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1.  Genome-wide modeling of complex phenotypes in Caenorhabditis elegans and Drosophila melanogaster 
BMC Genomics  2013;14:580.
The genetic and molecular basis for many intermediate and end stage phenotypes in model systems such as C. elegans and D. melanogaster has long been known to involve pleiotropic effects and complex multigenic interactions. Gene sets are groups of genes that contribute to multiple biological or molecular phenomena. They have been used in the analysis of large molecular datasets such as microarray data, Next Generation sequencing, and other genomic datasets to reveal pleiotropic and multigenic contributions to phenotypic outcomes. Many model systems lack species specific organized phenotype based gene sets to enable high throughput analysis of large molecular datasets.
Results and discussion
Here, we describe two novel collections of gene sets in C. elegans and D. melanogaster that are based exclusively on genetically determined phenotypes and use a controlled phenotypic ontology. We use these collections to build genome-wide models of thousands of defined phenotypes in both model species. In addition, we demonstrate the utility of these gene sets in systems analysis and in analysis of gene expression-based molecular datasets and show how they are useful in analysis of genomic datasets connecting multigenic gene inputs to complex phenotypes.
Phenotypic based gene sets in both C. elegans and D. melanogaster are developed, characterized, and shown to be useful in the analysis of large scale species-specific genomic datasets. These phenotypic gene set collections will contribute to the understanding of complex phenotypic outcomes in these model systems.
PMCID: PMC3849582  PMID: 23984798
C. elegans; D. melanogaster; Worm; Fly; Aging; Gene set; Phenotype; Ontology; Network; Gene expression
2.  Extension of Lifespan in C. elegans by Naphthoquinones That Act through Stress Hormesis Mechanisms 
PLoS ONE  2011;6(7):e21922.
Hormesis occurs when a low level stress elicits adaptive beneficial responses that protect against subsequent exposure to severe stress. Recent findings suggest that mild oxidative and thermal stress can extend lifespan by hormetic mechanisms. Here we show that the botanical pesticide plumbagin, while toxic to C. elegans nematodes at high doses, extends lifespan at low doses. Because plumbagin is a naphthoquinone that can generate free radicals in vivo, we investigated whether it extends lifespan by activating an adaptive cellular stress response pathway. The C. elegans cap‘n’collar (CNC) transcription factor, SKN-1, mediates protective responses to oxidative stress. Genetic analysis showed that skn-1 activity is required for lifespan extension by low-dose plumbagin in C. elegans. Further screening of a series of plumbagin analogs identified three additional naphthoquinones that could induce SKN-1 targets in C. elegans. Naphthazarin showed skn-1dependent lifespan extension, over an extended dose range compared to plumbagin, while the other naphthoquinones, oxoline and menadione, had differing effects on C. elegans survival and failed to activate ARE reporter expression in cultured mammalian cells. Our findings reveal the potential for low doses of naturally occurring naphthoquinones to extend lifespan by engaging a specific adaptive cellular stress response pathway.
PMCID: PMC3135594  PMID: 21765926
3.  Regulation of Caenorhabditis elegans vitellogenesis by DAF-2/IIS through separable transcriptional and posttranscriptional mechanisms 
BMC Physiology  2011;11:11.
Evolutionary theories of aging propose that longevity evolves as a competition between reproduction and somatic maintenance for a finite pool of resources. Reproduction is thought to shorten lifespan by depleting resources from processes promoting somatic maintenance. Maternal yolk production, vitellogenesis, represents a significant maternal cost for reproduction and is suppressed under genetic and environmental conditions that extend lifespan. However, little is known about the pathways regulating vitellogenesis in response to prolongevity cues.
In order to identify mechanisms that suppress vitellogenesis under prolongevity conditions, we studied factors regulating vitellogenesis in C. elegans nematodes. In C. elegans, vitellogenesis is depressed in the absence of insulin-like signaling (IIS). We found that the C. elegans daf-2/IIS pathway regulates vitellogenesis through two mechanisms. vit-2 transcript levels in daf-2 mutants were indirectly regulated through a germline-dependent signal, and could be rescued by introduction of daf-2(+) sperm. However, yolk protein (YP) levels in daf-2 mutants were also regulated by germline-independent posttranscriptional mechanisms.
C. elegans vitellogenesis is regulated transcriptionally and posttranscriptionally in response to environmental and reproductive cues. The daf-2 pathway suppressed vitellogenesis through transcriptional mechanisms reflecting reproductive phenotypes, as well as distinct posttranscriptional mechanisms. This study reveals that pleiotropic effects of IIS pathway mutations can converge on a common downstream target, vitellogenesis, as a mechanism to modulate longevity.
PMCID: PMC3160409  PMID: 21749693
4.  Co-Regulation of the DAF-16 Target Gene, cyp-35B1/dod-13, by HSF-1 in C. elegans Dauer Larvae and daf-2 Insulin Pathway Mutants 
PLoS ONE  2011;6(3):e17369.
Insulin/IGF-I-like signaling (IIS) has both cell autonomous and non-autonomous functions. In some cases, targets through which IIS regulates cell-autonomous functions, such as cell growth and metabolism, have been identified. In contrast, targets for many non-autonomous IIS functions, such as C. elegans dauer morphogenesis, remain elusive. Here, we report the use of genomic and genetic approaches to identify potential non-autonomous targets of C. elegans IIS. First, we used transcriptional microarrays to identify target genes regulated non-autonomously by IIS in the intestine or in neurons. C. elegans IIS controls expression of a number of stress response genes, which were differentially regulated by tissue-restricted IIS. In particular, expression of sod-3, a MnSOD enzyme, was not regulated by tissue-restricted IIS on the microarrays, while expression of hsp-16 genes was rescued back to wildtype by tissue restricted IIS. One IIS target regulated non-autonomously by age-1 was cyp-35B1/dod-13, encoding a cytochrome P450. Genetic analysis of the cyp-35B1 promoter showed both DAF-16 and HSF-1 are direct regulators. Based on these findings, we propose that hsf-1 may participate in the pathways mediating non-autonomous activities of age-1 in C. elegans.
PMCID: PMC3052305  PMID: 21408062
5.  Behaviour of Telomere and Telomerase during Aging and Regeneration in Zebrafish 
PLoS ONE  2011;6(2):e16955.
Telomere length and telomerase activity are important factors in the pathobiology of human diseases. Age-related diseases and premature aging syndromes are characterized by short telomeres, which can compromise cell viability, whereas tumour cells can prevent telomere loss by aberrantly upregulating telomerase. The zebrafish (Danio rerio) offers multiple experimental manipulation advantages over other vertebrate models and, therefore, it has been recently considered as a potential model for aging, cancer, and regeneration studies. However, it has only partially been exploited to shed light on these fundamental biological processes. The aim of this study was, therefore, to investigate telomere length and telomerase expression and activity in different strains of zebrafish obtained from different stock centres to determine whether they undergo any changes during aging and regeneration. We found that although both telomerase expression and telomere length increased from embryo to adulthood stages, they drastically declined in aged fish despite telomerase activity was detected in different tissues of old fish. In addition, we observed a weaker upregulation of telomerase expression in regenerating fins of old fish, which well correlates with their impaired regeneration capacity. Strikingly, telomeres were elongated or maintained during the fin regeneration process at all ages and after repeated amputations, likely to support high cell proliferation rates. We conclude that the expression of telomerase and telomere length are closely related during the entire life cycle of the fish and that these two parameters can be used as biomarkers of aging in zebrafish. Our results also reveal a direct relationship between the expression of telomerase, telomere length and the efficiency of tissue regeneration.
PMCID: PMC3036734  PMID: 21347393
6.  Mitochondrial Function Is Required for Secretion of DAF-28/Insulin in C. elegans 
PLoS ONE  2011;6(1):e14507.
While insulin signaling has been extensively studied in Caenorhabditis elegans in the context of ageing and stress response, less is known about the factors underlying the secretion of insulin ligands upstream of the insulin receptor. Activation of the receptor governs the decision whether to progress through the reproductive lifecycle or to arrest growth and enter hibernation. We find that animals with reduced levels of the mitochondrial outer membrane translocase homologue TOMM-40 arrest growth as larvae and have decreased insulin signaling strength. TOMM-40 acts as a mitochondrial translocase in C. elegans and in its absence animals fail to import a mitochondrial protein reporter across the mitochondrial membrane(s). Inactivation of TOMM-40 evokes the mitochondrial unfolded protein response and causes a collapse of the proton gradient across the inner mitochondrial membrane. Consequently these broadly dysfunctional mitochondria render an inability to couple food abundance to secretion of DAF-28/insulin. The secretion defect is not general in nature since two other neuropeptides, ANF::GFP and INS-22::VENUS, are secreted normally. RNAi against two other putative members of the TOMM complex give similar phenotypes, implying that DAF-28 secretion is sensitive to mitochondrial dysfunction in general. We conclude that mitochondrial function is required for C. elegans to secrete DAF-28/insulin when food is abundant. This modulation of secretion likely represents an additional level of control over DAF-28/insulin function.
PMCID: PMC3022011  PMID: 21264209
7.  Root-Knot Nematodes Exhibit Strain-Specific Clumping Behavior That Is Inherited as a Simple Genetic Trait 
PLoS ONE  2010;5(12):e15148.
Root-knot nematodes are obligate parasites of a wide range of plant species and can feed only on the cytoplasm of living plant cells. In the absence of a suitable plant host, infective juveniles of strain VW9 of the Northern root-knot nematode, Meloidogyne hapla, when dispersed in Pluronic F-127 gel, aggregate into tight, spherical clumps containing thousands of worms. Aggregation or clumping behavior has been observed in diverse genera in the phylum Nematoda spanning free-living species such as Caenorhabditis elegans as well as both plant and animal parasites. Clumping behavior differs between strains of M. hapla and occurs with other species within this genus where strain-specific differences in clumping ability are also apparent. Exposure of M. hapla juveniles to a gradient formed using low levels of cyanide promotes formation of clumps at a preferred cyanide level. Analysis of F2 lines from a cross of M. hapla strains that differ in clump-forming behavior reveals that the behavior segregates as a single, major locus that can be positioned on the genetic map of this nematode. Clumping behavior may be a survival strategy whose importance and function depend on the niche of the nematode strain or species.
PMCID: PMC3000325  PMID: 21151553
8.  Quantitative measurement of aging using image texture entropy 
Bioinformatics  2009;25(23):3060-3063.
Motivation: A key element in understanding the aging of Caenorhabditis elegans is objective quantification of the morphological differences between younger and older animals. Here we propose to use the image texture entropy as an objective measurement that reflects the structural deterioration of the C.elegans muscle tissues during aging.
Results: The texture entropy and directionality of the muscle microscopy images were measured using 50 animals on Days 0, 2, 4, 6, 8, 10 and 12 of adulthood. Results show that the entropy of the C.elegans pharynx tissues increases as the animal ages, but a sharper increase was measured between Days 2 and 4, and between Days 8 and 10. These results are in agreement with gene expression findings, and support the contention that the process of C.elegans aging has several distinct stages. This can indicate that C.elegans aging is driven by developmental pathways, rather than stochastic accumulation of damage.
Availability: The image data are freely available on the Internet at, and the Haralick and Tamura texture analysis source code can be downloaded at
PMCID: PMC3167693  PMID: 19808878
9.  Quantifiable Biomarkers of Normal Aging in the Japanese Medaka Fish (Oryzias latipes) 
PLoS ONE  2010;5(10):e13287.
Small laboratory fish share many anatomical and histological characteristics with other vertebrates, yet can be maintained in large numbers at low cost for lifetime studies. Here we characterize biomarkers associated with normal aging in the Japanese medaka (Oryzias latipes), a species that has been widely used in toxicology studies and has potential utility as a model organism for experimental aging research.
Principal Findings
The median lifespan of medaka was approximately 22 months under laboratory conditions. We performed quantitative histological analysis of tissues from age-grouped individuals representing young adults (6 months old), mature adults (16 months old), and adults that had survived beyond the median lifespan (24 months). Livers of 24-month old individuals showed extensive morphologic changes, including spongiosis hepatis, steatosis, ballooning degeneration, inflammation, and nuclear pyknosis. There were also phagolysosomes, vacuoles, and residual bodies in parenchymal cells and congestion of sinusoidal vessels. Livers of aged individuals were characterized by increases in lipofuscin deposits and in the number of TUNEL-positive apoptotic cells. Some of these degenerative characteristics were seen, to a lesser extent, in the livers of 16-month old individuals, but not in 6-month old individuals. The basal layer of the dermis showed an age-dependent decline in the number of dividing cells and an increase in senescence-associated β-galactosidase. The hearts of aged individuals were characterized by fibrosis and lipofuscin deposition. There was also a loss of pigmented cells from the retinal epithelium. By contrast, age-associated changes were not apparent in skeletal muscle, the ocular lens, or the brain.
The results provide a set of markers that can be used to trace the process of normal tissue aging in medaka and to evaluate the effect of environmental stressors.
PMCID: PMC2952620  PMID: 20949019
10.  A Naturally Occurring Polymorphism at Drosophila melanogaster Lim3 Locus, a Homolog of Human LHX3/4, Affects Lim3 Transcription and Fly Lifespan 
PLoS ONE  2010;5(9):e12621.
Lim3 encodes an RNA polymerase II transcription factor with a key role in neuron specification. It was also identified as a candidate gene that affects lifespan. These pleiotropic effects indicate the fundamental significance of the potential interplay between neural development and lifespan control. The goal of this study was to analyze the causal relationships between Lim3 structural variations, and gene expression and lifespan changes, and to provide insights into regulatory pathways controlling lifespan. Fifty substitution lines containing second chromosomes from a Drosophila natural population were used to analyze the association between lifespan and sequence variation in the 5′-regulatory region, and first exon and intron of Lim3A, in which we discovered multiple transcription start sites (TSS). The core and proximal promoter organization for Lim3A and a previously unknown mRNA named Lim3C were described. A haplotype of two markers in the Lim3A regulatory region was significantly associated with variation in lifespan. We propose that polymorphisms in the regulatory region affect gene transcription, and consequently lifespan. Indeed, five polymorphic markers located within 380 to 680 bp of the Lim3A major TSS, including two markers associated with lifespan variation, were significantly associated with the level of Lim3A transcript, as evaluated by real time RT-PCR in embryos, adult heads, and testes. A naturally occurring polymorphism caused a six-fold change in gene transcription and a 25% change in lifespan. Markers associated with long lifespan and intermediate Lim3A transcription were present in the population at high frequencies. We hypothesize that polymorphic markers associated with Lim3A expression are located within the binding sites for proteins that regulate gene function, and provide general rather than tissue-specific regulation of transcription, and that intermediate levels of Lim3A expression confer a selective advantage and longer lifespan.
PMCID: PMC2935391  PMID: 20838645
11.  Transcriptional Regulation of Human and Rat Hepatic Lipid Metabolism by the Grapefruit Flavonoid Naringenin: Role of PPARα, PPARγ and LXRα 
PLoS ONE  2010;5(8):e12399.
Disruption of lipid and carbohydrate homeostasis is an important factor in the development of prevalent metabolic diseases such as diabetes, obesity, and atherosclerosis. Therefore, small molecules that could reduce insulin dependence and regulate dyslipidemia could have a dramatic effect on public health. The grapefruit flavonoid naringenin has been shown to normalize lipids in diabetes and hypercholesterolemia, as well as inhibit the production of HCV. Here, we demonstrate that naringenin regulates the activity of nuclear receptors PPARα, PPARγ, and LXRα. We show it activates the ligand-binding domain of both PPARα and PPARγ, while inhibiting LXRα in GAL4-fusion reporters. Using TR-FRET, we show that naringenin is a partial agonist of LXRα, inhibiting its association with Trap220 co-activator in the presence of TO901317. In addition, naringenin induces the expression of PPARα co-activator, PGC1α. The flavonoid activates PPAR response element (PPRE) while suppressing LXRα response element (LXRE) in human hepatocytes, translating into the induction of PPAR-regulated fatty acid oxidation genes such as CYP4A11, ACOX, UCP1 and ApoAI, and inhibition of LXRα-regulated lipogenesis genes, such as FAS, ABCA1, ABCG1, and HMGR. This effect results in the induction of a fasted-like state in primary rat hepatocytes in which fatty acid oxidation increases, while cholesterol and bile acid production decreases. Our findings explain the myriad effects of naringenin and support its continued clinical development. Of note, this is the first description of a non-toxic, naturally occurring LXRα inhibitor.
PMCID: PMC2928300  PMID: 20811644
12.  Vibrio cholerae Hemolysin Is Required for Lethality, Developmental Delay, and Intestinal Vacuolation in Caenorhabditis elegans 
PLoS ONE  2010;5(7):e11558.
Cholera toxin (CT) and toxin-co-regulated pili (TCP) are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood.
Methodology/Principal Findings
To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations.
Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.
PMCID: PMC2903476  PMID: 20644623
13.  Evidence for an Essential Deglycosylation-Independent Activity of PNGase in Drosophila melanogaster 
PLoS ONE  2010;5(5):e10545.
Peptide:N-glycanase (PNGase) is an enzyme which releases N-linked glycans from glycopeptides/glycoproteins. This enzyme plays a role in the ER-associated degradation (ERAD) pathway in yeast and mice, but the biological importance of this activity remains unknown.
Principal Findings
In this study, we characterized the ortholog of cytoplasmic PNGases, PNGase-like (Pngl), in Drosophila melanogaster. Pngl was found to have a molecular weight of ∼74K and was mainly localized in the cytosol. Pngl lacks a CXXC motif that is critical for enzymatic activity in other species and accordingly did not appear to possess PNGase activity, though it still retains carbohydrate-binding activity. We generated microdeletions in the Pngl locus in order to investigate the functional importance of this protein in vivo. Elimination of Pngl led to a serious developmental delay or arrest during the larval and pupal stages, and surviving mutant adult males and females were frequently sterile. Most importantly, these phenotypes were rescued by ubiquitous expression of Pngl, clearly indicating that those phenotypic consequences were indeed due to the lack of functional Pngl. Interestingly, a putative “catalytic-inactive” mutant could not rescue the growth-delay phenotype, indicating that a biochemical activity of this protein is important for its biological function.
Pngl was shown to be inevitable for the proper developmental transition and the biochemical properties other than deglycosylation activity is important for its biological function.
PMCID: PMC2866665  PMID: 20479940
14.  Bone T-Scores and Functional Status: A Cross-Sectional Study on German Elderly 
PLoS ONE  2009;4(12):e8216.
We explore the association between bone T-scores, used in osteoporosis diagnosis, and functional status since we hypothesized that bone health can impact elderly functional status and indirectly independence.
In a cross-sectional study (2005–2006) on community dwelling elderly (> = 75 years) from Herne, Germany we measured bone T-scores with Dual-energy X-ray Absorptiometry, and functional status indexed by five geriatric tests: activities of daily living, instrumental activities of daily living, test of dementia, geriatric depression score and the timed-up-and-go test, and two pooled indexes: raw and standardized. Generalized linear regression was used to determine the relationship between T-scores and functional status.
From 3243 addresses, only 632 (19%) completed a clinical visit, of which only 440 (male∶female, 243∶197) could be included in analysis. T-scores (−0.99, 95% confidence interval [CI], −1.1–0.9) predicted activities of daily living (95.3 CI, 94.5–96.2), instrumental activities of daily living (7.3 CI, 94.5–96.2), and timed-up-and-go test (10.7 CI, 10.0–11.3) (P< = 0.05). Pooled data showed that a unit improvement in T-score improved standardized pooled functional status (15 CI, 14.7–15.3) by 0.41 and the raw (99.4 CI, 97.8–101.0) by 2.27 units. These results were limited due to pooling of different scoring directions, selection bias, and a need to follow-up with evidence testing.
T-scores associated with lower functional status in community-dwelling elderly. Regular screening of osteoporosis as a preventive strategy might help maintain life quality with aging.
PMCID: PMC2784940  PMID: 20011039
15.  Examination of the requirement for ucp-4, a putative homolog of mammalian uncoupling proteins, for stress tolerance and longevity in C. elegans 
Mechanisms of ageing and development  2005;126(10):1090-1096.
Reactive oxygen species (ROS) are generated by mitochondrial respiration and can react with and damage cellular components. According to the free radical theory of aging, oxidative damage from mitochondrial ROS is a major cause of cellular decline during aging. Mitochondrial uncoupling proteins (UCPs) uncouple ATP production from electron transport and can be stimulated by free radicals, suggesting UCPs may perform a cytoprotective function. The nematode, Caenorhabditis elegans, contains one UCP-like protein, encoded by the ucp-4 gene. We have investigated the genetic requirement for ucp-4 in normal aging and stress resistance. Consistent with the hypothesis that ucp-4 encodes a putative uncoupling protein, animals lacking ucp-4 function contained elevated ATP levels. However, the absence of ucp-4 function did not affect adult lifespan or survival in the presence of thermal or oxidative stress. Together, these results demonstrate that ucp-4 is a negative regulator of ATP production in C. elegans, but is not required for normal lifespan.
PMCID: PMC2553218  PMID: 15893362
Caenorhabditis elegans; Mitochondria; Oxidative stress; Lifespan
16.  Sarcopenia in the Caenorhabditis elegans pharynx correlates with muscle contraction rate over lifespan 
Experimental gerontology  2006;41(3):252-260.
In muscles, sarcopenia, the loss of muscle mass, is the major cause of aging-related functional decline and frailty. Several factors are correlated with sarcopenia during aging, including contraction-related cellular injury, oxidative stress, endocrine changes and reduced regenerative potential. However the involvement of these factors has not been experimentally investigated. Here, we report that contraction-related injury may significantly promote the progression of sarcopenia in the pharynx of the nematode, Caenorhabditis elegans, a model of aging in non-regenerative tissues. Both functional and structural declines in the pharynx during aging were significantly delayed in mutants with reduced muscle contraction rates. We also examined the role of bacteria in pharynx muscle decline during aging, as previous studies reported that antimicrobial treatments could extend C. elegans lifespan. Although microbial infection may have enhanced functional decline in the pharynx during aging, it was not the sole cause of decreased pumping rates in old animals. This study identifies contraction-related injury as a factor affecting the initiation and progression of sarcopenia during aging. Further, characterization of the specific types of damage induced by muscle contraction will be helpful for understanding the underlying causes of sarcopenia.
PMCID: PMC2553216  PMID: 16446070
Caenorhabditis elegans; Pharynx; Sarcopenia; Muscle; Aging; Insulin; Acetylcholine; Bacteria; Microbial stress
17.  Uncoupling protein homologs may provide a link between mitochondria, metabolism and lifespan 
Ageing research reviews  2006;5(2):196-208.
Uncoupling proteins (UCPs), which dissipate the mitochondrial proton gradient, have the ability to decouple mitochodrial respiration from ATP production. Since mitochondrial electron transport is a major source of free radical production, it is possible that UCP activity might impact free radical production. Free radicals can react with and damage cellular proteins, DNA and lipids. Accumulated damage from oxidative stress is believed to be a major contributor to cellular decline during aging. If UCP function were to impact mitochondrial free radical production, then one would expect to find a link between UCP activity and aging. This theory has recently been tested in a handful of organisms whose genomes contain UCP1 homologs. Interestingly, these experiments indicate that UCP homologs can affect lifespan, although they do not support a simple relationship between UCP activity and aging. Instead, UCP-like proteins appear to have a variety of effects on lifespan, and on pathways implicated in lifespan regulation. One possible explanation for this complex picture is that UCP homologs may have tissue-specific effects that complicate their effects on aging. Furthermore, the functional analysis of UCP1 homologs is incomplete. Thus, these proteins may perform functions in addition to, or instead of, mitochondrial uncoupling. Although these studies have not revealed a clear picture of UCP effects on aging, they have contributed to the growing knowledge base for these interesting proteins. Future biochemical and genetic investigation of UCP-like proteins will do much to clarify their functions and to identify the regulatory networks in which they are involved.
PMCID: PMC2553214  PMID: 16707280
UCP; Mitochondria; Lifespan; Oxidative stress; Insulin; Caloric restriction
18.  Dietary deprivation extends lifespan in Caenorhabditis elegans 
Aging cell  2006;5(6):515-524.
Dietary restriction (DR) is well known as a nongenetic intervention that robustly extends lifespan in a variety of species; however, its underlying mechanisms remain unclear. We have found in Caenorhabditis elegans that dietary deprivation (DD) during adulthood, defined as removal of their food source Escherichia coli after the completion of larval development, increased lifespan and enhanced thermotolerance and resistance to oxidative stress. DD-induced longevity was independent of one C. elegans SIRTUIN, sir-2.1, which is required for the effects of DR, and was independent of the daf-2/insulin-like signaling pathway that independently regulates longevity and larval diapause in C. elegans. DD did not significantly alter lifespan of fem-1(hc17); eat-2(ad465) worms, a genetic model of DR. These findings suggest that DD and DR share some downstream effectors. In addition, DD was detrimental for longevity when imposed on reproductively active young adults, suggesting that DD may only be beneficial in the absence of competing metabolic demands, such as fertility. Adult-onset DD offers a new paradigm for investigating dietary regulation of longevity in C. elegans. This study presents the first evidence that long-term DD, instead of being detrimental, can extend lifespan of a multicellular adult organism.
PMCID: PMC2546582  PMID: 17096674
ad libitum; aging; Caenorhabditis elegans; calorie restriction; dietary restriction; reproduction
19.  Methoxylation enhances stilbene bioactivity in Caenorhabditis elegans 
BMC Pharmacology  2008;8:15.
Stilbenes are 1,2-diphenylethylene congeners produced by plants in response to stress. Many stilbenes also exhibit xenobiotic activities in animal cells, such as inhibition of cancer cell growth, neuroprotection, and immune modulation. In vivo, hydroxylated stilbenes are metabolized by glucuronidation to facilitate excretion. Methoxylated stilbenes are metabolized more slowly, which may have a positive effect on in vivo bioactivity. Here, we have directly compared in vivo bioactivities of methoxylated and hydroxylated stilbenes in a whole organism using the roundworm Caenorhabditis elegans, an advantageous experimental system for such studies due to its rapid lifecycle, genetic amenability and relatively low-cost.
Toxicity towards C. elegans adults was observed for trimethoxylated and dimethoxylated stilbenes, as well as the monomethoxylated stilbene desoxyrhapontigenin. Toxicity was not observed for the monomethoxylated stilbene, pinostilbene, nor for hydroxylated stilbenes. The methoxylated stilbenes that exhibited toxicity also showed stronger inhibitory effects than the hydroxylated stilbenes on germline tumor growth in gld-1(q485) adults. However, steady-state levels of three inhibitory methoxylated stilbenes did not directly correlate to their relative bioactivities.
These findings demonstrate that, for the group of stilbenes investigated, methoxylation generally increased bioactivity in vivo in a whole organism, with the exception of pinostilbene. Differences in bioactivity in C. elegans adults did not appear to correlate with differential uptake. Rather, we speculate that methoxylated stilbenes may have increased interactions with biological targets in vivo or may interact with specific targets unaffected by hydroxylated stilbenes. The potent activities of methoxylated stilbenes provide a basis for further investigations to identify in vivo targets for these compounds.
PMCID: PMC2532997  PMID: 18700960
20.  Quantitative Image Analysis Reveals Distinct Structural Transitions during Aging in Caenorhabditis elegans Tissues 
PLoS ONE  2008;3(7):e2821.
Aging is associated with functional and structural declines in many body systems, even in the absence of underlying disease. In particular, skeletal muscles experience severe declines during aging, a phenomenon termed sarcopenia. Despite the high incidence and severity of sarcopenia, little is known about contributing factors and development. Many studies focus on functional aspects of aging-related tissue decline, while structural details remain understudied. Traditional approaches for quantifying structural changes have assessed individual markers at discrete intervals. Such approaches are inadequate for the complex changes associated with aging. An alternative is to consider changes in overall morphology rather than in specific markers. We have used this approach to quantitatively track tissue architecture during adulthood and aging in the C. elegans pharynx, the neuromuscular feeding organ. Using pattern recognition to analyze aged-grouped pharynx images, we identified discrete step-wise transitions between distinct morphologies. The morphology state transitions were maintained in mutants with pharynx neurotransmission defects, although the pace of the transitions was altered. Longitudinal measurements of pharynx function identified a predictive relationship between mid-life pharynx morphology and function at later ages. These studies demonstrate for the first time that adult tissues undergo distinct structural transitions reflecting postdevelopmental events. The processes that underlie these architectural changes may contribute to increased disease risk during aging, and may be targets for factors that alter the aging rate. This work further demonstrates that pattern analysis of an image series offers a novel and generally accessible approach for quantifying morphological changes and identifying structural biomarkers.
PMCID: PMC2483734  PMID: 18665238
21.  Insulin signaling in Caenorhabditis elegans regulates both endocrine-like and cell-autonomous outputs 
Developmental biology  2006;303(2):434-447.
In C. elegans, insulin signaling affects development, lifespan and stress resistance. Several studies have shown that insulin signaling affects lifespan in an endocrine-like manner from different cells, while the major downstream target of insulin, the FOXO transcription factor encoded by daf-16, may act preferentially in intestinal cells to prolong lifespan. This discrepancy raised the possibility that insulin may have both endocrine and cell-intrinsic outputs. Here, we further investigated the types of cells capable of producing endocrine outputs of insulin and also identified a new cell-intrinsic insulin output. We found that insulin signaling within groups of neurons promoted wildtype lifespan, showing that the endocrine outputs of insulin were not restricted to specific cells. In contrast, DAF-16 appeared to have a greater effect on lifespan when expressed in a combination of tissues. These results suggest that insulin signaling may regulate DAF-16 through cell-intrinsic and endocrine pathways. We also found that an insulin-dependent response to fasting in intestinal cells was preferentially regulated by intestinal insulin signaling and was less responsive to insulin signaling from non-intestinal cells. Together, these results show that C. elegans insulin signaling has endocrine as well as tissue-specific outputs which could influence lifespan in a combinatorial fashion.
PMCID: PMC2262836  PMID: 17257591
Insulin; Phosphoinositide 3-kinase; FOXO; Caenorhabditis elegans; Aging; Lifespan; Dauer; Diapause
22.  Behavioral deficits during early stages of aging in Caenorhabditis elegans result from locomotory deficits possibly linked to muscle frailty. 
Many behavioral responses require the coordination of sensory inputs with motor outputs. Aging is associated with progressive declines in both motor function and muscle structure. However, the consequences of age-related motor deficits upon behavior have not been clearly defined. Here, we examined the effects of aging on behavior in the nematode, Caenorhabditis elegans. As animals aged, mild locomotory deficits appeared that were sufficient to impair behavioral responses to sensory cues. In contrast, sensory ability appeared well-maintained during aging. Age-related behavioral declines were delayed in animals with mutations in the daf-2/insulin-like pathway governing longevity. A decline in muscle tissue integrity was correlated with the onset of age-related behavioral deficits, although significant muscle deterioration did not. Treatment with a muscarinic agonist significantly improved locomotory behavior in aged animals, indicating that improved neuromuscular signaling may be one strategy for reducing the severity of age-related behavioral impairments.
PMCID: PMC1458366  PMID: 15699524
23.  Comparative approaches to facilitate the discovery of prolongevity interventions: Effects of tocopherols on lifespan of three invertebrate species 
Many compounds hold promise for pharmacologic manipulation of aging. However, such claims are difficult to investigate due to time and budget constraints. Here, we took a comparative approach, using short-lived invertebrate species, to directly test the effects of two tocopherols (vitamin E) on longevity. γ-tocopherol represents the most abundant tocopherol in the Western diet, while α-tocopherol is selectively enriched in human plasma. Both isoforms demonstrate antioxidant activity and are proposed to have anti-aging activities. We compared the effects of α- and γ-tocopherol supplementation on lifespan in three invertebrate species. γ-, but not α-, tocopherol slightly extended lifespan in nematodes, but neither significantly affected lifespan in two fly species. This study shows that a comparative approach, utilizing multiple invertebrate species, can increase the robustness of invertebrate-based pilot screens for prolongevity interventions.
PMCID: PMC1820625  PMID: 17169403
24.  DAF-2/Insulin-Like Signaling in C. elegans Modifies Effects of Dietary Restriction and Nutrient Stress on Aging, Stress and Growth 
PLoS ONE  2007;2(11):e1240.
Dietary restriction (DR) and reduced insulin/IGF-I-like signaling (IIS) are two regimens that promote longevity in a variety of organisms. Genetic analysis in C. elegans nematodes has shown that DR and IIS couple to distinct cellular signaling pathways. However, it is not known whether these pathways ultimately converge on overlapping or distinct targets to extend lifespan.
Principal Findings
We investigated this question by examining additional effects of DR in wildtype animals and in daf-2 mutants with either moderate or severe IIS deficits. Surprisingly, DR and IIS had opposing effects on these physiological processes. First, DR induced a stress-related change in intestinal vesicle trafficking, termed the FIRE response, which was suppressed in daf-2 mutants. Second, DR did not strongly affect expression of a daf-2- and stress-responsive transcriptional reporter. Finally, DR-related growth impairment was suppressed in daf-2 mutants.
These findings reveal that an important biological function of DAF-2/IIS is to enhance growth and survival under nutrient-limited conditions. However, we also discovered that levels of DAF-2 pathway activity modified the effects of DR on longevity. Thus, while DR and IIS clearly affect lifespan through independent targets, there may also be some prolongevity targets that are convergently regulated by these pathways.
PMCID: PMC2080776  PMID: 18043747
25.  Activated AKT/PKB signaling in C. elegans uncouples temporally distinct outputs of DAF-2/insulin-like signaling 
In the nematode, Caenorhabditis elegans, a conserved insulin-like signaling pathway controls larval development, stress resistance and adult lifespan. AGE-1, a homolog of the p110 catalytic subunit of phosphoinositide 3-kinases (PI3K) comprises the major known effector pathway downstream of the insulin receptor, DAF-2. Phospholipid products of AGE-1/PI3K activate AKT/PKB kinase signaling via PDK-1. AKT/PKB signaling antagonizes nuclear translocation of the DAF-16/FOXO transcription factor. Reduced AGE-1/PI3K signaling permits DAF-16 to direct dauer larval arrest and promote long lifespan in adult animals. In order to study the downstream effectors of AGE-1/PI3K signaling in C. elegans, we conducted a genetic screen for mutations that suppress the constitutive dauer arrest phenotype of age-1(mg109) animals.
This report describes mutations recovered in a screen for suppressors of the constitutive dauer arrest (daf-C) phenotype of age-1(mg109). Two mutations corresponded to alleles of daf-16. Two mutations were gain-of-function alleles in the genes, akt-1 and pdk-1, encoding phosphoinositide-dependent serine/threonine kinases. A fifth mutation, mg227, located on chromosome X, did not correspond to any known dauer genes, suggesting that mg227 may represent a new component of the insulin pathway. Genetic epistasis analysis by RNAi showed that reproductive development in age-1(mg109);akt-1(mg247) animals was dependent on the presence of pdk-1. Similarly, reproductive development in age-1(mg109);pdk-1(mg261) animals was dependent on akt-1. However, reproductive development in age-1(mg109); mg227 animals required only akt-1, and pdk-1 activity was dispensable in this background. Interestingly, while mg227 suppressed dauer arrest in age-1(mg109) animals, it enhanced the long lifespan phenotype. In contrast, akt-1(mg247) and pdk-1(mg261) did not affect lifespan or stress resistance, while both daf-16 alleles fully suppressed these phenotypes.
A screen for suppressors of PI3K mutant phenotypes identified activating mutations in two known pathway components, providing insights into their regulation. In particular, the interdependence of akt-1 and pdk-1, even in activated forms, supports the existence of AGE-1-independent pathways for these phospholipid-dependent kinases. Phenotypic analysis of these alleles shows that the larval and adult outputs of AGE-1/PI3K are fully separable in these mutants.
PMCID: PMC1609106  PMID: 17020605

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