Stupp, Gregory S. et al. (2011) Worm Breeder's Gazette "WormBox: a parallelized webcam recording system for nematode bioassays"

Stupp, Gregory S., Edison, Arthur S.

[Worm Breeder's Gazette, 2011]

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.

Goldberg, Hailey V. et al. (2019) International Worm Meeting "Development of a UGT Mutant Library in Caenorhabditis elegans."

Edison, Arthur S., Asif, Muhammad Z., Doll, Jack, Roth, Julia, Goldberg, Hailey V.

[International Worm Meeting, 2019]

The purpose of this study is to create a Caenorhabditis elegans (C. elegans) mutant library by using CRISPR/Cas9 to knock out UGT genes. This library will be comprised of the existing UGT mutants in order to provide us with the needed information to peruse other non-explored UGT genes to knock out in the future. In C. elegans, UGT genes regulate the glycosylation of environmental toxins allowing for survival of the nematode[1]. CRISPR/Cas9 is a powerful gene-editing system allowing for a Cas9 endonuclease to induce a double strand break in the DNA, rendering non-homologous end joining between the broken DNA[2]. As a result, that particular gene in the DNA is knocked out and a mutant is created. As part of the Vertically Integrated Projects (VIP) undergraduate research team at UGA, we have developed a workflow that will allow us to create this mutant library[3]. Upon completion, this library will allow us to test the effects of different xenobiotics and natural compounds on UGT knockout mutants which will allow us to better understand the role of these genes and their associated proteins in the glycosylation and drug resistance pathways of C. elegans; this provides us with a model which can be later be tested in parasitic nematodes. Additionally, the CRISPR/Cas9 protocols established for UGT knockouts will allow future undergraduate students to partake in CRISPR/Cas9 genetic research through the VIP program in the Edison Lab to continue producing UGT mutants for metabolomics analysis.

Stupp, Gregory et al. (2011) International Worm Meeting "Responses of C. elegans to Pathogenic Challenge."

Stupp, Gregory, Edison, Arthur S., Ajredini, Ramadan, Gulig, Paul A., Robinette, Steven L.

[International Worm Meeting, 2011]

C. elegans releases and responds to many different chemicals necessary for regulating important behaviors such as mating attraction, dauer formation, aggregation, and recognition and differentiation of food and pathogens. The nematode can sense bacterial populations through small-molecule messengers such as acyl-homoserine lactones, and are able to interfere with certain quorum sensing systems. Additionally, C. elegans has a complex olfactory system which allows the nematode to avoid detrimental conditions such as areas of high population density or osmolarity and areas containing pathogenic bacteria. Moreover, this system, which is affected by released pheromones, exhibits behavioral plasticity that allows the nematode to learn and adapt, for example, to avoid an odorant associated with harmful conditions. This study seeks to determine changes in C. elegans behavior and exometabolome, the set of small molecules released into the environment by the worms, in the presence of a pathogen. We collected exudates from a synchronous population of C. elegans under standard conditions and in the presence of synthetic compounds produced by Pseudomonas aeruginosa, for the purpose of identifying worm responses to pathogenic conditions. We acquired 2D NMR spectra of the exudates, and used a novel method developed in our lab for 2D NMR alignment and pattern recognition (Robinette et al., 2011) to identify NMR peaks correlated with worm responses. We also quantified behavioral responses to these pathogen-challenged exudates using custom software that tracks individual nematodes, quantifies reversal frequency and calculates average speed for a set of nematodes on an agar plate in order to identify potential alarm responses. Additionally, we are working on a high-throughput version of the bioassay apparatus that consists of six webcams mounted on a custom lighting unit which will enable recording and analysis of six bioassays simultaneously. The availability of libraries of mutants for both C. elegans and P. aeruginosa makes this an exciting project to probe mechanisms of interspecies chemical interactions. 1. Edison AS. Current opinion in neurobiology. 2009;19(4):378-88. 2. Beale E, et al. Applied and environmental microbiology. 2006;72(7):5135-7. 3. Kaplan F, et al. Journal of chemical ecology. 2009;35(8):878-92. 4. Schulenburg H, Ewbank JJ. Molecular microbiology. 2007;66(3):563-70. 5. Yamada K, et al. Science. 2010;329(5999):1647-1650. 6. Robinette SL, et al. In Press, Analytical Chemistry (2011).

Schroeder F C et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Endogenous Small Molecules Increase Lifespan of Caenorhabditis elegans via SIR-2.1"

Fox B W, Bethke A, Pungaliya C, Malik R U, Schroeder F C, Ludewig A H

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Lifespan in metazoans is regulated by several conserved signaling pathways, including the insulin/insulin-like growth factor and sirtuin pathways. W e have found that components of the dauer pheromone, the ascarosides (Edison 2009), regulate C. elegans adult lifespan and stress resistance. Ascarosides increased lifespan and thermotolerance of wild-type worms by up to 56% and 25%, respectively, without reducing fecundity or feeding rate. These lifespan increases are completely abolished by loss of the histone deacetylase SIR-2.1 or loss of components of peroxisomal fatty acid beta-oxidation, but do not require insulin signaling via the FOXO-homolog DAF-16 or TGF-beta signaling. Our findings establish endogenous small molecules as modulators of sirtuin-dependent pathways that connect longevity and stress resistance with peroxisomal fat metabolism. A. S. Edison, Curr. Opin. Neurobiol. 19(4), 378 (2009).

Malik R U et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Ascarosides ascr#2 and ascr#3 Increase Thermotolerance of Adult C. elegans"

Fox B W, Malik R U, Pungaliya C, Schroeder F C, Ludewig A H

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

We investigated whether the ascarosides, major components of the C. elegans dauer pheromone (Edison, 2009), affect stress resistance of adult worms. We found that ascarosides markedly increased survival under oxidative stress and resistance to heat stress (thermotolerance at 35 degC). We further measured pharyngeal pumping rates under heat stress and found that pumping rates of worms on ascaroside plates were significantly higher than on control plates. Next, we asked whether nutritional conditions influence the observed ascaroside-mediated increases of stress resistance. For thermotolerance assays under caloric restriction (CR) conditions, we transferred worms to plates without bacteria before exposure to heat stress. Mean heat stress survival time under CR conditions was higher than for worms with bacteria, in accordance with previous studies demonstrating increased stress resistance under starvation conditions. Notably, addition of ascarosides did not further increase thermotolerance of CR worms. These results show that the worms' metabolic state influences the efficacy of ascarosides in increasing thermotolerance. A. S. Edison, Curr. Opin. Neurobiol. 19(4), 378 (2009).

Kaplan, Fatma et al. (2011) International Worm Meeting "Density-based regulation of ascr"

Teal, Peter, Schroeder, Frank C., Kaplan, Fatma, Alborn, Hans T., Edison, Arthur S.

[International Worm Meeting, 2011]

The ascarosides are a family of nematode small molecules, many of which induce formation of long-lived and highly stress resistant dauer larvae. More recent studies have shown that ascarosides serve additional functions as social signals and mating pheromones. For example, the male attracting pheromone is composed of a blend of at least four ascarosides, ascr#2, ascr#3, ascr#4 and ascr#8. Although many of the ascarosides have been shown to induce dauer formation at high concentrations, ascr#2 has been shown to be the most potent dauer-inducing compound. By using developmentally synchronized nematodes under starvation conditions we recently showed that ascr#2 content in growth media increased dramatically in conjunction with dauer formation. In addition to being a component of the mating pheromone, ascr#2 may also function as a density pheromone for C. elegans. In that case, its concentration in growth media would increase with increasing worm density. To test this hypothesis we grew nematodes in standard media and incubated at densities of 5000, 10000, 20000, 30000 and 40000 worms/ml in water for 1 h. Exudates were analyzed by LC-MS as previously described. As expected, total ascr#2 and ascr#4 content increased; however, ascr#2 release positively and ascr#4 release was negatively correlated with the increasing nematode density. Because ascr#2 is the non-glucosylated form of ascr#4, these data suggested that ascr#4 may be converted to ascr#2. Deuterium labeled ascr#2 or ascr#4 was incubated in water either with nematodes or nematode exudates. Labeled ascr#4 was converted to labeled ascr#2 wheras no ascr#2 was converted to ascr#4 in the presence of nematodes suggesting ascr#4 as a precursor of ascr#2. Furthermore, incubating ascr#4 with worm exudates did not result in conversion to ascr#2, suggesting that ascr#4-to-ascr#2 conversion does not happen in the media but instead after ascr#4-uptake by the worms. In conclusion, ascr#2 secretion per worm increases with increasing worm density, and this increase may in part result from enzymatic conversion of ascr#4 to ascr#2 by the worms. Ascr#4 might mainly function as an inactive form of low nutrients or high density alarm pheromone, easily converted to its active form by the nematodes. The function of ascr#4 or the ascr#2/ascr#4 ratio remains unknown. However, the conversion appears to be highly nematode specific, thus knowledge of enzymes that regulate production of nematode density pheromones may reveal new drug targets for controlling nematode parasites in humans and plants.

Clendinen, Chaevien S et al. (2011) International Worm Meeting "Mass Spectrometric Imaging and Behavioral Assays of Pristionchus pacificus."

Clendinen, Chaevien S, Menger, Robert, Ajredini, Ramadan, Edison, Arthur S, Yost, Richard, Khan, Ghulam

[International Worm Meeting, 2011]

Nematodes are one of the most numerous and diverse multicellular organisms on earth. They have a profound impact on human health, agriculture, and economy. Like many other organisms, they synthesize and use small molecules to communicate within and between their species. In Caenorhabditis elegans, ascarosides have been found to control dauer formation (Butcher, et al., 2007), mating (Srinivasan, et al., 2008), aggregation (Macosko, et al., 2009), and olfaction (Yamada, et al., 2010). Pristionchus pacificus is a useful satellite organism for comparative studies with C. elegans (Hong & Sommer, 2006). We are developing a bioassay for gender specific chemotaxis (e.g. mating behavior) in P. pacificus for activity-guided fractionation of the mating cue. The mating assays are complicated by aggregation behavior in males under some conditions. We are trying to understand the chemical and environmental factors that control these different behaviors. Several behaviors, including aggregation may be mediated or enhanced by chemical cues on the cuticle surface of the nematodes. Therefore, we are trying to develop an alternative approach to chemical ecology studies in nematodes using matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) (Garrett & Yost, 2006). This technique could potentially not only allow us to detect metabolites on the surface of nematodes, but also visualize the spatial distribution of these small molecules. Using a combination of MALDI MSI and principal component analysis (PCA), we can compare nematodes of different species and find differences in mass spectral profiles resulting from gender, developmental stage, strain or species. We are currently examining C. elegans strain N2 (a standard wild-type) and C. elegans daf-22 (dauer-defective mutants). Daf-22 mutants are unable to produce short chain ascarosides such as the dauer pheromones common in the N2 strain. Once this technique is developed by the use of C. elegans, we will begin to analyze the surface molecules of P. pacificus nematodes. Bibliography: Yamada, K., et al. (2010). Science, 329 (5999), 1647-1650. Butcher, R. et al. (2007). Nature Chemical Biology, 3 (7), 420-422. Garrett, T., & Yost, R. (2006). Analytical Chemistry, 78, 2465-2469. Hong, R., & Sommer, R. (2006). BioEssays, 28, 651-659. Macosko, E., et al. (2009). Nature (458), 1171-1175. Srinivasan, J., et al. (2008). Nature, 1115-18.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.

Fanning S et al. (2018) Mol Cell "Lipidomic Analysis of -Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment."

Lou Y, Haque A, Freyzon Y, Farese RV, Terry-Kantor E, Hofbauer HF, Termine D, Welte MA, Barrasa MI, Imberdis T, Noble T, Lindquist S, Clish CB, Jaenisch R, Pincus D, Nuber S, Sandoe J, Kohlwein SD, Kim TE, Ho GPH, Ramalingam N, Walther TC, Baru V, Selkoe D, Srinivasan S, Landgraf D, Soldner F, Dettmer U, Fanning S, Becuwe M, Newby G

[Mol Cell, 2018]

In Parkinson's disease (PD), -synuclein (S) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in S or lipid/fattyacid homeostasis affect each other. Lipidomic profiling of human S-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of S dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased S yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in S-overexpressing rat neurons. In a C.elegans model, SCD knockout prevented S-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on S homeostasis: in human neural cells, excess OA caused S inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for S-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.