R Lee et al. (1999) International C. elegans Meeting "Extension of C. elegans lifespan by a mutation in daf-21/hsp9"

G Ruvkun, R Lee

[International C. elegans Meeting, 1999]

Longevity often associates with resistance to environmental stress. In C. elegans , long-lived (Age) mutants such as age-1 and daf-2 are more resistant to oxidative, UV-radiation, or thermal stresses. To get at the proximal causes for this longevity-associated stress resistance, we are focusing on the worm stress-response gene hsp90 because it is a molecular chaperone; in contrast to many other 'house-keeping' genes, its mRNA level is dramatically increased in the dauer compared to other developmental stages (Dalley & Golomb 1992, Dev Biol 151: 80). The Thomas lab found that daf-21(p673) has a missense mutation in the worm hsp90 gene (Malone and Thomas, WBG14.4: 52). daf-21(p673) has a temperature-sensitive dauer constitutive (Daf-c) phenotype. Based on this phenotype, genetic analysis showed that p673 is a recessive, possibly neomorphic mutation that encourages dauer formation by affecting chemosensory neuron function (Malone et al, IWM93: 297). We found that daf-21(p673) mutant lived about 40-50% longer (mean and maximal adult lifespan) than wild-type animals when shifted from the permissive (15deg) to the restrictive (25deg) temperature at the L4 or young adult stages. Whereas either daf-16(mgDf47) or daf-12(m20) efficiently suppressed the Daf-c phenotype (Thomas et al 1993, Genetics 134: 1105), only daf-16 suppressed Age, suggesting that daf-21 may affect dauer formation and lifespan independantly. daf-16 is a member of the Forkhead class of transcription factors; and it mediates all of the effects caused by daf-2 insulin-like signaling, including lifespan (Ogg et al 1997, Nature 389: 994; Lin et al 1997, Science 278: 1319). One possible explanation for the observation that a daf-16 null mutation is epistatic to daf-21 is that daf-21 expression is positively regulated by daf-16 . We are testing this hypothesis. We are also asking if Age mutants in general have an elevated hsp90 expression or function. We thank Jim Thomas for providing daf-21 mutant strains.

Revtovich, A. et al. (2017) International Worm Meeting "Diet Influences Stress and Innate Immune Resistance in C. elegans."

Revtovich, A., Kirienko, N., Lee, R.

[International Worm Meeting, 2017]

The opportunistic, multi-host pathogen Pseudomonas aeruginosa causes a wide-variety of hospital acquired infections, as well as being a chief contributor to morbidity and mortality in cystic fibrosis patients. Unfortunately, this bacterium is notoriously resistant to antimicrobials and readily acquires additional resistance mechanisms. To simplify identification of novel anti-Pseudomonal compounds, a liquid-based C. elegans-P. aeruginosa pathogenesis assay (called Liquid Killing) was developed. In previous publications, we have reported on the characterization of this assay, and the identification of pyoverdine, the main siderophore (or iron-acquiring molecule) of P. aeruginosa, as the key determinant of pathogenesis. During our characterization of pyoverdine-mediated pathogenesis and C. elegans' defense against it, we performed conventional RNAi studies using the HT115(DE3) strain. To our surprise, we noted a substantial increase in resistance to Liquid Killing when compared to OP50. Since pyoverdine-mediated killing involves the removal of host iron, we initially hypothesized that HT115 more effectively mobilized and provided iron to C. elegans. Mass spectrometric and fluorometric analyses disproved this hypothesis. We also ruled out weak OP50 pathogenesis; we compared pathogenesis using RNAi targeting innate immune genes in either the OP50 or HT115 backgrounds and saw no differences from wild-type worms. Further experimentation revealed that worms reared on HT115 also show increased resistance to other abiotic stresses (e.g., heat shock, oxidative stress) as well. In the absence of any other obvious candidates, we turned to microarray transcriptional profiling to gain an unbiased picture of the differences that result from feeding C. elegans with HT115 instead of OP50. To our surprise, there were a relatively small number of genes differentially expressed between worms fed OP50 and HT115. Amongst them were several mitochondrially-associated genes, suggesting that the relevant difference may involve this organelle. Using a variety of approaches, we determined that worms fed HT115 exhibit increased mitochondrial health than their OP50 counterparts. Furthermore, supplementation of growing OP50 with cobalamin recapitulated virtually all of the HT115 phenotypes.

Lee SS et al. (2000) East Coast Worm Meeting "Isolation of genetic suppressors of daf-16, a forkhead class transcription factor mediating insulin-like signaling"

Lee R, Lee SS, Ruvkun GB

[East Coast Worm Meeting, 2000]

Insulin and insulin-like signaling pathways are phylogenetically conserved in animals. Biochemical analysis done in mammalian cells and genetic analysis in worms indicated that insulin (-like) signals are transduced via a lipid and protein kinase cascade that negatively regulates specific forkhead class transcription factors: DAF-16 in C. elegans and AFX, FKHR, FKHRL1 in mammals. In worms, reduced insulin signaling causes pleitropic effects including constitutive dauer larval development, metabolic shift towards fat storage, and extended adult lifespan. We are taking a genetic approach to identify targets of daf-16. Loss-of-function daf-16 mutants have a short adult lifespan. We sought long-lived animals among F3 progeny of (semi) clonal lines of EMS-mutagenized daf-16(null). In a pilot screen of 320 cohorts, each established from 10 F2 parents, we recovered two suppressor mutants. We are expanding the scale of the screen. We are also screening for suppressors of phenotype associated with forced expression of daf-16. Animals carrying transgenes that express DAF-16 under the control of the hsp16 heatshock promoter (hsp::daf-16) show a slow and kinky uncoordinated movement phenotype (Unc) even without any heat shock treatment. In a screen for suppressors of Unc, we recovered five mutants among 30,000 EMS-mutagenized F2s. Four of them showed reduced expression of a GFP-encoding co-injection marker. The remaining mutant is still unable to move normally but is significantly more active than the starting strain. Furthermore, whereas hsp::daf-16 transgenic animals inappropriately form dauers at high temperatures, the suppressed animals do not. Nonetheless, normal dauers do form on starved plates. Because daf-16(null) mutants do not form normal dauers under similar conditions, this result suggests that the suppressor affects daf-16 function only partially. Alternatively, the suppressor affects specifically the transgene expression of daf-16. We are testing these possibilities by asking if the suppressor affects the dauer-constitutive phenotype caused by either reduced insulin signaling or a different daf-16 transgene.

AR Winnier et al. (1999) International C. elegans Meeting "Mutations in the L-type calcium channel EGL-19 affect UNC-4 function"

DM Miller, AR Winnier

[International C. elegans Meeting, 1999]

Wildtype movement depends on precisely defined connections between interneurons and motor neurons. In unc-4 mutant animals, the VA motor neurons, which are required for backward locomotion, are miswired to interneurons that are normally reserved for their sister cells, the VBs. As a result, unc-4 mutant animals cannot move backward. unc-4 encodes a homeoprotein that is required for the repression of VB-specific traits in the VA motor neurons. This UNC-4-mediated repression is dependent on interactions with the Groucho-like co-repressor protein UNC-37 (1). We would like to identify other genes which function with unc-4 . Using genetic screens that resemble strategies used to study unc-4/unc-37 interactions, we have uncovered mutations in the EGL-19 calcium channel which appear to modify UNC-4 function. The semi-dominant wd29 (gf) allele was isolated in an Unc-4 suppressor screen. wd29 is an allele-specific suppressor of unc-4 missense mutations and maps to the stp44-unc-24 (IV) interval. A revertant allele, wd29wd45 , results in the loss of wd29 -dependent Unc-4 suppression. Homozygous wd29wd45 enhances the Unc-4 phenotype of unc-4(e2322ts) but does not result in a backward movement defect in a wildtype background. wd29wd45 fails to complement the egg-laying defective and long phenotypes of the n582 , ad1015 , and ad1006 egl-19(lf) mutants. Furthermore, the egl-19 mutations n582 and ad1006 enhance the unc-4(e2322ts) phenotype. Therefore, we propose that wd29wd45 represents an egl-19(lf) mutation. egl-19 , which encodes an L-type calcium channel, is expressed in muscle and neurons although a function for EGL-19 in unc-4 -expressing motor neurons has not been described (2). Currently, we are testing the idea that egl-19 is a downstream target of UNC-4 by evaluating egl-19::gfp in wildtype and unc-4 mutant backgrounds. (1) Winnier, A. et al. , this meeting. (2) Lee, R. et al. (1997) EMBO J. 16 : 6066-6076.

Akwasi Anyanful et al. (2005) International Worm Meeting "Paralysis and killing of C. elegans by enteropathogenic E. coli requires the bacterial tryptophanase gene"

Daniel Kalman, Jennifer Dolan-Livengood, Lisa Kalman, Melanie Sherman, Mark DeZalia, Taiesha Lewis, Guy Benian, Akwasi Anyanful, Seema Sheth

[International Worm Meeting, 2005]

Enteropathogenic E. coli (EPEC), a major cause of food and water-borne disease causes high morbidity and mortality in developing countries. We have developed a model of EPEC virulence based on our observation that these bacteria paralyze and kill C. elegans. Paralysis and killing of C. elegans by EPEC did not require direct contact, suggesting mediation by a secreted toxin. Virulence however did require tryptophan and bacterial tryptophanase because lack of tryptophan in growth media or deletion of tryptophanase gene failed to paralyze or kill C. elegans. While known tryptophan metabolites failed to complement an EPEC tryptophanase mutant when presented extracellularly, complementation was achieved with the enzyme itself expressed either within the pathogen or within a co-cultured K12 strains. Thus, an unknown metabolite of tryptophanase, derived from EPEC or from commensal nonpathogenic strains, appears to directly or indirectly regulate toxin production within EPEC. EPEC strains containing mutations in the locus of enterocyte effacement (LEE), a pathogenicity island required for virulence in humans, also displayed attenuated capacity to paralyze and kill the nematodes. Furthermore, tryptophanase activity was required for full activation of LEE promoters, and for efficient formation of actin-filled membranous protrusions (attaching and effacing lesions) that form on the surface of mammalian epithelial cells following attachment and which depends on LEE genes. Finally, several C. elegans genes, including daf-2, age-1, hif-1 and egl-9, rendered C. elegans less susceptible to EPEC when mutated, suggesting their involvement in mediating toxin effects. Other genes, including daf-16, mev-1, mek-1, pgp-1,3 and vhl-1, rendered C. elegans more susceptible to EPEC effects when mutated, suggesting their involvement in protecting the worms. Together, these data suggest that this C. elegans/EPEC system will be valuable in elucidating novel factors relevant to human disease that regulate virulence in the pathogen or susceptibility to infection in the host.

Lee, Inhwan et al. (2013) International Worm Meeting "Epigentic regulation of L1 longevity."

You, Young-jai, Lee, Inhwan

[International Worm Meeting, 2013]

Animals encounter food infrequently and starvation is a common physiological state in their natural circumstance [1]. Hatching in the absence of food, worms arrest their development at the L1 stage and survive starvation more than two weeks [2]. It was shown that adult longevity is regulated epigenetically by chromatin alterations and the genes that regulate epigenetics [3]. Here, we investigate whether L1 longevity is also regulated epigenetically. When we measured various histone modifications using Western blot, we found histone 3 lysine 4 tri-methylation, known to be a modification to activate gene transcription, is increased in L1 starvation. Moreover, mutants of set-2, which encodes a histone 3 lysine 4 tri-methyl transferase that functions in adult longevity [3], has reduced L1 longevity. There are several genes essential for normal L1 longevity, such as aak-2 and daf-16. Based on our data, we hypothesize that chromatin remodeling through histone 3 lysine 4 tri-methylation regulates transcription of genes involved in L1 longevity. Currently we are testing this hypothesis by measuring expression levels these genes by ChIP-qPCR during L1 starvation in set-2 mutant. References [1] Brian H. Lee, Plus Genetics, 2008 [2] Inhwan Lee, Plus One, 2012 [3] Eric L. Greer, Nature, 2010.

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Reis-Rodrigues, Pedro et al. (2015) International Worm Meeting "N-acylethanolamines modulate C. elegans longevity at warm temperatures."

S. Gill, Matthew, Harrison, Neale, Reis-Rodrigues, Pedro

[International Worm Meeting, 2015]

The relationship between environment and longevity is perhaps best illustrated by the effect of temperature on lifespan across multiple species. Contrary to previous thought, it has been shown that temperature mediates its effects on lifespan in part through highly coordinated genetic pathways (Lee et al. 2009, Xiao et al. 2013). We have recently reported that the N-acylethanolamine (NAE) system in worms reduces C. elegans lifespan at 25oC but not at 15oC (Harrison et al. 2014), phenocopying the effect of ablation of the AFD thermosensory neuron (Lee et al. 2009). Here we show that deletion of faah-1, an NAE hydrolyzing enzyme, recapitulates the effect of over-expression of the biosynthetic enzyme nape-1. We also find that the effect of faah-1 deletion on lifespan is dependent on daf-9 and daf-12, indicating that the NAE signal acts via the same mechanism as that described for thermosensory mutants (Lee et al. 2009). In other systems, NAEs act as short-range signaling molecules and thus we hypothesized that NAEs directly influence neuronal activity to control lifespan cell non-autonomously. In accordance with this, we observed that genetic ablation of the ASJ neuron by cell-specific caspase expression rescues the effect of faah-1 on lifespan at 25oC. We are currently evaluating the requirement for thermosensory neurons in mediating the effects of faah-1 deletion.We are also interested in determining which specific NAEs are responsible for signaling this effect. NAEs are synthesized from phospholipid precursors and differ in their fatty acid moiety. We have previously reported that eicosapentaenoyl ethanolamine (EPEA), a C20:5n3-containing NAE, influences dauer entry and suppresses DR-induced longevity in worms. To test if this molecule could be responsible for the temperature-dependent effects of NAEs, we crossed nape-1 over-expressers into a fat-1 mutant, which lacks the fatty acid precursor needed for EPEA production. In this background we found that fat-1 is not required for the effects of nape-1 over-expression on lifespan, indicating that the effect of the NAE system on temperature-dependent longevity is independent of EPEA. We are currently trying to identify the specific NAE(s) responsible for this effect, as well as further defining the mechanism by which the NAE system affects lifespan in a temperature dependent manner.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.