Schroeder, Frank C. (2013) International Worm Meeting "Worms as chemical masterminds: complete control with small molecules."

Schroeder, Frank C.

[International Worm Meeting, 2013]

Worms are amazingly skilled chemists: using simple building blocks from conserved primary metabolism and a strategy of modular assembly, they create complex molecular architectures to regulate almost every aspect of their development and behavior. Phenotypes regulated by this combinatorial library of small-molecule signals include dauer formation, adult phenotypic plasticity, adult lifespan, attraction of the other sex, aggregation, dispersal, and other behaviors. Many, but not all of the identified compounds are based on the dideoxysugar ascarylose, which serves as a scaffold for attachment of moieties from amino acid, carbohydrate, neurotransmitter, lipid, and nucleoside metabolism, creating signaling molecules that can be active at femtomolar concentrations, such that encountering just a few molecules per minute is sufficient for worms to respond. Motivated by this unexpected structural and functional diversity, we have embarked on a systematic characterization of the C. elegans metabolome. We find that most small molecules in C. elegans remain undescribed and that even important primary metabolite classes may include non-canonical compounds. For example, up to a quarter of the entire C. elegans lipid content is not represented by triglycerides, phospholipids or fatty acids, but instead consists of very long-chain ascaroside lipids in which the ascarylose sugar takes the place of conventional glycerol. Our screen has produced evidence for several thousand different metabolites of yet undetermined structures, ranging from simple lipids, amino acid derivatives, and nucleosides to complex modular assemblies. Their identification and subsequent quantification in genome-wide mutant screens will, akin to transcriptional profiling, provide a new basis for the study of C. elegans metabolism and associated signaling pathways, enabling re-interpretation of many metabolic, developmental, and behavioral phenotypes.

Schroeder, Frank C. et al. (2009) International Worm Meeting "Caenorhabditis elegans chemical biology: lessons from small molecules."

Kaplan, Fatma, Sternberg, Paul, Edison, Arthur, Schroeder, Frank C., Pungaliya, Chirag, Srinivasan, Jagan

[International Worm Meeting, 2009]

How can we complement C. elegans genomics and proteomics with a comprehensive structural and functional annotation of its metabolome? Several lines of evidence indicate that small molecules of largely undetermined structure play important roles in C. elegans biology, including key pathways regulating lifespan, development, and metabolism. We have developed NMR spectroscopic methodology that enables linking small molecule metabolites directly with corresponding mutant phenotypes and probable biological functions. Application of this approach to identifying the C. elegans mating and dauer pheromones revealed a complex signaling system based on a bi-functional group of signaling molecules, the ascarosides ascr#1-ascr#8. Low concentrations of ascarosides attract males and thus appear to be part of the C. elegans sex pheromone, whereas higher concentrations induce developmental arrest at the dauer stage. Intriguingly, only mixtures of several ascarosides produce strong phenotypes at physiologically relevant concentrations, and individual ascarosides exhibit different though overlapping activity profiles. For example, the ascaroside ascr#2 is a stronger dauer inducer than ascaroside ascr#3, whereas ascr#3 is a stronger male attractant than ascr#2. However, mixtures of ascr#2 and ascr#3 are much more active than either compound alone, and addition of a third component, ascr#8, further increases activity, indicating synergistic modes of action. Cellular and genetic analyses of ascr#3 suggest the role of both core and sex-specific sensory neurons in regulating the response to this metabolite. Additional studies indicate that different ascarosides act through different neuronal and genetic pathways. These findings present a significant departure from the one-compound one-phenotype paradigm and emphasize the need to develop systemic approaches for correlating genome, phenotype, and metabolome. 1. J. Srinivasan et al. (2008). A blend of small molecules regulates both mating and development in C. elegans. Nature 454:1115-1118. 2. C. Pungaliya, J. Srinivasan, B. W. Fox, R. U. Malik, H. A. Ludewig, P. W. Sternberg, F. C. Schroeder (2009). A shortcut to identifying small molecule signals that regulate behavior and development in C. elegans. PNAS, in press.

Schroeder, Frank C. et al. (2015) International Worm Meeting "How sirtuins regulate lifespan: endogenous small molecules trigger ROS signaling."

Booth, Lauren, Izrayelit, Yegeniy, Brunet, Anne, Schroeder, Frank C., Ludewig, Andreas, Campbell, Sydney

[International Worm Meeting, 2015]

Sirtuins, a family of NAD+ dependent histone deacetylases, have been implied as conserved regulators of lifespan and stress resistance; however, the underlying mechanisms are unclear. Previous work showed that chemosensation of hermaphrodite-produced pheromones ascr#2 and ascr#3 increases C. elegans adult lifespan and stress resistance (Ludewig 2013), whereas male-produced pheromones of unknown identity shorten hermaphrodite lifespan (Gems 2000, Maures 2014).We show that the male-produced ascaroside ascr#10 decreases hermaphrodite lifespan, and that pheromone-mediated lifespan increase and decrease depend on two different sirtuins, SIR-2.1 and SIR-2.3. Whereas SIR-2.1 mutants are defective in pheromone mediated lifespan increase, the ascr#10-dependent lifespan phenotype is reversed in SIR-2.3 worms: male-produced ascr#10 decreases lifespan in wild-type worms but increases lifespan in SIR-2.3 mutants. We further show that lifespan regulation via sirtuins primarily depends on metabolism of co-factor (NAD+)-derived nicotinamide, which triggers generation of reactive oxygen species (ROS) in the mitochondria and activation of the transcription factor skn-1, a homolog of mammalian Nrf. Similar to the effects of SIR-2.1 over expression (Schmeisser 2013), ascaroside perception transiently increases ROS in mitochondria, followed by a long-term decrease of ROS activity for lifespan-increasing compounds. In sir-2.1 mutants, longevity-promoting ascarosides do not increase ROS, indicating a direct connection between sirtuin-dependent mitochondrial ROS increase and longevity. Our work demonstrates that sirtuins regulate lifespan in a non-over expression background (i.e WT) via ROS generation, and that sex-specific endogenous small molecules modulate lifespan through this mechanism. Furthermore, our results link SIR-2.1, the homolog of mammalian SIRT1, to increased lifespan, whereas the SIRT4 homolog SIR-2.3 appears to promote shorter lifespan.Lastly, our work shows that a seemingly minor modification of the chemical structure of an endogenous small molecule, namely the addition of a double bond, can drastically change its signaling properties: a lifespan-decreasing (ascr#10, no double bond) signal turns into a lifespan extending (ascr#3, with double bond) signal. Both ascr#3 and ascr#10 accumulate on C. elegans plates, suggesting confounding effects of population density on C. elegans lifespan studies.

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.

Rodrigues, Pedro R. et al. (2019) International Worm Meeting "A Draft Metabolome for C. elegans."

Fox, Bennett, Rodrigues, Pedro R., Schroeder, Frank C., Helf, Maximilian J.

[International Worm Meeting, 2019]

Progress in ascribing phenotypic characteristics to genetic identity has moved at a fast pace since the annotation of genomes. However, a comprehensive annotation of metabolomes has moved at a woefully slow pace. Recent advances made in high resolution accurate mass spectrometry (HRAM) now provide the opportunity for small molecule research to adopt that same discovery rate that has been seen for genomes. While traditional metabolomic analysis measures dozens or, at best, hundreds of metabolites, common HRAM analysis detects tens of thousands of features with largely unknown identities. In this work, we are pursuing a comprehensive annotation of the entire C. elegans metabolome. By using Furrier Transform Mass Spectrometry (FTMS), we have developed the methods to systematically annotate molecular formulae and structural characteristics of the C. elegans metabolome across all developmental stages. We have evidence for the presence of more than 10,000 different compounds, and thus genome and metabolome of C. elegans may be of similar numerical sizes. Among several thousand novel molecules we have identified or partially characterized, some represent compound classes that have not previously been described in C. elegans but are known from other organisms, and others are entirely novel. Many of the detected compounds suggest the presence of yet unexplored biosynthetic networks. For example, we identified over 80 N-acyl amino acids, a class of endogenous molecules not previously described in worms, whereas in mammals these compounds are well known for their anti-inflammatory and analgesic functions. Biosynthesis and function of these small molecules and many other new compound families in C. elegans we will present on is currently unknown and remains a focus of this work. In parallel, we are developing a timeline for the production of all metabolites throughout larval stages and adulthood. We believe that our comprehensive structural and functional annotation of the C. elegans metabolome will become an invaluable resource to the worm community and beyond, allowing researchers to connect metabolomic information with genetic tractability. Lastly, the first draft annotation of the C. elegans metabolome may become an example for other metazoan model systems, akin to the sequencing of the C. elegans genome over 20 years ago.

O'Doherty, Inish M et al. (2009) International Worm Meeting "Receptor trapping in C. elegans: identifying the targets of ascarosides and other endogenous small molecules."

O'Doherty, Inish M, Schroeder, Frank C, Ludewig, Andreas H

[International Worm Meeting, 2009]

Recent studies have led to the identification of several families of small molecule signals in C. elegans1. These compounds affect key pathways regulating lifespan, development, and metabolism. Further knowledge of corresponding receptors will be essential for understanding their biological role. We are developing a chemical approach to identifying small-molecule receptors in C. elegans. This approach is based on designing probes that are structural analogs of the endogenous small-molecule ligands but feature additional structural elements. These chemical modifications of the ligand allow covalent tagging of the small molecule to its receptor; with this linkage established it may then be possible to pull down and isolate the specific receptor in question. Design of these probes takes advantage of recently developed methods for in-vivo photocrosslinking of ligands to their corresponding receptors2,3 and further introduces a bioorthogonal handle for subsequent pull down of the ligand-receptor complex for mass spectrometric analysis4,5. Our current investigations focus on receptors of the ascarosides, ascr#1-ascr#8, a recently discovered family of small molecules that play important roles in male mating, and dauer formation. We have designed ascaroside-derived probes featuring a receptor-binding motif based on the pheromone component ascr#8, a photolabile receptor-tagging group, and a third functionality for subsequent pull down. Results from bioactivity studies of synthetic model compounds will be reported. 1. Srinivasan et al.(2008) Nature 454:1115-1118. 2. Gubbens et al. (2009) Chemistry & Biology 16, 3-14. 3. Wang et al.(2004) J. AM. CHEM. SOC. 126, 14435-14446. 4. Cravatt et al.(2007) QSAR Comb. Sci. 26, No. 11-12, 1229-1238 5. ACS Chem. Bio. 2006,Vol.1, No.10, 644-648.

Cohen, Sarah M et al. MicroPubl Biol

Schroeder, Frank C, Cohen, Sarah M, Sun, Jessica J, Sternberg, Paul W

[MicroPubl Biol, 2021]

The dauer diapause stage in C. elegans is a non-feeding alternative to the L3 larval stage that is highly resistant to harsh environmental conditions. The decision to enter dauer is a two-step process. First, L1 larvae encounter adverse conditions such as lack of food or overcrowding and decide to enter the L2d rather than the L2 stage. Second, L2d worms that continue to experience disadvantageous conditions decide to enter dauer instead of L3. Here, we have used RNA-seq to characterize the transcriptional response to a cocktail of dauer-inducing ascaroside pheromones at the late L1 stage as worms enter the L2d phase. We find that, in response to ascarosides, C. elegans L1 larvae preparing to enter the L2d stage begin upregulating genes involved in stress response and downregulating genes associated with growth and metabolism.

Chuman, Tatsuji et al. (2011) International Worm Meeting "Identification of a New Ascaroside, Female Attracting Pheromone in Panagrellus redivivus."

Edison, Arthur S., Choe, Andrea, von Reuss, Stephan, Schroeder, Frank C., Chuman, Tatsuji, Sternberg, Paul W., Kaplan, Fatma, Ajredini, Ramadan, Alborn, Hans

[International Worm Meeting, 2011]

Panagrellus redivivus, a free-living nematode related to the well-known model organism, Caenorhabiditis elegans, has been studied in the laboratory for decades and is therefore useful for comparative biological studies with C. elegans. P. redivivus can be easily cultured in the laboratory using conditions similar to those used for C. elegans, and the two species share many desirable traits such as short generation time. Whereas C. elegans has self-fertilizing hermaphrodites and males, P. redivivus has females and males and requires mating for reproduction. P. redivivus females can specifically attract males and males can specifically attract females but the chemical nature of this attraction has until now not been known. We used a protocol, previously developed for C. elegans, to collect large volume liquid co-cultures with bacterial food as well as biologically active worm water samples of P. redivivus. In addition we developed a robust bioassay to test for female attraction using the worm water samples. By activity-guided fractionation, in combination with NMR and LC-MS analyses, we found a pheromone component, component-1, as a female attractant from its worm water sample. Component-1 is a new ascaroside compound and its structure is elucidated by MS and NMR analyses after purification. The synthesis of component-1 for confirmation of the proposed structure is now undergoing. These results suggest a highly conserved and complex system of nematode pheromones and may one day lead to new approaches to the control of parasitic species1,2). References 1.Srinivasan, J., Kaplan, F., Ajredini, R., Zachariah, C., Alborn, H. T., Teal, P. E., Malik, R. U., Edison, A. S., Sternberg, P. W., and Schroeder, F. C. 2008. A blend of small molecules regulates both mating and development in Caenorhabditits elegans. Nature. 454:1115-1118. 2.Edison, A. S. 2009. Caenorhabditis elegans pheromones regulate multiple complex behaviors, Curr Opin Neurobiol 19, 378-388.

Ilbay, Orkan et al. (2013) International Worm Meeting "Ascaroside signals suppress heterochronic phenotypes of the daf-12(rh61) mutant."

Schroeder, Frank C., Ilbay, Orkan, Ren, Zhiji, Srinivasan, Jagan, Ambros, Victor

[International Worm Meeting, 2013]

During animal development, cells divide and adopt specific fates and progressively differentiate into distinct cell types. The relative timing of a series of developmental events determines morphological endpoints and may affect the physiology of an organism. In Caenorhabditis elegans development, there are four larval stages (L1-L4), and all cell division and differentiation events at all stages have been mapped. Heterochronic genes, whose products include developmentally-expressed microRNAs and transcription factors, control the temporal patterning of developmental events, and contribute to developmental robustness. One of the components of the heterochronic pathway, DAF-12, promotes L2-to-L3 cell fate transitions by positively regulating the transcription of certain let-7 family microRNAs. Under unfavorable conditions, such as crowding, scant food supplies, or elevated temperature, C. elegans L2 larvae can commit to developmental arrest as the stress-resistant dauer larva. In such unfavorable conditions, DAF-12 negatively regulates progression to L3 cell fates, in part by repressing expression of let-7 family microRNAs, and promotes dauer formation. Several daf-12 mutations have been characterized as causing defective dauer regulation or heterochronic phenotypes, or both. In particular daf-12(rh61) results in expression of a truncated form of DAF-12. daf-12(rh61) mutant animals are dauer-defective and have penetrant heterochronic phenotypes. Dauer formation is induced by ascarosides, signaling molecules produced by C. elegans that serve as measures of population status for individual worms. Here, we report that a combination of three dauer-inducing ascarosides, namely ascr#2, ascr#3, and ascr#5, suppresses heterochronic phenotype of daf-12(rh61). Interestingly, ascr#5 alone can suppress the rh61 heterochronic phenotype, while only moderately inducing dauer formation in N2 animals. This suggests that while signaling from all three ascarosides contributes to the dauer program, suppression of rh61 heterochronic phenotype is mainly regulated by ascr#5 signaling.