Ludewig, Andreas et al. (2009) International Worm Meeting "Ascaroside Signalling and Lifespan in C. elegans."

Pungaliya, Chirag, Malik, Rabia, Schroeder, Frank, Fox, Bennett, Ludewig, Andreas

[International Worm Meeting, 2009]

In the nematode Caenorhabditis elegans the endogenously produced ascarosides differentially regulate development and behaviour. At low concentrations, ascarosides act as mating signal (Srinivasan, 2008), whereas at higher concentrations, they induce developmental arrest at the dauer stage (Butcher, 2007, Butcher 2008). Here we report that the ascarosides ascr#2 and ascr#3 influence adult lifespan and stress tolerance of C. elegans. Combinations of ascr#2 and ascr#3 increased lifespan of wild type animals by 30 % and increased thermotolerance up to 70%. Thermotolerance and ageing assays in different mutant background revealed complex regulatory networks for the activity of ascr#2 and 3. Notably, only ascr#3-, but not ascr#2-, mediated heat stress and tolerance and longevity are abolished in reduced insulin signalling background indicating that ascr#2 and ascr#3 act through different pathways. Ascr#2 and ascr#3-mediated increases in ageing and thermotolerance are dependent on the histone deacetylases SIR2.1 and SIR2.3, which also mediate caloric restriction-dependent increases in lifespan. These studies provide the first examples for endogenous small molecules that strongly increase lifespan and thermotolerance.

Ludewig, Andreas et al. (2011) International Worm Meeting "Identification of evolutionary conserved regulators of dietary restriction using the "Ortho2ExpressMatrix"."

Meinel, Thomas, Doering, Frank, Ludewig, Andreas

[International Worm Meeting, 2011]

During the past decades, increasing amounts of data derived from high throughput approaches assaying all kinds of species, tissues and diseases have been accumulating into gigantic pools of data. However, the usability and the accessibility of these data for the generation of new scientific information is sometimes difficult. Here, we apply a novel developed bioinformatic tool - the Ortho2ExpressMatrix (Meinel et al., 2011) to summarize, compare and interpret gene expression data from various dietary restriction (DR) experiments performed in the model organisms mouse and C. elegans. We use the Ortho2ExpressMatrix to integrate complex gene family information, computed from sequence similarity with gene expression profiles of the two species exposed to DR and ad libidum feeding conditions. We come up with a comprehensive list of genes that are co- regulated in both species under DR. All in all, huge cohorts of data have been condensed to a list of 18 mouse genes and 24 assigned putative functional C. elegans orthologs, that can be directly used for experimental set ups further elucidating the molecular mechanisms that control DR related phenomena.

Ludewig, Andreas H. et al. (2013) International Worm Meeting "In silico identification and functional analysis of genes responsive to dietary restriction in Caenorhabditis elegans ."

Ludewig, Andreas H., Doering, Frank, Meinel, Thomas, Bruns, Meike, Klapper, Maja

[International Worm Meeting, 2013]

Increasing mounds of data derived from high throughput approaches assaying a huge number of species, tissues and diseases have been generated over the past 2 decades. However, the integration of those data sets to generate useful biological information can be challenging. Here, we introduce two approaches to translate data derived from extensive microarray analysis into a manageable set of candidate genes according to biological relevant questions. In a meta-analysis of microarray based screens for differentially regulated genes under dietary restriction (DR) from 8 independent experimental set ups in C. elegans, we extracted 177 strongly overlapping differentially regulated genes. Among those, the sub class of CUB like proteins was significantly enriched (12,4%). Notably, most of those CUB like proteins have not been linked to DR. In another approach we applied a novel developed bioinformatic tool - the Ortho2Express Matrix (Meinel et al., 2011) - to summarize, compare and interpret gene expression data performed in mouse and C. elegans under DR and ad libidum feeding conditions. We use this tool to combine those gene expression profiles with complex gene family information, derived from sequence similarity. We end up with a list of 18 mouse genes and 24 assigned putative functional C. elegans orthologs that are regulated in both species in the same direction in response to DR. In conclusion, based on data mining we identified a set of genes which likely play a role in the regulation of the DR response and DR related phenomena such as improved stress resistance and long life. We discriminate between genes with already known functions in DR regulation, genes that have been connoted with other functional context and genes of complete unknown function. First functional assays for heat stress resistance under DR with selected pre-evaluated mutant C. elegans strains confirmed our in silico analysis.

Ludewig, Andreas H. et al. (2013) International Worm Meeting "Low population density increases lifespan and delays egg laying of C. elegans hermaphrodites."

Doering, Frank, Schroeder, Frank C., Ludewig, Andreas H.

[International Worm Meeting, 2013]

In C. elegans, high population density (overcrowding) has been shown to trigger developmental arrest and dauer formation within early larval stages (Golden and Riddle 1982). The actual population density signal sensed by the worms includes a synergistic mixture of ascaroside pheromones as well as small signalling peptides (Ludewig and Schroeder, 2013; Yamada et al., 2010). However, many aspects of population density-dependent signaling events remain poorly understood. Here, we compared low and intermediate dense C. elegans populations grown under ad libidum feeding conditions. We found that the number of worms per plate strictly determined their average lifespan whereat the number of worms was inverse correlated to mean lifespan. Isolated worms lived around 4 (!) days longer than animals grown up in larger groups. In 1999, Gems and Riddle demonstrated strong dependence of lifespan on population density for males but not for hermaphrodites. However, in those assays worms were placed on experimental plates as L4 larvae, whereas our assays started with eggs, hinting on some defining events during developmental stages L1-L3 in hermaphrodites. We also found that the time point of first egg lay was correlated with the number of worms per plate. Worms that grew up in isolation laid their first egg 9-11 hours later than worms grown up in groups of 25 or more animals per plate. The overall time course of egg laying from isolated worms was shifted towards later time points and the total number of eggs was reduced by 45%. This population dependent early egg laying (= eel) phenotype is completely abolished in daf-16(mu86) mutants and partially abolished in daf-12(rh61; rh411) mutants. Unexpectedly, we also found precocious first egg laying under mild dietary restriction (DR). Evidence from previous studies suggests that small-molecule signalling may trigger DR early egg laying and population dependent longevity (Ludewig and Schroeder, 2013; Yamada et al., 2010; Yzraelit, 2012). To test this hypothesis, we currently examine candidate small molecules for their influence on those phenomena.

Ludewig, Andreas H. et al. (2021) International Worm Meeting "A super-long lived mutant and a multi-omics discovery platform for new regulators of lifespan."

Fajardo Palomino, Diana C., Ludewig, Andreas H., Pulido, Dania C., Schroeder, Frank C.

[International Worm Meeting, 2021]

The Insulin like signaling (ILS) pathway and the peroxisomal beta oxidation (PBO) are highly conserved central metabolic gateways that regulate the uptake of glucose and amino acids and the breakdown of fatty acids in all animals. ILS has been widely associated with lifespan control across species (1); PBO has not been directly connected with lifespan modifications but our and other laboratories work showed that small molecular products of C. elegans PBO can trigger pro-health and anti-ageing effects (ascarosides #2, #3, #8, ncas#3 (2, unpublished)). Crossing daf-22(ok693) worms lacking the CoA C-acetyltransferase DAF-22 and daf-2(e1370) mutants in which the function of the insulin receptor tyrosine kinase domain is compromised, we generated a d2d22 double mutant, which exhibits an extremely high long lifespan, almost double the lifespan of the already long-lived daf-2(e1370) worms. d2d22 double mutants further exhibit slow development, an extended fertility period, reduced number of progeny, and formation of large triglyceride containing droplets during adulthood. Comparative metabolomic and transcriptomic analyses of wild type, daf-2, daf-22 and d2d22 mutants at different ages reveal small molecules and transcripts with starkly altered abundances in the long-lived strains. Accompanying proteomic analysis will provide further insight into the biosynthesis and regulatory aspects of those compounds. Selected compounds and mutants are tested in bioassays for developmental pace and lifespan. Integration of the all-omics data will facilitate the creation of a C. elegans pathway map of age-related small molecules. 1. Michelangela Barbieri, ... Giuseppe Paolisso. Am J Physiol Endocrinol Metab. 2003 Nov;285(5): E1064-71. Insulin/IGF-I-signaling pathway: an evolutionarily conserved mechanism of longevity from yeast to humans. DOI: 10.1152/ ajpendo.00296.2003 2. Ludewig AH, ... Schroeder FC. Proc Natl Acad Sci U S A. 2013 Apr 2;110(14):5522-7. Pheromone sensing regulates Caenorhabditis elegans lifespan and stress resistance via the deacetylase SIR-2.1. doi: 10.1073/pnas.1214467110.

Andreas Ludewig et al. (2007) International Worm Meeting "C. elegans - a model for studies of human diseases."

Andreas Ludewig, Hans Lehrach

[International Worm Meeting, 2007]

In the past few years, studies in C. elegans have been greatly expanded our knowledge of molecular mechanisms underlying human diseases as complex as cancer, diabetes and neurodegenerative diseases. In our present project, we aim for the establishment of suitable C. elegans models for detailed studies of human diseases. Importantly, this will be achieved in tight connection with collaboration partners exploring different aspects of the particular disease. By merging our results from C. elegans studies with data derived from cell culture assays, patient studies, mammalian animal models and bioinformatic approaches, we hope for new insight into molecular mechanisms of diseases. Initially, we focus on the effects of transient anoxia on C. elegans. Although the global neuronal structure differs widely in nematodes and mammals, there is good evidence that basic molecular circuits following neuronal damage are conserved throughout species. By examining the processes accompanying anoxia and recovery in C. elegans in molecular detail, we wish for a better understanding of the mechanisms promoting stroke and neuronal self-healing processes in men. In our preliminary studies, C. elegans wild type, adult hermaphrodites, when exposed to anaerobic atmosphere for 4 hours completely stopped moving. Re-exposure to oxygen reconstituted vitality of a large subset of animals. We currently determine global gene expression profiles and morphologic changes at different time points of anoxia and recovery. In a reciprocal approach, we explore C. elegans orthologs of mammalian genes that have been linked with stroke by our collaboration partners in a mouse stroke model (Nietfeld et al., unpublished). Currently, we score knock down strains of each C. elegans ortholog for differential gene expression, for morphologic changes and for changes in the survival- and motility rate under anoxic conditions.

Andreas Ludewig et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Ascaroside Signalling and Lifespan in C. elegans"

Andreas Ludewig, Rabia Malik, Frank Schroeder, Chirag Pungaliya, Bennett Fox

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Key developmental pathways in C. elegans appear to be regulated by several sets of endogenous small molecule signals. Recent analyses in our laboratory characterized eight C. elegans-produced glycosides of the dideoxysugar ascarylose. Among these, the three ascarosides ascr#1, ascr#2, and ascr#3 had previously been shown to induce dauer formation in C. elegans; in addition, ascr#2 and ascr#3 synergize with ascr#4 as sex pheromones (Kawano et al., 2005; Butcher et al., 2007, Srinivasan et al., in press). Identification of these endogenous compounds presents a unique opportunity by providing small-molecule tools for dissecting developmental pathways. Here, we show that ascarosides ascr#2 and ascr#3 strongly increase thermo tolerance in C. elegans. Wild type worms grown on NGM medium containing nanomolar concentrations of these ascarosides survived up to six times longer at 35degC compared to untreated controls. We also found that animals exposed to ascr#2 and ascr#3 on minimal growth medium had up to three-fold increased lifespan. Notably, 50 day old adult worms grown on minimal medium containing ascr#2 and ascr#3 carried small numbers of fully developed or over-developed embryos. When re-exposed to favourableconditions, these animals gave rise to few, but viable and reproductive progeny. We currently investigate the impact of these endogenous signaling molecules on thermotolerance and life span extension in a comprehensive set of mutant strains.

Andreas H Ludewig et al. (2002) European Worm Meeting "DIN-1, a putative DAF-12 coregulator important for the C.elegans dauer diapause"

Andreas H Ludewig, Alexander Stein, Adam Antebi, Corinna Kober-Eisermann, Cindy Weitzel

[European Worm Meeting, 2002]

The C. elegans orphan nuclear receptor DAF-12 acts as key control gene at the intersection of heterochronic, dauer and aging pathways. A putative steroid hormone, metabolized by DAF-9/ cytochrome P450 likely regulates DAF-12. In addition, distinct transcriptional ensembles could mediate DAF-12 functional complexity. Some vertebrate nuclear receptors activate gene expression in the presence of cognate hormone, and repress in its absence, by assembling coactivator and corepressor complexes, respectively. This mechanism allows nuclear receptors to act as ligand-inducible logic switches. To define such transcriptional complexes, we screened for DAF-12 interacting proteins by the Yeast-Two Hybrid method. Eight candidate interactors were identified. One of these, DIN-1 (DAF-12 Interacting Protein 1) is predicted to encode two large nuclear proteins of 2713 and 2784 amino acids based on cDNAs isolated by RT-PCR. DIN-1 contains several nuclear localization sites and three RNA recognition motifs. It also has several motifs typical of nuclear receptor corepressors, and most of these are located within the receptor interaction domain (RID). Recently, the human homolog, SHARP, was shown to act as a corepressor with a number of nuclear receptors. In C. elegans, DIN-1(+) is important for the dauer diapause. A knockdown of DIN-1(+) activity by RNAi or conventional mutations produces no evident phenotype in an otherwise wild type background. However, din-1 partly or completelv suppresses the dauer constitutive phenotypes (Daf-c) of various dauer pathway mutants, e.g. insulin like receptor daf-2 (class I) alleles. It also suppresses the delayed heterochronic and Daf-c phenotypes of certain daf-12 alleles. Interestingly, all din-1 alleles identified to date map within the RID. din-1 does not affect the expression or localization of daf-12::GFP. Taken together, these structural and functional data strongly suggest that DIN-1 is a DAF-12 coregulator. We propose that in the absence of reproductive hormone, DAF-12 assembles a corepressor complex containing DIN-1 and other components to mediate developmental arrest and diapause. Moreover, these complexes must be functionally redundant because DIN-1(+)is not essential for diapause under dauer inducing conditions.

Andreas H Ludewig et al. (2001) International C. elegans Meeting "DIN-1, a putative DAF-12 cofactor that regulates the C.elegans dauer diapause"

Alexander Stein, Andreas H Ludewig, Adam Antebi

[International C. elegans Meeting, 2001]

The nuclear receptor daf-12 acts as a control gene at the intersection of heterochronic and dauer pathways. To further understand the molecular mechanisms of DAF-12 regulated pathways, we screened for DAF-12 interacting proteins by the Yeast-Two Hybrid method. Three candidate interactors were identified. One of these, called DIN-1 (DAF-12 interacting Protein 1) is predicted to be a nuclear protein of 2328 amino acids, containing several nuclear localisation sites, an RNA recognition motif, and regions of homology to the mammalian Msx-2 interacting target protein (MINT). In the C-terminus, there are several motifs characteristic of nuclear receptor corepressors in the region that interacts with DAF-12. These structural data suggest that DIN-1 might be a DAF-12 cofactor. din-1 was shown to have clear effects in functional knock out assays done by RNAi feeding experiments. din-1 partly or completely suppressed the dauer constitutive (Daf-c) phenotypes of mutants that act upstream of daf-12 in the dauer pathway, including insulin-like ( daf-2 ), TGF-beta ( daf-7 ), cGMP ( daf-11 ) and hormonal ( daf-9 ) signaling. Moreover, din-1 dramatically suppressed the delayed heterochronic and Daf-c phenotypes of daf-12 gain-of-function alleles, but had no effect on daf-12 null mutants. Consistent with two-hybrid interaction, these genetic data strongly suggest that DAF-12 and DIN-1act at the same step. We think that DIN-1could function in a transcriptional complex with DAF-12, perhaps as a corepressor or coactivator controlling developmental arrest and dauer formation.

Andreas H Ludewig et al. (2003) International Worm Meeting "The nuclear coregulator DIN-1 interacts with nuclear receptor DAF-12 to specify developmental arrest and dauer diapause"

Kerstin Neubert, Andreas H Ludewig, Axel Bethke, Harald Hutter, Corinna Kober-Eisermann, Birgit Gerisch, Adam Antebi, Cindy Weitzel

[International Worm Meeting, 2003]

In response to hormonal signals, C. elegans steroid receptor DAF-12 promotes either developmental arrest at L3 dauer diapause or continuous larval development. In adults, DAF-12 promotes long life or reproduction. How these choices are molecularly specified is not known. To address this issue, we performed yeast two-hybrid screens to find DAF-12 interactors and identified nuclear coregulator DIN-1 (daf-12 interactor 1). din-1 encodes a protein homologous to the human SHARP corepressor and the Drosophila Split ends (SPEN). SHARP has been shown to function as corepressor with a variety of transcription factors, including nuclear receptors, by recruiting components of the Nurd complex. We have shown that DIN-1 and DAF-12 physically interact in yeast two-hybrid and GST pull-down assays. The din-1 locus gives rise to 4 long and 1 short isoform (L and S) that evidently originate from two transcriptional units. Long and short isoforms are nuclear localized and widely expressed. However, din-1L and din-1S are essentially functionally independent since they differ in phenotype, and complement. Mutations in din-1L result in low penetrance body morphology defects, gonadal arm displacement, uncoordination and egg retention. Mutations in din-1S alone have no obvious phenotype, but suppress the dauer constitutive (Daf-c) phenotypes of mutants representing all the major branches of dauer pathways, including insulin receptor (daf-2), TGF- (daf-7), guanyl cyclase (daf-11), and cytochrome P450 (daf-9) mutants. Moreover, din-1S suppresses daf-9 and daf-12 heterochronic gonadal Mig phenotypes, as well as daf-12 heterochronic phenotypes in the epidermis. Finally, din-1S suppresses daf-9 longevity. These results place din-1S downstream or parallel to the known dauer signaling pathways, at the same point as daf-12. Interestingly, all of the din-1S mutations cluster in an isoform specific exon that corresponds to the DAF-12 interaction domain, suggesting that association with DAF-12 is largely abolished. We propose that in the presence of a DAF-9 produced hormone, DIN-1S is inactive. In the absence of hormone, a repressor complex comprised of DIN-1S and unliganded DAF-12 nuclear receptor specifies developmental arrest, dauer formation and long life.