense research in part because they might provide a window into the pathology of normal ageing. Werner syndrome and HutchinsonGilford progeria syndrome are two of the best characterized human progeroid diseases. Mutated genes that are associated with these syndromes have been identified, mouse models of disease have been developed, and molecular studies have implicated decreased cell proliferation and altered DNAdamage responses as common causal mechanisms in the pathogenesis of both diseases. http://www.ncbi.nlm.nih.gov/pubmed/17450177 Sat, 31 Mar 2007 00:00:00 -0700 The peroxisome, sole site of betaoxidation in Saccharomyces cerevisiae, is known to be required for optimal growth in the presence of fatty acid. Screening of the haploid yeast deletion collection identified approximately 130 genes, 23 encoding peroxisomal proteins, necessary for normal growth on oleic acid. Oleate slightly enhances growth of wildtype yeast and inhibits growth of all strains identified by the screen. Nonperoxisomal processes, among them chromatin modification by H2AZ, Pol II mediator function, and cellwallassociated activities, also prevent oleate toxicity. The most oleateinhibited strains lack Sap190, a putative adaptor for the PP2Atype protein phosphatase Sit4 (which is also required for normal growth on oleate) and Ilm1, a protein of unknown function. Palmitoleate, the other main unsaturated fatty acid of Saccharomyces, fails to inhibit growth of the sap190delta, sit4delta, and ilm1delta strains. Data that suggest that oleate inhibition of the growth of a peroxisomal mutant is due to an increase in plasma membrane porosity are presented. We propose that yeast deficient in peroxisomal and other functions are sensitive to oleate perhaps because of an inability to effectively control the fatty acid composition of membrane phospholipids. http://www.ncbi.nlm.nih.gov/pubmed/17151231 Sun, 31 Dec 2006 00:00:00 -0800 Technological advancements in invertebrate model organisms have recently made it possible to survey many or all of the genes in the genome for phenotypes of interest. In both C. elegans and S. cerevisiae, genomewide searches for hypomorphic mutations that extend life span have been performed. The results from these screens are starting to provide a more complete view of the range of life span determinants in eukaryotes. In addition, it is becoming possible to test the premise that conserved aging genes and pathways regulate aging in disparate eukaryotic species. Here we compare and contrast the results from genomewide aging screens and assess the likelihood that there are "public" aging mechanisms. http://www.ncbi.nlm.nih.gov/pubmed/17126379 Sun, 31 Dec 2006 00:00:00 -0800 Two models have been proposed for how calorie restriction (CR) enhances replicative longevity in yeast: (i) suppression of rDNA recombination through activation of the sirtuin protein deacetylase Sir2 or (ii) decreased activity of the nutrientresponsive kinases Sch9 and TOR. We report here that CR increases lifespan independently of all Sir2family proteins in yeast. Furthermore, we demonstrate that nicotinamide, an inhibitor of Sir2mediated deacetylation, interferes with lifespan extension from CR, but does so independent of Sir2, Hst1, Hst2, and Hst4. We also find that 5 mm nicotinamide, a concentration sufficient to inhibit other sirtuins, does not phenocopy deletion of HST3. Thus, we propose that lifespan extension by CR is independent of sirtuins and that nicotinamide has sirtuinindependent effects on lifespan extension by CR. http://www.ncbi.nlm.nih.gov/pubmed/17129213 Tue, 31 Oct 2006 00:00:00 -0800 A partial reduction in food intake has been found to increase lifespan in many different organisms. We report here a new dietary restriction regimen in the nematode Caenorhabditis elegans, based on the standard agar plate lifespan assay, in which adult worms are maintained in the absence of a bacterial food source. These findings represent the first report in any organism of lifespan extension in response to prolonged starvation. Removal of bacterial food increases lifespan to a greater extent than partial reduction of food through a mechanism that is distinct from insulin/IGFlike signaling and the Sir2family deacetylase, SIR2.1. Removal of bacterial food also increases lifespan when initiated in postreproductive adults, suggesting that dietary restriction started during middle age can result in a substantial longevity benefit that is independent of reproduction. http://www.ncbi.nlm.nih.gov/pubmed/17081160 Tue, 31 Oct 2006 00:00:00 -0800 The relationship between progeriasdiseases that resemble premature agingand the normal aging process has been a source of debate in the aging research community. A recent study finds that LMNA, a gene targeted for mutation in Hutchinson Gilford Progeria Syndrome, may control the onset of agingassociated decline in normal fibroblasts. http://www.ncbi.nlm.nih.gov/pubmed/16920618 Mon, 31 Jul 2006 00:00:00 -0700 Sirtuins have been the focus of intense scrutiny since the discovery of Sir2 as a yeast longevity factor. Functioning as either deacetylases or ADP ribosylases, Sirtuins are regulated by the cofactor NAD and thus may serve as sensors of the metabolic state of the cell and organism. Here we examine the roles of Sirtuins in diverse eukaryotic species, with special emphasis on their links to aging and agerelated diseases including cancer, diabetes, and neurodegenerative disorders. http://www.ncbi.nlm.nih.gov/pubmed/16873059 Fri, 30 Jun 2006 00:00:00 -0700 Mutations in the LMNA gene, which encodes all Atype lamins, including lamin A and lamin C, cause a variety of tissuespecific degenerative diseases termed laminopathies. Little is known about the pathogenesis of these disorders. Previous studies have indicated that Atype lamins interact with the retinoblastoma protein (pRB). Here we probe the functional consequences of this association and further examine links between nuclear structure and cell cycle control. Since pRB is required for cell cycle arrest by p16(ink4a), we tested the responsiveness of multiple lamin A/Cdepleted cell lines to overexpression of this CDK inhibitor and tumor suppressor. We find that the loss of Atype lamin expression results in marked destabilization of pRB. This reduction in pRB renders cells resistant to p16(ink4a)mediated G(1) arrest. Reintroduction of lamin A, lamin C, or pRB restores p16(ink4a)responsiveness to Lmna(/) cells. An array of lamin A mutants, representing a variety of pathologies as well as lamin A processing mutants, was introduced into Lmna(/) cells. Of these, a mutant associated with mandibuloacral dysplasia (MAD R527H), as well as two lamin A processing mutants, but not other diseaseassociated mutants, failed to restore p16(ink4a) responsiveness. Although our findings do not rule out links between altered pRB function and laminopathies, they fail to support such an assertion. These findings do link lamin A/C to the functional activation of a critical tumor suppressor pathway and further the possibility that somatic mutations in LMNA contribute to tumor progression. http://www.ncbi.nlm.nih.gov/pubmed/16809772 Wed, 31 May 2006 00:00:00 -0700 Calorie restriction (CR) increases life span in yeast independently of Sir2. Lamming et al. (Reports, 16 September 2005, p. 1861) recently proposed that Sir2independent lifespan extension by CR is mediated by the Sir2 paralogs Hst1 and Hst2. Contradictory to this, we find that CR greatly increases life span in cells lacking Sir2, Hst1, and Hst2, which suggests that CR is not mediated by Sir2, Hst2, or Hst1. http://www.ncbi.nlm.nih.gov/pubmed/16741098 Wed, 31 May 2006 00:00:00 -0700 A model for replicative life span extension by calorie restriction (CR) in yeast has been proposed whereby reduced glucose in the growth medium leads to activation of the NADdependent histone deacetylase Sir2. One mechanism proposed for this putative activation of Sir2 is that CR enhances the rate of respiration, in turn leading to altered levels of NAD or NADH, and ultimately resulting in enhanced Sir2 activity. An alternative mechanism has been proposed in which CR decreases levels of the Sir2 inhibitor nicotinamide through increased expression of the gene coding for nicotinamidase, PNC1. We have previously reported that life span extension by CR is not dependent on Sir2 in the longlived BY4742 strain background. Here we have determined the requirement for respiration and the effect of nicotinamide levels on life span extension by CR. We find that CR confers robust life span extension in respiratorydeficient cells independent of strain background, and moreover, suppresses the premature mortality associated with loss of mitochondrial DNA in the shortlived PSY316 strain. Addition of nicotinamide to the medium dramatically shortens the life span of wild type cells, due to inhibition of Sir2. However, even in cells lacking both Sir2 and the replication fork block protein Fob1, nicotinamide partially prevents life span extension by CR. These findings (1) demonstrate that respiration is not required for the longevity benefits of CR in yeast, (2) show that nicotinamide inhibits life span extension by CR through a Sir2independent mechanism, and (3) suggest that CR acts through a conserved, Sir2independent mechanism in both PSY316 and BY4742. http://www.ncbi.nlm.nih.gov/pubmed/16311627 Wed, 31 May 2006 00:00:00 -0700 Mutations within LMNA, encoding Atype nuclear lamins, are associated with multiple tissuespecific diseases, including EmeryDreifuss (EDMD2/3) and LimbGirdle muscular dystrophy (LGMD1B). Xlinked EDMD results from mutations in emerin, a lamin Aassociated protein. The mechanisms through which these mutations cause muscular dystrophy are not understood. Here we show that most, but not all, cultured muscle cells from lamin A/C knockout mice exhibit impaired differentiation kinetics and reduced differentiation potential. Similarly, normal muscle cells that have been RNA interference (RNAi) downregulated for either Atype lamins or emerin have impaired differentiation potentials. Replicative myoblasts lacking Atype lamins or emerin also have decreased levels of proteins important for muscle differentiation including pRB, MyoD, desmin, and Mcadherin upregulated Myf5 but no changes in Pax3, Pax7, MEF2C, MEF2D, cmet, and betacatenin. To determine whether impaired myogenesis is linked to reduced MyoD or desmin levels, these proteins were individually expressed in Lmna(/) myoblasts that were then induced to undergo myogenesis. Expression of either MyoD or, more surprisingly, desmin in Lmna(/) myoblasts resulted in increased differentiation potential. These studies indicate roles for Atype lamins and emerin in myogenic differentiation and also suggest that these effects are at least in part due to decreased endogenous levels of other critical myoblast proteins. The delayed differentiation kinetics and decreased differentiation potential of lamin A/Cdeficient and emerindeficient myoblasts may in part underlie the dystrophic phenotypes observed in patients with EDMD. http://www.ncbi.nlm.nih.gov/pubmed/16481476 Tue, 31 Jan 2006 00:00:00 -0800 Chronological life span (CLS) in Saccharomyces cerevisiae, defined as the time cells in a stationary phase culture remain viable, has been proposed as a model for the aging of postmitotic tissues in mammals. We developed a highthroughput assay to determine CLS for approximately 4800 singlegene deletion strains of yeast, and identified longlived strains carrying mutations in the conserved TOR pathway. TOR signaling regulates multiple cellular processes in response to nutrients, especially amino acids, raising the possibility that decreased TOR signaling mediates life span extension by calorie restriction. In support of this possibility, removal of either asparagine or glutamate from the media significantly increased stationary phase survival. Pharmacological inhibition of TOR signaling by methionine sulfoximine or rapamycin also increased CLS. Decreased TOR activity also promoted increased accumulation of storage carbohydrates and enhanced stress resistance and nuclear relocalization of the stressrelated transcription factor Msn2. We propose that upregulation of a highly conserved response to starvationinduced stress is important for life span extension by decreased TOR signaling in yeast and higher eukaryotes. http://www.ncbi.nlm.nih.gov/pubmed/16418483 Sat, 31 Dec 2005 00:00:00 -0800 Calorie restriction increases life span in many organisms, including the budding yeast Saccharomyces cerevisiae. From a largescale analysis of 564 singlegenedeletion strains of yeast, we identified 10 gene deletions that increase replicative life span. Six of these correspond to genes encoding components of the nutrientresponsive TOR and Sch9 pathways. Calorie restriction of tor1D or sch9D cells failed to further increase life span and, like calorie restriction, deletion of either SCH9 or TOR1 increased life span independent of the Sir2 histone deacetylase. We propose that the TOR and Sch9 kinases define a primary conduit through which excess nutrient intake limits longevity in yeast. http://www.ncbi.nlm.nih.gov/pubmed/16293764 Mon, 31 Oct 2005 00:00:00 -0800 In this issue of Cell, Longo and colleagues (Fabrizio et al., 2005) examine the role of Sir2, a histone deacetylase, in chronological aging in yeast by measuring the longterm survival of nondividing cells. In contrast to measurements of aging for mitotic cells, cell survival in the nonmitotic state is decreased by Sir2 activity under conditions that mimic calorie restriction. http://www.ncbi.nlm.nih.gov/pubmed/16286003 Mon, 31 Oct 2005 00:00:00 -0800 OBJECTIVES: To determine the importance of lamin A/C for fat cell differentiation in vitro and for the antiadipogenic activity of HIV protease inhibitors such as indinavir. METHODS: Lipodystrophyassociated and processingdefective mutants of lamin A were stably expressed at high levels in 3T3L1 preadipocytes. Additionally, 3T3L1 preadipocytes with stable reduction of lamin A/C or emerin were derived. The cells were differentiated for 8 days into mature adipocytes in the presence or absence of indinavir or nelfinavir. RESULTS: 3T3L1 cells stably expressing high levels of lipodystrophyassociated or processingdefective mutants of lamin A differentiated with comparable efficiencies to control cells. Similarly, cells with dramatically reduced lamin A levels differentiated as efficiently as controls. Although indinavir stimulated the accumulation of unprocessed lamin A, cells with dramatically reduced lamin A/C levels and no detectable prelamin A remained responsive to an indinavirinduced inhibition of adipogenesis. CONCLUSIONS: The ability of HIV protease inhibitor to stimulate the accumulation of unprocessed lamin A is neither necessary nor sufficient to explain their antiadipogenic activity. Furthermore, lamin A/C plays a minimal role in the differentiation of 3T3L1. http://www.ncbi.nlm.nih.gov/pubmed/16184025 Wed, 31 Aug 2005 00:00:00 -0700 Numerous studies have identified key binding partners and functional activities of nuclear tumorsuppressor proteins such as the retinoblastoma protein, p53 and BRCA1. Historically, less attention has been given to the subnuclear locations of these proteins. Here, we describe several recent studies that promote the view that regulated association with subcompartments of the nucleus is inherent to tumorsuppressor function. http://www.ncbi.nlm.nih.gov/pubmed/15936946 Thu, 30 Jun 2005 00:00:00 -0700 Resveratrol, a small molecule found in red wine, is reported to slow aging in simple eukaryotes and has been suggested as a potential calorie restriction mimetic. Resveratrol has also been reported to act as a sirtuin activator, and this property has been proposed to account for its antiaging effects. We show here that resveratrol is a substratespecific activator of yeast Sir2 and human SirT1. In particular, we observed that, in vitro, resveratrol enhances binding and deacetylation of peptide substrates that contain Fluor de Lys, a nonphysiological fluorescent moiety, but has no effect on binding and deacetylation of acetylated peptides lacking the fluorophore. Consistent with these biochemical data we found that in three different yeast strain backgrounds, resveratrol has no detectable effect on Sir2 activity in vivo, as measured by rDNA recombination, transcriptional silencing near telomeres, and life span. In light of these findings, the mechanism accounting for putative longevity effects of resveratrol should be reexamined. http://www.ncbi.nlm.nih.gov/pubmed/15684413 Thu, 31 Mar 2005 00:00:00 -0800 Segmental progeroid syndromes are disorders in which affected individuals. present various features that suggest accelerated ageing. The two bestknown examples are HutchinsonGilford progeria syndrome (HGPS, 'Progeria of childhood') and Werner syndrome (WS, 'Progeria of the adult'). A novel, recurrent de novo mutation in the LMNA gene, responsible for the majority of HGPS cases, results in an inframe deletion of 50 amino acids, including endoproteolytic sites required for processing of prelamin A to mature lamin A protein. Another mutation results in a 35 amino acid inframe deletion with a milder HGPS phenotype. WRN, the gene responsible for the majority of WS cases, encodes a multifunctional nuclear protein with exonuclease and helicase activities and may participate in optimizing DNA repair/recombination. A subset of WS patients do not show mutations at the WRN locus (atypical WS), but show heterozygous amino acid substitutions in the heptad repeat region of lamin A. Structural analysis suggests that mutations in atypical WS may interfere with proteinprotein interactions. When compared to WRNmutant WS, LMNAmutant atypical WS patients appear to show earlier onset and possibly more severe ageingrelated symptoms. http://www.ncbi.nlm.nih.gov/pubmed/15773755 Mon, 28 Feb 2005 00:00:00 -0800 Here we describe the replicative life spans of more than 50 congenic Saccharomyces cerevisiae strains, each carrying a mutation previously implicated in yeast aging. This analysis provides a direct comparison, in a single, longlived strain background, of a majority of reported yeast aging genes. Of the eleven deletion mutations previously reported to increase yeast life span, we find that deletion of FOB1, deletion of SCH9, and deletion of GPA2, GPR1, or HXK2 (three genetic models of calorie restriction) significantly enhanced longevity. In addition, overexpression of SIR2 or growth on low glucose increased life span. These results define a limited number of genes likely to regulate replicative life span in a strainindependent manner, and create a basis for future epistasis analysis to determine genetic pathways of aging. http://www.ncbi.nlm.nih.gov/pubmed/15722108 Mon, 31 Jan 2005 00:00:00 -0800 Nuclear lamins were identified as core nuclear matrix constituents over 20 years ago. They have been ascribed structural roles such as maintaining nuclear integrity and assisting in nuclear envelope formation after mitosis, and have also been linked to nuclear activities including DNA replication and transcription. Recently, Atype lamin mutations have been linked to a variety of rare human diseases including muscular dystrophy, lipodystrophy, cardiomyopathy, neuropathy and progeroid syndromes (collectively termed laminopathies). Most diseases arise from dominant, missense mutations, leading to speculation as to how different mutations in the same gene can give rise to such a diverse set of diseases, some of which share little phenotypic overlap. Understanding the cellular dysfunctions that lead to laminopathies will almost certainly provide insight into specific roles of Atype lamins in nuclear organization. Here, we compare and contrast the LMNA mutations leading to laminopathies with emphasis on progerias, and discuss possible functional roles for Atype lamins in the maintenance of healthy tissues. http://www.ncbi.nlm.nih.gov/pubmed/15722103 Mon, 31 Jan 2005 00:00:00 -0800 Recent advances have suggested the existence of phylogenetically conserved pathways regulating ageing in eukaryotes. At least two of these "public" longevitydetermining pathways appear to have been evolutionarily conserved from yeast through mammals. We have developed a highthroughput, genomewide approach to identify a large fraction of the nonessential, singlegene deletion mutations that confer increased longevity in yeast. The identification and characterization of conserved genes that regulate the ageing process across eukaryotic species is likely to result in an improved understanding of the causes of human ageing and provide potential therapeutic targets for drug discovery. http://www.ncbi.nlm.nih.gov/pubmed/15610758 Tue, 30 Nov 2004 00:00:00 -0800 Calorie restriction slows aging and increases life span in many organisms. In yeast, a mechanistic explanation has been proposed whereby calorie restriction slows aging by activating Sir2. Here we report the identification of a Sir2independent pathway responsible for a majority of the longevity benefit associated with calorie restriction. Deletion of FOB1 and overexpression of SIR2 have been previously found to increase life span by reducing the levels of toxic rDNA circles in aged mother cells. We find that combining calorie restriction with either of these genetic interventions dramatically enhances longevity, resulting in the longestlived yeast strain reported thus far. Further, calorie restriction results in a greater life span extension in cells lacking both Sir2 and Fob1 than in cells where Sir2 is present. These findings indicate that Sir2 and calorie restriction act in parallel pathways to promote longevity in yeast and, perhaps, higher eukaryotes. http://www.ncbi.nlm.nih.gov/pubmed/15328540 Tue, 31 Aug 2004 00:00:00 -0700 The retinoblastoma protein (pRB) is a critical regulator of cell proliferation and differentiation and an important tumor suppressor. In the G(1) phase of the cell cycle, pRB localizes to perinucleolar sites associated with lamin A/C intranuclear foci. Here, we examine pRB function in cells lacking lamin A/C, finding that pRB levels are dramatically decreased and that the remaining pRB is mislocalized. We demonstrate that Atype lamins protect pRB from proteasomal degradation. Both pRB levels and localization are restored upon reintroduction of lamin A. Lmna(/) cells resemble Rb(/) cells, exhibiting altered cellcycle properties and reduced capacity to undergo cellcycle arrest in response to DNA damage. These findings establish a functional link between a core nuclear structural component and an important cellcycle regulator. They further raise the possibility that altered pRB function may be a contributing factor in dystrophic syndromes arising from LMNA mutation. http://www.ncbi.nlm.nih.gov/pubmed/15210943 Mon, 31 May 2004 00:00:00 -0700 In primary mammalian cells, DNA replication initiates in a small number of perinucleolar, lamin A/Cassociated foci. During Sphase progression in proliferating cells, replication foci distribute to hundreds of sites throughout the nucleus. In contrast, we find that the limited perinucleolar replication sites persist throughout S phase as cells prepare to exit the cell cycle in response to contact inhibition, serum starvation, or replicative senescence. Proteins known to be involved in DNA synthesis, such as PCNA and DNA polymerase delta, are concentrated in perinucleolar foci throughout S phase under these conditions. Moreover, chromosomal loci are redirected toward the nucleolus and overlap with the perinucleolar replication foci in cells poised to undergo cell cycle exit. These same loci remain in the periphery of the nucleus during replication under highly proliferative conditions. These results suggest that mammalian cells undergo a largescale reorganization of chromatin during the rounds of DNA replication that precede cell cycle exit. http://www.ncbi.nlm.nih.gov/pubmed/14701733 Sun, 30 Nov 2003 00:00:00 -0800 BACKGROUND: Werner's syndrome is a progeroid syndrome caused by mutations at the WRN helicase locus. Some features of this disorder are also present in laminopathies caused by mutant LMNA encoding nuclear lamin A/C. Because of this similarity, we sequenced LMNA in individuals with atypical Werner's syndrome (wildtype WRN). METHODS: Of 129 index patients referred to our international registry for molecular diagnosis of Werner's syndrome, 26 (20) had wildtype WRN coding regions and were categorised as having atypical Werner's syndrome on the basis of molecular criteria. We sequenced all exons of LMNA in these individuals. Mutations were confirmed at the mRNA level by RTPCR sequencing. In one patient in whom an LMNA mutation was detected and fibroblasts were available, we established nuclear morphology and subnuclear localisation. FINDINGS: In four (15) of 26 patients with atypical Werner's syndrome, we noted heterozygosity for novel missense mutations in LMNA, specifically A57P, R133L (in two people), and L140R. The mutations altered relatively conserved residues within lamin A/C. Fibroblasts from the patient with the L140R mutation had a substantially enhanced proportion of nuclei with altered morphology and mislocalised lamins. Individuals with atypical Werner's syndrome with mutations in LMNA had a more severe phenotype than did those with the disorder due to mutant WRN. INTERPRETATION: Our findings indicate that Werner's syndrome is molecularly heterogeneous, and a subset of the disorder can be judged a laminopathy. http://www.ncbi.nlm.nih.gov/pubmed/12927431 Thu, 31 Jul 2003 00:00:00 -0700 http://www.ncbi.nlm.nih.gov/pubmed/12438146 Mon, 30 Sep 2002 00:00:00 -0700 Many transcription factors in human cells have functional orthologues in yeast, and a common experimental theme has been to define the function of the yeast protein and then test whether the mammalian version behaves similarly. Although, at first glance, this approach does not seem feasible for factors that do not have yeast counterparts, mammalian transcriptional activators or repressors can be expressed directly in yeast. Often, the mammalian factor retains function in yeast, and this allows investigators to exploit the experimental tractability of yeast to ask a diverse set of questions. http://www.ncbi.nlm.nih.gov/pubmed/11823797 Thu, 31 Jan 2002 00:00:00 -0800