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1.  The effect of different ubiquinones on lifespan in Caenorhabditis elegans 
Ubiquinone (UQ, Coenzyme Q, CoQ) transfers electrons from complexes I and II to complex III in the mitochondrial electron transport chain. Depending on the degree of reduction, UQ can act as either a pro-or an antioxidant. Mutations disrupting ubiquinone synthesis increase lifespan in both the nematode (clk-1) and the mouse (mclk-1). The mutated nematodes survive using exogenous ubiquinone from bacteria, which has a shorter isoprenyl tail length (UQ8) than the endogenous nematode ubiquinone (UQ9).
The mechanism underlying clk-1’s increased longevity is not clear. Here we directly measure the effect of different exogenous ubiquinones on clk-1 lifespan and mitochondrial function. We fed clk-1 engineered bacteria that produced UQ6, UQ7, UQ8, UQ9 or UQ10, and measured clk-1’s lifespan, mitochondrial respiration, ROS production, and accumulated ROS damage to mitochondrial protein. Regardless of dietary UQ, clk-1 animals have increased lifespan, decreased mitochondrial respiration, and decreased ROS damage to mitochondrial protein than N2. However clk-1 mitochondria did not produce less ROS than N2. The simplest explanation of our results is that clk-1 mitochondria scavenge ROS more effectively than wildtype due to the presence of DMQ9. Moreover, when compared to other dietary quinones, UQ10 further decreased mitochondrial oxidative damage and extended adult lifespan in clk-1.
doi:10.1016/j.mad.2009.03.003
PMCID: PMC2684812  PMID: 19428456
ubiquinone; mitochondria; respiration; reactive oxygen species; C. elegans; life span; genetics
2.  The submitochondrial distribution of ubiquinone affects respiration in long-lived Mclk1+/− mice 
The Journal of Cell Biology  2012;199(2):215-224.
MCLK1 and COQ3 are mitochondrial enzymes necessary for ubiquinone biosynthesis, but only MCLK1 also regulates the partitioning of ubiquinone between mitochondrial membranes and affects longevity in mice.
Mclk1 (also known as Coq7) and Coq3 code for mitochondrial enzymes implicated in the biosynthetic pathway of ubiquinone (coenzyme Q or UQ). Mclk1+/− mice are long-lived but have dysfunctional mitochondria. This phenotype remains unexplained, as no changes in UQ content were observed in these mutants. By producing highly purified submitochondrial fractions, we report here that Mclk1+/− mice present a unique mitochondrial UQ profile that was characterized by decreased UQ levels in the inner membrane coupled with increased UQ in the outer membrane. Dietary-supplemented UQ10 was actively incorporated in both mitochondrial membranes, and this was sufficient to reverse mutant mitochondrial phenotypes. Further, although homozygous Coq3 mutants die as embryos like Mclk1 homozygous null mice, Coq3+/− mice had a normal lifespan and were free of detectable defects in mitochondrial function or ubiquinone distribution. These findings indicate that MCLK1 regulates both UQ synthesis and distribution within mitochondrial membranes.
doi:10.1083/jcb.201203090
PMCID: PMC3471228  PMID: 23045551
3.  Mclk1+/- mice are not resistant to the development of atherosclerosis 
Background
Mice with a single copy of Mclk1 (a.k.a. Coq7), a gene that encodes a mitochondrial enzyme required for the biosynthesis of ubiquinone and other functions, live longer than wild-type mice. The prolonged survival implies a decreased mortality from age-dependent lethal pathologies. Atherosclerosis is one of the main age-dependent pathologies in humans and can be modeled in mice that lack Apolipoprotein E (ApoE-/-) or mice that lack the Low Density Lipoprotein Receptor (LDLr-/-) in addition to being fed an atherosclerosis-inducing diet. We sought to determine if Mclk1 heterozygosity protects against atherosclerosis and dyslipidemia in these models.
Results
We found that Mclk1 heterozygosity did not protect against dyslipidemia, oxidative stress, or atherosclerosis in young (6 or 10 months) or older (18 months) mice. Furthermore, the absence of ApoE suppressed the lifespan-promoting effects of Mclk1 heterozygosity.
Conclusion
These findings indicate that although Mclk1 heterozygosity can extend lifespan of mice, it does not necessarily protect against atherosclerosis. Moreover, in the presence of hyperlipidemia and chronic inflammation, Mclk1 heterozygosity is incapable of extending lifespan.
doi:10.1186/1476-511X-8-16
PMCID: PMC2683836  PMID: 19416523
4.  Longevity in mice: is stress resistance a common factor? 
Age  2006;28(2):145-162.
A positive relationship between stress resistance and longevity has been reported in a multitude of studies in organisms ranging from yeast to mice. Several mouse lines have been discovered or developed that exhibit extended longevities when compared with normal, wild-type mice of the same genetic background. These long-living lines include the Ames dwarf, Snell dwarf, growth hormone receptor knockout (Laron dwarf), IGF-1 receptor heterozygote, Little, α-MUPA knockout, p66shc knockout, FIRKO, mClk-1 heterozygote, thioredoxin transgenic, and most recently the Klotho transgenic mouse. These mice are described in terms of the reported extended lifespans and studies involving resistance to stress. In addition, caloric restriction (CR) and stress resistance are briefly addressed for comparison with genetically altered mice. Although many of the long-living mice have GH/IGF-1/insulin signaling-related alterations and enhanced stress resistance, there are some that exhibit life extension without an obvious link to this hormone pathway. Resistance to oxidative stress is by far the most common system studied in long-living mice, but there is evidence of enhancement of resistance in other systems as well. The differences in stress resistance between long-living mutant and normal mice result from complex interrelationships among pathways that appear to coordinate signals of growth and metabolism, and subsequently result in differences in lifespan.
doi:10.1007/s11357-006-9003-y
PMCID: PMC2464727  PMID: 19943136
mutant mice; lifespan; oxidative stress; hormesis
5.  Insulin/IGF-1-mediated longevity is marked by reduced protein metabolism 
Quantitative proteomics, lifespan analysis, and biochemical assays were utilized to show that Insulin/IGF-1-mediated longevity in C. elegans is strongly associated with a daf-16 dependent global reduction in protein metabolism.
A daf-16 dependent global reduction in protein translation is observed in daf-2 long-lived mutant.The reduction in active translation is independent of germline activityA role for protein metabolism is identified in the Insulin/IGF-1-mediated extension of life.
Mutations in the daf-2 gene of the conserved Insulin/Insulin-like Growth Factor (IGF-1) pathway double the lifespan of the nematode Caenorhabditis elegans. This phenotype is completely suppressed by deletion of Forkhead transcription factor daf-16. To uncover regulatory mechanisms coordinating this extension of life, we employed a quantitative proteomics strategy with daf-2 mutants in comparison with N2 and daf-16; daf-2 double mutants. This revealed a remarkable longevity-specific decrease in proteins involved in mRNA processing and transport, the translational machinery, and protein metabolism. Correspondingly, the daf-2 mutants display lower amounts of mRNA and 20S proteasome activity, despite maintaining total protein levels equal to that observed in wild types. Polyribosome profiling in the daf-2 and daf-16;daf-2 double mutants confirmed a daf-16-dependent reduction in overall translation, a phenotype reminiscent of Dietary Restriction-mediated longevity, which was independent of germline activity. RNA interference (RNAi)-mediated knockdown of proteins identified by our approach resulted in modified C. elegans lifespan confirming the importance of these processes in Insulin/IGF-1-mediated longevity. Together, the results demonstrate a role for the metabolism of proteins in the Insulin/IGF-1-mediated extension of life.
doi:10.1038/msb.2013.35
PMCID: PMC3734508  PMID: 23820781
ageing; high-throughput analysis; metabolism; protein metabolism; translation
6.  A Regulated Response to Impaired Respiration Slows Behavioral Rates and Increases Lifespan in Caenorhabditis elegans 
PLoS Genetics  2009;5(4):e1000450.
When mitochondrial respiration or ubiquinone production is inhibited in Caenorhabditis elegans, behavioral rates are slowed and lifespan is extended. Here, we show that these perturbations increase the expression of cell-protective and metabolic genes and the abundance of mitochondrial DNA. This response is similar to the response triggered by inhibiting respiration in yeast and mammalian cells, termed the “retrograde response”. As in yeast, genes switched on in C. elegans mitochondrial mutants extend lifespan, suggesting an underlying evolutionary conservation of mechanism. Inhibition of fstr-1, a potential signaling gene that is up-regulated in clk-1 (ubiquinone-defective) mutants, and its close homolog fstr-2 prevents the expression of many retrograde-response genes and accelerates clk-1 behavioral and aging rates. Thus, clk-1 mutants live in “slow motion” because of a fstr-1/2–dependent pathway that responds to ubiquinone. Loss of fstr-1/2 does not suppress the phenotypes of all long-lived mitochondrial mutants. Thus, although different mitochondrial perturbations activate similar transcriptional and physiological responses, they do so in different ways.
Author Summary
Mitochondrial respiration generates energy in the form of adenosine triphospate (ATP), a molecule that powers many cellular processes. When respiration is inhibited in C. elegans, rates of behavior and growth are slowed and, interestingly, lifespan is extended. In this study, we investigated the mechanism of this response. We find that inhibiting respiration increases the expression of genes predicted to protect and metabolically remodel the animal. This pattern of gene expression is reminiscent of the expression profile of long-lived respiration-defective yeast, suggesting ancient evolutionary conservation. Mutations in clk-1, which inhibit the synthesis of the respiratory-chain factor ubiquinone, produce gene expression, longevity, and behavioral phenotypes similar to those produced by inhibiting components of the respiratory chain. We find that knocking down the activities of two similar genes—fsrt-1 and fstr-2—accelerates the behaviors and aging rates of clk-1 mutants and inhibits the clk-1(−) transcriptional response. Thus, fstr-1/2, which encode potential signaling proteins, appear to be part of a mechanism that actively slows rates of growth, behavior, and aging in response to altered ubiquinone synthesis. Unexpectedly, fsrt-1/2 are not required for the longevity and behavioral phenotypes produced by inhibiting the gene isp-1, which encodes a different component of the respiratory chain. Our findings suggest that different types of mitochondrial perturbations activate distinct pathways that converge on similar downstream processes to slow behavioral rates and extend lifespan.
doi:10.1371/journal.pgen.1000450
PMCID: PMC2660839  PMID: 19360127
7.  The Aging-Associated Enzyme CLK-1 is a Member of the Carboxylate-Bridged Diiron Family of Proteins 
Biochemistry  2010;49(45):9679-9681.
The aging-associated enzyme CLK-1 is proposed to be a member of the carboxylate-bridged diiron family of proteins. To evaluate this hypothesis and characterize the protein, we expressed soluble mouse CLK-1 (MCLK1) in E. coli as a heterologous host. Using Mössbauer and EPR spectroscopy, we established that MCLK1 indeed belongs to this protein family. Biochemical analyses for in vitro activity of MCLK1 with quinone substrates revealed that NADH can serve directly as a reductant for catalytic activation of dioxygen and substrate oxidation by the enzyme, with no requirement for an additional reductase protein component. The direct reaction of NADH with a diiron-containing oxidase enzyme has not previously been encountered for any member of the protein superfamily.
doi:10.1021/bi101475z
PMCID: PMC2976817  PMID: 20923139
8.  Uncoupling the Pleiotropic Phenotypes of clk-1 with tRNA Missense Suppressors in Caenorhabditis elegans 
Molecular and Cellular Biology  2006;26(10):3976-3985.
clk-1 encodes a demethoxyubiquinone (DMQ) hydroxylase that is necessary for ubiquinone biosynthesis. When Caenorhabditis elegans clk-1 mutants are grown on bacteria that synthesize ubiquinone (UQ), they are viable but have a pleiotropic phenotype that includes slowed development, behaviors, and aging. However, when grown on UQ-deficient bacteria, the mutants arrest development transiently before growing up to become sterile adults. We identified nine suppressors of the missense mutation clk-1(e2519), which harbors a Glu-to-Lys substitution. All suppress the mutant phenotypes on both UQ-replete and UQ-deficient bacteria. However, each mutant suppresses a different subset of phenotypes, indicating that most phenotypes can be uncoupled from each other. In addition, all suppressors restore the ability to synthesize exceedingly small amounts of UQ, although they still accumulate the precursor DMQ, suggesting that the presence of DMQ is not responsible for the Clk-1 phenotypes. We cloned six of the suppressors, and all encode tRNAGlu genes whose anticodons are altered to read the substituted Lys codon of clk-1(e2519). To our knowledge, these suppressors represent the first missense suppressors identified in any metazoan. The pattern of suppression we observe suggests that the individual members of the tRNAGlu family are expressed in different tissues and at different levels.
doi:10.1128/MCB.26.10.3976-3985.2006
PMCID: PMC1488993  PMID: 16648490
9.  The GATA Transcription Factor egl-27 Delays Aging by Promoting Stress Resistance in Caenorhabditis elegans 
PLoS Genetics  2012;8(12):e1003108.
Stress is a fundamental aspect of aging, as accumulated damage from a lifetime of stress can limit lifespan and protective responses to stress can extend lifespan. In this study, we identify a conserved Caenorhabditis elegans GATA transcription factor, egl-27, that is involved in several stress responses and aging. We found that overexpression of egl-27 extends the lifespan of wild-type animals. Furthermore, egl-27 is required for the pro-longevity effects from impaired insulin/IGF-1 like signaling (IIS), as reduced egl-27 activity fully suppresses the longevity of worms that are mutant for the IIS receptor, daf-2. egl-27 expression is inhibited by daf-2 and activated by pro-longevity factors daf-16/FOXO and elt-3/GATA, suggesting that egl-27 acts at the intersection of IIS and GATA pathways to extend lifespan. Consistent with its role in IIS signaling, we found that egl-27 is involved in stress response pathways. egl-27 expression is induced in the presence of multiple stresses, its targets are significantly enriched for many types of stress genes, and altering levels of egl-27 itself affects survival to heat and oxidative stress. Finally, we found that egl-27 expression increases between young and old animals, suggesting that increased levels of egl-27 in aged animals may act to promote stress resistance. These results identify egl-27 as a novel factor that links stress and aging pathways.
Author Summary
Stress is a fundamental aspect of aging, but it is unclear whether the molecular mechanisms underlying stress response become altered during normal aging and whether these alterations can affect the aging process. In this study, we found a GATA transcription factor called egl-27, whose targets are significantly enriched for age-dependent genes and stress response genes, and whose expression increases with age. In contrast to previous work describing factors that are causal for aging, we found that egl-27 activity is likely beneficial for survival since egl-27 overexpression extends lifespan. egl-27 promotes longevity by enhancing stress response; specifically, increased levels of egl-27 protect animals against heat stress, while reduced egl-27 activity impairs survival following heat and oxidative stress. These results suggest that aging is not simply a process of constant decline. Some factors, such as egl-27, are more active in old animals, working to restore organismal function and to improve survival.
doi:10.1371/journal.pgen.1003108
PMCID: PMC3521710  PMID: 23271974
10.  Metabolic mechanisms of longevity: Caloric restriction in mammals and longevity mutations in Caenorhabditis elegans; a common pathway?? 
Several recent studies in Caenorhabditis elegans have reported significant extension of the lifespan by probable loss of function mutations in various genes. When sequenced, many of these genes exhibited significant homology to genes in the mammalian insulin signaling cascade. For example, the daf-2 gene that has been shown to regulate lifespan in C elegans shares significant sequence homology with the insulin and IGF-1 receptor genes in mammals. Another longevity gene in the nematode, age-1, is homologous with the p110 subunit of phosphatidylinositol 3-kinase in mammals. This enzyme functions early in the mammalian insulin response cascade to influence many important cellular growth and metabolic processes. These findings and others have led to the suggestion that lifespan regulation in nematodes is controlled by a mechanism similar to that involved in lifespan extension by caloric restriction in mammals. Many intriguing similarities exist between these two model systems providing some support for this idea. However, at present there is insufficient data to conclude that similar genes or mechanisms regulate lifespan determination in nematodes and in mammals.
doi:10.1007/s11357-000-0001-1
PMCID: PMC3455360  PMID: 23604793
11.  Inhibition of respiration extends C. elegans’ lifespan via reactive oxygen species that increase HIF-1 activity 
Current biology : CB  2010;20(23):2131-2136.
Summary
A mild inhibition of mitochondrial respiration extends the lifespan of many organisms, including yeast, worms, flies and mice [1–10], but the underlying mechanism is unknown. One environmental condition that reduces rates of respiration is hypoxia (low oxygen). Thus it is possible that mechanisms that sense oxygen play a role in the longevity response to reduced respiration. The hypoxia-inducible factor HIF-1 is a highly-conserved transcription factor that activates genes that promote survival during hypoxia [11–12]. In this study, we show that inhibiting respiration in C. elegans can promote longevity by activating HIF-1. Through genome-wide screening, we found that RNAi knockdown of many genes encoding respiratory-chain components induced hif-1-dependent transcription. Moreover, HIF-1 was required for the extended lifespans of clk-1 and isp-1 mutants, which have reduced rates of respiration [1, 4, 13]. Inhibiting respiration appears to activate HIF-1 by elevating the level of reactive oxygen species (ROS). We found that ROS is increased in respiration mutants, and that mild increases in ROS can stimulate HIF-1 to activate gene expression and promote longevity. In this way, HIF-1 appears to link respiratory stress in the mitochondria to a nuclear transcriptional response that promotes longevity.
doi:10.1016/j.cub.2010.10.057
PMCID: PMC3058811  PMID: 21093262
12.  Heat shock proteins in long-lived worms and mice with insulin/insulin-Like signaling mutations 
Aging  2009;1(6):573-577.
Heat shock proteins (HSPs) have proven to be effective tools for extending invertebrate lifespan, and inC. elegans daf-2 mutants, longevity resulting from loss of insulin / insulin-like signals is at least partly dependent upon elevated HSP expression. In mice, inhibition of the orthologous growth hormone / insulin-like growth factor I (GH / IGF-I) pathway has similar pro-longevity effects. A recent study, however, suggests that loss of GH / IGF-I signals in long-lived mice does not broadly elevate HSP expression, but in fact decreases HSP expression in many tissue types, such as liver and kidney. The contribution of chaperones to the longevity of long-lived mice with altered GH / IGF-I signals may therefore differ from that described in C. elegans daf-2 mutants. This result, in combination with other recent findings, underscores the possibility that systemic overexpression of chaperones will have dissimilar effects on longevity in vertebrate and invertebrate systems.
PMCID: PMC2806032  PMID: 20157538
Aging; chaperone; growth hormone; longevity; Snell; stress
13.  Patterns of metabolic activity during aging of the wild type and longevity mutants of Caenorhabditis elegans 
At least three mechanisms determine life span in Caenorhabditis elegans. An insulin-like signaling pathway regulates dauer diapause, reproduction and longevity. Reduction-or loss-of-function mutations in this pathway can extend longevity substantially, suggesting that the wild-type alleles shorten life span. The mutations extend life span by activating components of a dauer longevity assurance program in adult life, resulting in altered metabolism and enhanced stress resistance. The Clock (Clk) genes regulate many temporal processes, including life span. Mutation in the Clk genes clk-1 and gro-1 mildly affect energy production, but repress energy consumption dramatically, thereby reducing the rate of anabolic metabolism and lengthening life span. Dietary restriction, either imposed by mutation or by the culture medium increases longevity and uncovers a third mechanism of life span determination. Dietary restriction likely elicits the longevity assurance program. There is still uncertainty as to whether these pathways converge on daf-16 to activate downstream longevity effector genes such as ctl-1 and sod-3.
There is overwhelming evidence that the interplay between reactive oxygen species (ROS) and the capacity to resist oxidative stress controls the aging process and longevity. It is as yet not clear whether metabolic homeostasis collapses with age as a direct result of ROS-derived damage or is selectively repressed by longevity-determining genes. The dramatic decline of protein turnover during senescence results in the accumulation of altered enzymes and in a gradual decline of metabolic performance eventually followed by fatal failure of the system.
doi:10.1007/s11357-000-0007-8
PMCID: PMC3455783  PMID: 23604840
14.  A Novel Role for the SMG-1 Kinase in Lifespan and Oxidative Stress Resistance in Caenorhabditis elegans 
PLoS ONE  2008;3(10):e3354.
The PTEN tumour suppressor encodes a phosphatase, and its daf-18 orthologue in Caenorhabditis elegans negatively regulates the insulin/IGF-1 DAF-2 receptor pathway that influences lifespan in worms and other species. In order to identify new DAF-18 regulated pathways involved in aging, we initiated a candidate RNAi feeding screen for clones that lengthen lifespan. Here, we report that smg-1 inactivation increases average lifespan in a daf-18 dependent manner. Genetic analysis is consistent with SMG-1 acting at least in part in parallel to the canonical DAF-2 receptor pathway, but converging on the transcription factor DAF-16/FOXO. SMG-1 is a serine-threonine kinase which plays a conserved role in nonsense-mediated mRNA decay (NMD) in worms and mammals. In addition, human SMG-1 has also been implicated in the p53-mediated response to genotoxic stress. The effect of smg-1 inactivation on lifespan appears to be unrelated to its NMD function, but requires the p53 tumour suppressor orthologue cep-1. Furthermore, smg-1 inactivation confers a resistance to oxidative stress in a daf-18-, daf-16- and cep-1-dependent manner. We propose that the role of SMG-1 in lifespan regulation is at least partly dependent on its function in oxidative stress resistance. Taken together, our results unveil a novel role for SMG-1 in lifespan regulation.
doi:10.1371/journal.pone.0003354
PMCID: PMC2556085  PMID: 18836529
15.  Studies of Caenorhabditis elegans DAF-2/insulin signaling reveal targets for pharmacological manipulation of lifespan 
Aging Cell  2006;5(1):31-37.
Summary
Much excitement has arisen from the observation that decrements in insulin-like signaling can dramatically extend lifespan in the nematode, Caenorhabditis elegans, and fruitfly, Drosophila melanogaster. In addition, there are tantalizing hints that the IGF-I pathway in mice may have similar effects. In addition to dramatic effects on lifespan, invertebrate insulin-like signaling also promotes changes in stress resistance, metabolism and development. Which, if any, of the various phenotypes of insulin pathway mutants are relevant to longevity? What are the genes that function in collaboration with insulin to prolong lifespan? These questions are at the heart of current research in C. elegans longevity. Two main theories exist as to the mechanism behind insulin's effects on invertebrate longevity. One theory is that insulin programs metabolic parameters that prolong or reduce lifespan. The other theory is that insulin determines the cell's ability to endure oxidative stress from respiration, thereby determining the rate of aging. However, these mechanisms are not mutually exclusive and several studies seem to support a role for both. Here, we review recently published reports investigating the mechanisms behind insulin's dramatic effect on longevity. We also spotlight several C. elegans genes that are now known to interact with insulin signaling to determine lifespan. These insights into pathways affecting invertebrate lifespan may provide a basis for developing strategies for pharmacological manipulation of human lifespan.
doi:10.1111/j.1474-9726.2006.00188.x
PMCID: PMC1413578  PMID: 16441841
Aging; C. elegans; FOXO; insulin; lifespan; phosphoinositol 3-kinase
16.  The Evolutionarily Conserved Longevity Determinants HCF-1 and SIR-2.1/SIRT1 Collaborate to Regulate DAF-16/FOXO 
PLoS Genetics  2011;7(9):e1002235.
The conserved DAF-16/FOXO transcription factors and SIR-2.1/SIRT1 deacetylases are critical for diverse biological processes, particularly longevity and stress response; and complex regulation of DAF-16/FOXO by SIR-2.1/SIRT1 is central to appropriate biological outcomes. Caenorhabditis elegans Host Cell Factor 1 (HCF-1) is a longevity determinant previously shown to act as a co-repressor of DAF-16. We report here that HCF-1 represents an integral player in the regulatory loop linking SIR-2.1/SIRT1 and DAF-16/FOXO in both worms and mammals. Genetic analyses showed that hcf-1 acts downstream of sir-2.1 to influence lifespan and oxidative stress response in C. elegans. Gene expression profiling revealed a striking 80% overlap between the DAF-16 target genes responsive to hcf-1 mutation and sir-2.1 overexpression. Subsequent GO-term analyses of HCF-1 and SIR-2.1-coregulated DAF-16 targets suggested that HCF-1 and SIR-2.1 together regulate specific aspects of DAF-16-mediated transcription particularly important for aging and stress responses. Analogous to its role in regulating DAF-16/SIR-2.1 target genes in C. elegans, the mammalian HCF-1 also repressed the expression of several FOXO/SIRT1 target genes. Protein–protein association studies demonstrated that SIR-2.1/SIRT1 and HCF-1 form protein complexes in worms and mammalian cells, highlighting the conservation of their regulatory relationship. Our findings uncover a conserved interaction between the key longevity determinants SIR-2.1/SIRT1 and HCF-1, and they provide new insights into the complex regulation of FOXO proteins.
Author Summary
The nematode C. elegans has been instrumental in identifying and characterizing genetic components that influence aging. Studies in worms have been successfully extended to complex mammalian organisms allowing for the identification of genetic factors that impact longevity in mammals. DAF-16/FOXO transcription factors are among the best characterized longevity factors, and their increased activity leads to a longer lifespan and improved stress resistance in many organisms. Elucidating how the activities of DAF-16/FOXO are regulated will provide new insights into the basic biology of aging and will aid future therapeutic developments aiming to improve healthy aging and alleviate age-related diseases in humans. We utilized both C. elegans and mammalian cell culture systems to dissect the functional and molecular interactions between two important DAF-16 regulators, HCF-1 and SIR-2.1/SIRT1. We demonstrated that HCF-1 and SIR-2.1/SIRT1 physically associate and antagonize each other to properly regulate DAF-16/FOXO-mediated expression of genes important for longevity and stress response. We further showed that the functional relationships among these three proteins are conserved in mammals. Our work implicates HCF-1 as an important player in the regulation of FOXO by SIRT1, and thereby a potential longevity determinant in humans, and prompts further characterization of HCF-1's functions in aging and age-related pathologies.
doi:10.1371/journal.pgen.1002235
PMCID: PMC3164695  PMID: 21909281
17.  A conserved ubiquitination pathway determines longevity in response to diet restriction 
Nature  2009;460(7253):396-399.
Dietary Restriction (DR) extends longevity in diverse species suggesting that there is a conserved mechanism for nutrient regulation and prosurvival responses1. We have discovered a role for the HECT E3 ubiquitin ligase WWP-1 as a positive regulator of lifespan in C. elegans in response to diet restriction. We find that overexpression of wwp-1 in worms extends lifespan up to 20% under conditions of ad libitum feeding. This extension is dependent upon the FoxA transcription factor pha-4, and independent of the FoxO transcription factor, daf-16. Reduction of wwp-1 completely suppresses the extended longevity of diet-restricted animals. However, loss of wwp-1 does not affect the long lifespan of animals with compromised mitochondrial function or reduced insulin/IGF-1 signaling. Overexpression of a mutant form of WWP-1 lacking catalytic activity suppresses the increased lifespan of diet-restricted animals, indicating that WWP-1 ubiquitin ligase activity is essential for longevity. Additionally, we find that the E2 ubiquitin conjugating enzyme, UBC-18, is essential and specific for DR induced longevity. UBC-18 interacts with WWP-1 and is required for the ubiquitin ligase activity of WWP-1 and the extended longevity of worms overexpressing wwp-1. Taken together, our results indicate that WWP-1 and UBC-18 function to ubiquitinate substrates that regulate DR induced longevity.
doi:10.1038/nature08130
PMCID: PMC2746748  PMID: 19553937
18.  Positive Feedback between Transcriptional and Kinase Suppression in Nematodes with Extraordinary Longevity and Stress Resistance 
PLoS Genetics  2009;5(4):e1000452.
Insulin/IGF-1 signaling (IIS) regulates development and metabolism, and modulates aging, of Caenorhabditis elegans. In nematodes, as in mammals, IIS is understood to operate through a kinase-phosphorylation cascade that inactivates the DAF-16/FOXO transcription factor. Situated at the center of this pathway, phosphatidylinositol 3-kinase (PI3K) phosphorylates PIP2 to form PIP3, a phospholipid required for membrane tethering and activation of many signaling molecules. Nonsense mutants of age-1, the nematode gene encoding the class-I catalytic subunit of PI3K, produce only a truncated protein lacking the kinase domain, and yet confer 10-fold greater longevity on second-generation (F2) homozygotes, and comparable gains in stress resistance. Their F1 parents, like weaker age-1 mutants, are far less robust—implying that maternally contributed trace amounts of PI3K activity or of PIP3 block the extreme age-1 phenotypes. We find that F2-mutant adults have <10% of wild-type kinase activity in vitro and <60% of normal phosphoprotein levels in vivo. Inactivation of PI3K not only disrupts PIP3-dependent kinase signaling, but surprisingly also attenuates transcripts of numerous IIS components, even upstream of PI3K, and those of signaling molecules that cross-talk with IIS. The age-1(mg44) nonsense mutation results, in F2 adults, in changes to kinase profiles and to expression levels of multiple transcripts that distinguish this mutant from F1 age-1 homozygotes, a weaker age-1 mutant, or wild-type adults. Most but not all of those changes are reversed by a second mutation to daf-16, implicating both DAF-16/ FOXO–dependent and –independent mechanisms. RNAi, silencing genes that are downregulated in long-lived worms, improves oxidative-stress resistance of wild-type adults. It is therefore plausible that attenuation of those genes in age-1(mg44)-F2 adults contributes to their exceptional survival. IIS in nematodes (and presumably in other species) thus involves transcriptional as well as kinase regulation in a positive-feedback circuit, favoring either survival or reproduction. Hyperlongevity of strong age-1(mg44) mutants may result from their inability to reset this molecular switch to the reproductive mode.
Author Summary
Insulin/IGF-1 signaling (IIS) impacts development, metabolism, and longevity in Caenorhabditis elegans. It has been viewed as a cascade of kinase reactions, chiefly phosphorylation of other kinases, leading to inactivation of the DAF-16/FOXO transcription factor. PI3K, a phosphatidylinositol kinase at the center of this pathway, converts PIP2 to PIP3, instrumental to kinase docking and activation. Here we show that PI3K deficiency elicits transcriptional inhibition of many kinases, including those of IIS itself. This creates a positive-feedback loop, wherein DAF-16/FOXO silences expression of the very kinases that would have inactivated it. In the resulting “flip-flop” genetic switch, either kinase signaling or transcriptional silencing may predominate. We discovered the transcriptional arm of this switch in infertile age-1(mg44) mutants, defective for PI3K activity. The absence of PIP3 and PIP3-dependent kinase activity gives free rein to gene silencing by DAF-16/FOXO. This two-tiered response could scarcely have evolved for the benefit of a sterile mutant; some components presumably serve regulatory functions in normal animals, reinforcing a switch responsive to environmental and internal signals. In age-1(mg44) mutants, complete inactivation of PI3K “fuses” the switch, locking worms into longevity mode. With signaling profoundly silenced, they cannot resume reproduction, but instead acquire a remarkable capacity for individual survival.
doi:10.1371/journal.pgen.1000452
PMCID: PMC2661368  PMID: 19360094
19.  Structural and functional characterisation of the fork head transcription factor-encoding gene, Hc-daf-16, from the parasitic nematode Haemonchus contortus (Strongylida) 
Despite their phylogenetic diversity, parasitic nematodes share attributes of longevity and developmental arrest (=hypobiosis) with free-living nematodes at key points in their life cycles, particularly in larval stages responsible for establishing infection in the host. Insulin-like signalling plays crucial roles in the regulation of life span and arrest (=dauer formation) in the free-living nematode, Caenorhabditis elegans. Insulin-like signalling in C. elegans negatively regulates the fork head boxO (FoxO) transcription factor encoded by daf-16, which is linked to initiating a dauer-specific pattern of gene expression. Orthologues of daf-16 have been identified in several species of parasitic nematode. Although function has been demonstrated for an orthologue from the parasitic nematode Strongyloides stercoralis (Rhabditida), the functional capabilities of homologues/orthologues in bursate nematodes (Strongylida) are unknown. In the present study, we used a genomic approach to determine the structures of two complete daf-16 orthologues (designated Hc-daf-16.1 and Hc-daf-16.2) and their transcripts in the parasitic nematode Haemonchus contortus, and assessed their function(s) using C. elegans as a genetic surrogate. Unlike the multiple isoforms of Ce-DAF-16 and Ss-DAF-16, which are encoded by a single gene and produced by alternative splicing, mRNAs encoding the proteins Hc-DAF-16.1 and Hc-DAF-16.2 are transcribed from separate and distinct loci. Both orthologues are transcribed in all developmental stages and both sexes of H. contortus, and the inferred proteins (603 and 556 amino acids) each contain a characteristic, highly conserved fork head domain. In spite of distinct differences in genomic organisation compared with orthologues in C. elegans and S. stercoralis, genetic complementation studies demonstrated here that Hc-daf-16.2, but not Hc-daf-16.1, could restore daf-16 function to a C. elegans strain carrying a null mutation at this locus. These findings are consistent with previous results for S. stercoralis and demonstrate functional conservation of the daf-16b orthologue between key parasitic nematodes from two different taxonomic orders and C. elegans. We conclude from these experiments that the fork head transcription factor DAF-16 and, by inference, other insulin-like signalling elements, are conserved in H. contortus, a parasitic nematode of paramount economic importance. We demonstrate that functionality is sufficiently conserved in Hc-DAF-16.2 that it can replace Ce-DAF-16 in promoting dauer arrest in C. elegans.
doi:10.1016/j.ijpara.2009.09.005
PMCID: PMC2853935  PMID: 19796644
Daf-16; Fork head transcription factor; Dauer; Haemonchus contortus; Caenorhabditis elegans; Transgenesis
20.  TDP-1/TDP-43 Regulates Stress Signaling and Age-Dependent Proteotoxicity in Caenorhabditis elegans 
PLoS Genetics  2012;8(7):e1002806.
TDP-43 is a multifunctional nucleic acid binding protein linked to several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia. To learn more about the normal biological and abnormal pathological role of this protein, we turned to Caenorhabditis elegans and its orthologue TDP-1. We report that TDP-1 functions in the Insulin/IGF pathway to regulate longevity and the oxidative stress response downstream from the forkhead transcription factor DAF-16/FOXO3a. However, although tdp-1 mutants are stress-sensitive, chronic upregulation of tdp-1 expression is toxic and decreases lifespan. ALS–associated mutations in TDP-43 or the related RNA binding protein FUS activate the unfolded protein response and generate oxidative stress leading to the daf-16–dependent upregulation of tdp-1 expression with negative effects on neuronal function and lifespan. Consistently, deletion of endogenous tdp-1 rescues mutant TDP-43 and FUS proteotoxicity in C. elegans. These results suggest that chronic induction of wild-type TDP-1/TDP-43 by cellular stress may propagate neurodegeneration and decrease lifespan.
Author Summary
TAR DNA Binding Protein 43 (TDP-43) is implicated in several human age-dependent neurodegenerative disorders, but until now little was known about TDP-43's role in the aging process. Here we used the nematode Caenorhabditis elegans to study the role of the TDP-43 orthologue tdp-1 in aging and neurodegeneration. In this study we discovered that tdp-1 is a stress-responsive gene acting within the Insulin/IGF signaling pathway to regulate lifespan and the response to oxidative stress. We found that, although worms missing tdp-1 were stress-sensitive, elevated expression of tdp-1 was toxic. We asked if tdp-1 also responded to the stress caused by toxic proteins found in Amyotrophic Lateral Sclerosis (ALS). Using worm models for ALS, we discovered that mutant TDP-43 generated oxidative stress and induced tdp-1 expression with negative consequences on neuronal function and lifespan. Consistently, removing tdp-1 rescued toxicity in our worm ALS models. tdp-1's role in the cellular stress response likely reflects an ancient adaptation to deal with unfavorable environmental conditions that is inappropriately activated and maintained by genetic mutations leading to proteotoxic and oxidative stress. We predict that similar mechanisms may exist in humans, helping explain the involvement of TDP-43 in a growing number of neurodegenerative disorders.
Author Summary
TAR DNA Binding Protein 43 (TDP-43) is implicated in several human age-dependent neurodegenerative disorders, but until now little was known about TDP-43's role in the aging process. Here we used the nematode Caenorhabditis elegans to study the role of the TDP-43 orthologue tdp-1 in aging and neurodegeneration. In this study we discovered that tdp-1 is a stress-responsive gene acting within the Insulin/IGF signaling pathway to regulate lifespan and the response to oxidative stress. We found that, although worms missing tdp-1 were stress-sensitive, elevated expression of tdp-1 was toxic. We asked if tdp-1 also responded to the stress caused by toxic proteins found in Amyotrophic Lateral Sclerosis (ALS). Using worm models for ALS, we discovered that mutant TDP-43 generated oxidative stress and induced tdp-1 expression with negative consequences on neuronal function and lifespan. Consistently, removing tdp-1 rescued toxicity in our worm ALS models. tdp-1's role in the cellular stress response likely reflects an ancient adaptation to deal with unfavorable environmental conditions that is inappropriately activated and maintained by genetic mutations leading to proteotoxic and oxidative stress. We predict that similar mechanisms may exist in humans, helping explain the involvement of TDP-43 in a growing number of neurodegenerative disorders.
doi:10.1371/journal.pgen.1002806
PMCID: PMC3390363  PMID: 22792076
21.  Daf-2 Signaling Modifies Mutant SOD1 Toxicity in C. elegans 
PLoS ONE  2012;7(3):e33494.
The DAF-2 Insulin/IGF-1 signaling (IIS) pathway is a strong modifier of Caenorhabditis elegans longevity and healthspan. As aging is the greatest risk factor for developing neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS), we were interested in determining if DAF-2 signaling modifies disease pathology in mutant superoxide dismutase 1 (SOD1) expressing C. elegans. Worms with pan-neuronal G85R SOD1 expression demonstrate significantly impaired locomotion as compared to WT SOD1 expressing controls and they develop insoluble SOD1 aggregates. Reductions in DAF-2 signaling, either through a hypomorphic allele or neuronally targeted RNAi, decreases the abundance of aggregated SOD1 and results in improved locomotion in a DAF-16 dependant manner. These results suggest that manipulation of the DAF-2 Insulin/IGF-1 signaling pathway may have therapeutic potential for the treatment of ALS.
doi:10.1371/journal.pone.0033494
PMCID: PMC3308959  PMID: 22457769
22.  Caenorhabditis elegans HCF-1 Functions in Longevity Maintenance as a DAF-16 Regulator 
PLoS Biology  2008;6(9):e233.
The transcription factor DAF-16/forkhead box O (FOXO) is a critical longevity determinant in diverse organisms, however the molecular basis of how its transcriptional activity is regulated remains largely unknown. We report that the Caenorhabditis elegans homolog of host cell factor 1 (HCF-1) represents a new longevity modulator and functions as a negative regulator of DAF-16. In C. elegans, hcf-1 inactivation caused a daf-16-dependent lifespan extension of up to 40% and heightened resistance to specific stress stimuli. HCF-1 showed ubiquitous nuclear localization and physically associated with DAF-16. Furthermore, loss of hcf-1 resulted in elevated DAF-16 recruitment to the promoters of its target genes and altered expression of a subset of DAF-16-regulated genes. We propose that HCF-1 modulates C. elegans longevity and stress response by forming a complex with DAF-16 and limiting a fraction of DAF-16 from accessing its target gene promoters, and thereby regulates DAF-16-mediated transcription of selective target genes. As HCF-1 is highly conserved, our findings have important implications for aging and FOXO regulation in mammals.
Author Summary
One of the key molecules that modulate longevity in evolutionarily diverse organisms is the transcription factor DAF-16/FOXO. Despite its importance in aging and other biological processes, how DAF-16/FOXO activity is regulated in the nucleus is largely unknown. We report a new player important for aging modulation, the nematode homolog of host cell factor 1 (HCF-1), and show that it functions as a negative regulator of DAF-16. In worms, HCF-1 inactivation extends lifespan up to 40% and increases resistance to specific stress stimuli. To affect lifespan and stress response, HCF-1 requires the activity of DAF-16. We show that the HCF-1 protein is expressed in the nucleus and partners with DAF-16 in worms. Furthermore, we demonstrate that loss of HCF-1 results in elevated levels of DAF-16 at the promoters of its target genes and altered expression of a subset of DAF-16-regulated genes. We propose that HCF-1 modulates longevity and stress response by binding to DAF-16 and preventing the transcription factor from accessing its target gene promoters, thereby regulating the expression of DAF-16 target genes. As HCF-1 is highly conserved, our findings have important implications for aging and FOXO regulation in humans.
Caenorhabditis elegans HCF-1 is a new longevity factor that functions to antagonize the transcriptional activities of DAF-16/FOXO by forming a complex with DAF-16/FOXO and preventing a fraction of DAF-16/FOXO from accessing its target gene promoters.
doi:10.1371/journal.pbio.0060233
PMCID: PMC2553839  PMID: 18828672
23.  Altered bacterial metabolism, not coenzyme Q content, is responsible for the lifespan extension in Caenorhabditis elegans fed an Escherichia coli diet lacking coenzyme Q 
Aging cell  2008;7(3):291-304.
Summary
Coenzyme Qn is a fully substituted benzoquinone containing a polyisoprene tail of distinct numbers (n) of isoprene groups. Caenorhabditis elegans fed Escherichia coli devoid of Q8 have a significant lifespan extension when compared to C. elegans fed a standard ‘Q-replete’ E. coli diet. Here we examine possible mechanisms for the lifespan extension caused by the Q-less E. coli diet. A bioassay for Q uptake shows that a water-soluble formulation of Q10 is effectively taken up by both clk-1 mutant and wild-type nematodes, but does not reverse lifespan extension mediated by the Q-less E. coli diet, indicating that lifespan extension is not due to the absence of dietary Q per se. The enhanced longevity mediated by the Q-less E. coli diet cannot be attributed to dietary restriction, different Qn isoforms, reduced pathogenesis or slowed growth of the Q-less E. coli, and in fact requires E. coli viability. Q-less E. coli have defects in respiratory metabolism. C. elegans fed Q-replete E. coli mutants with similarly impaired respiratory metabolism due to defects in complex V also show a pronounced lifespan extension, although not as dramatic as those fed the respiratory deficient Q-less E. coli diet. The data suggest that feeding respiratory incompetent E. coli, whether Q-less or Q-replete, produces a robust life extension in wild-type C. elegans. We believe that the fermentation-based metabolism of the E. coli diet is an important parameter of C. elegans longevity.
doi:10.1111/j.1474-9726.2008.00378.x
PMCID: PMC3104051  PMID: 18267002
aging; Caenorhabditis elegans; clk-1; coenzyme Q or ubiquinone; dietary restriction; respiratory defective Escherichia coli
24.  TATN-1 Mutations Reveal a Novel Role for Tyrosine as a Metabolic Signal That Influences Developmental Decisions and Longevity in Caenorhabditis elegans 
PLoS Genetics  2013;9(12):e1004020.
Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR) signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people.
Author Summary
In people, elevated blood levels of the amino acid tyrosine are seen in obese individuals, and these elevations represent a novel risk factor for the development of diabetes. The enzyme tyrosine aminotransferase, which removes tyrosine from the body, has also been identified as a tumor suppressor gene, and this enzyme normally acts to prevent the development of liver cancer. In our work, we identify tyrosine aminotransferase as a regulator of larval development and adult longevity in the non-parasitic worm Caenorhabditis elegans. Worms with mutations impairing tyrosine aminotransferase activity show elevated levels of tyrosine, are prone to arresting development in a larval stage called a dauer, and show increased longevity. Part of the effect of tyrosine aminotransferase is due to inhibitory effects on an insulin-like signaling pathway in the worms. Our work suggests that levels of the amino acid tyrosine are sensed and can lead to changes in cell signaling. These results may provide insights into how tyrosine could be involved in obesity, diabetes, and cancer in people.
doi:10.1371/journal.pgen.1004020
PMCID: PMC3868569  PMID: 24385923
25.  Fitness cost of extended lifespan in Caenorhabditis elegans. 
An insulin/IGF-I-like signalling pathway determines the rate of aging of the adult nematode, Caenorhabditis elegans. Mutations in genes encoding this pathway can result in a doubling of lifespan. While such mutations may appear to have little effect on development or fertility, evolutionary theory predicts that large increases in lifespan will not be optimal for fitness. We demonstrate by laboratory natural selection that partial loss of function of the insulin receptor-like protein DAF-2 results in dramatically reduced fitness even under laboratory conditions. Despite long-lived mutants appearing healthy, they exhibit a heavy fitness cost consistent with an evolutionary theory of aging.
doi:10.1098/rspb.2004.2897
PMCID: PMC1440519  PMID: 15590605

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