Park, S. et al. (2019) International Worm Meeting "Lipid metabolism during axon regeneration."

Wu, Z., Park, S., Jin, Y., Chisholm, A.

[International Worm Meeting, 2019]

Emerging evidence supports a crucial link between lipids and health of the brain, yet it is unclear how neuronal lipids are regulated under various conditions. The axon of certain C. elegans adult neurons regrows after injury; multiple extrinsic and intrinsic factors have been identified to affect axon regrowth. As axon elongation likely demands extensive membrane reorganization and addition, a key unaddressed question is how regenerating axons acquire sufficient lipids under rapid regrowth. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant membrane lipids. De novo synthesis of phospholipids occurs via the highly conserved Kennedy pathway, and other pathways also contribute to their synthesis (1). To determine whether PC or PE supply is necessary for regrowth of injured axons, we systematically tested null and strong loss-of-function mutants in most genes encoding the enzymes of Kennedy pathway. We found decreased axon regeneration in a subset of mutants, suggesting phospholipid synthesis is important in axon regrowth (2). In addition to phospholipid synthesis, activation of fatty acid may also regulate axon regrowth. In collaboration with the Colavita lab (Univ. Ottawa), we have recently reported that DIP-2 (Disco-interacting-protein 2) inhibits axon regeneration (3). DIP-2 consists of two adenylate-forming domains (AFD), predicted to act in acyl CoA synthesis. While phospholipids are structural components of membranes, they are also substrates of lipid-based signaling molecules. As the analysis of DIP-2 suggests its role in acyl CoA synthesis, DIP-2 may generate signaling molecules using phospholipids as substrates. To further understand the function of DIP-2, we are investigating genetic interactions between dip-2 and the phospholipid synthesis pathways. We will report our additional investigations into how lipid metabolism may contribute to axon regeneration. (1) Watts and Ristow, 2017 Genetics (2) Kim et al., 2018 eLife (3) Noblett et al., 2018. J Cell Biol.

Park S et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Behavioral Thresholds for Force-sensation Determined by an Integrated Video-tracking and Force-clamp System"

Park S, Goodman M B, Fung J, Petzold B, Pruitt B L

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Neurosensory mechanotransduction, the conversion of a force into electrical signals, is the fundamental process governing touch sensation. Understanding how the touch receptor neurons (TRNs) of Caenorhabditis elegans mediate this conversion can unravel how touch works. The classical method for scoring touch sensitivity in C. elegans relies on manual delivery of mechanical stimuli delivered by an eyebrow hair glued to a toothpick (Hart AC, ed. Behavior (July 3, 2006), Wormbook doi/10.1895/wormbook.1.87.1, http://www.wormbook.org.). However, the forces delivered by eyebrow hairs are unknown and likely to be extremely variable between trials and among experimenters. To circumvent these limitations and to quantify touch responses, we developed a force clamp system capable of applying stimuli with high force resolution (nanoNewtons), a fast response (1 kHz), and a large dynamic range (10-10,000nN). To do this, we integrated a custom-made, piezoresistive micro-cantilever force probe with a piezoelectric actuator and a programmable real-time controller. We also implemented a computer vision-based x-y tracking system, which allows the desired force to be applied precisely at a selected location on a moving nematode. We used image processing techniques to measure stimulus-evoked changes in body kinematics and moving velocity. Touch responses were scored as positive if the direction, speed, or animal curvature patterns changed in response to mechanical stimulation. We used this system to determine the threshold and dynamic range of touch sensation for stimuli applied along the body wall. We find that response probability increases with applied force, saturates above 1 μN, and that the average force at which half probability was attained is 120 nN in wild-type worms. This value is three orders of magnitude lower than the threshold for human touch sensation (~500 μN), demonstrating that C. elegans is exquisitely sensitive to touch. As expected from the pivotal role of MEC-4 in the formation of force-gated ion channels in TRNs, we show that mutations in mec-4 disrupt responses to stimuli applied to the animal's body, but not its nose. Currently, we are using this force-clamp system to determine force sensitivity, response amplitude, and response latency in wild type worms. Future studies will use this system to quantify the touch sensitivity of mutants with weak phenotypes, enabling analysis of genetic enhancement and suppression.

Pinan-Lucarre, Berangere et al. (2009) International Worm Meeting "Transgenic C. elegans expressing the fungal prion protein HET-s as a model for the study of amyloid infectivity and toxicity."

Qiao, Yujie, Saupe, Sven, Pinan-Lucarre, Berangere, Driscoll, Monica

[International Worm Meeting, 2009]

Amyloids are protein fibers rich in beta sheet structure that are associated with numerous neurodegenerative diseases, including Alzheimer disease. Amyloids have two major biological properties. First, they can be infectious--meaning that they can spread the altered amyloid conformation to the same protein or even among proteins of distinct primary sequence (the latter phenomenon is known as cross-polymerization). Infectious amyloids are called prions. Second, amyloids can be toxic, whether they are infectious or not. Both properties have tremendous fundamental and biomedical impact. [Het-s] is a prion of the fungus Podospora anserina involved in a defense cell suicide mechanism named heterokaryon incompatibility, which disables mixing of different strains by somatic fusion, an ubiquitous feature in filamentous fungi. The HET-s protein exists in a soluble state or in an aggregated, prion state named [Het-s]. The [Het-s] prion is not toxic by itself in P. anserina or in yeast. However cell death is rapidly triggered by the lethal interaction between 2 alleles of the het-s locus: het-S (het-"large S") and het-s (het-"small s") when the HET-s protein adopts the prion form. This prion has been extensively characterized and it is the only prion for which tridimensional structure is available to date. The critical point is that in the [Het-s] system, transgenic expression of het-s allows propagating a non-toxic prion while co-expression of het-s and het-S generates toxicity. We constructed strains expressing het-s or the prion domain only het-s(218-289) (the 218-289 fragment of the protein) as GFP fusions under the control of the ubiquitous promoter dpy-30. The fluorescence of pdpy-30het-s::gfp appears to be mainly diffuse in the cytoplasm while it shows strong aggregation in pdpy-30het-s(218-289)::gfp strains. We constructed strains expressing het-s(218-289)::gfp in the touch neurons using mec-4 promoter and observed that the fusion protein is mostly aggregated into a large fiber spanning the cell body and the proximal part of the axon. These aggregates remain to be shown as infectious [Het-s] amyloids. In future experiments, we will aim to generate neuronal toxicity through the co-expression of het-s(218-289) and het-S. This C. elegans model for prion studies, filling a gap between simple fungal systems and highly complex mammalian systems, might allow a better understanding of the molecular and cellular basis of amyloid infectivity and toxicity.

Ahn, Byungchan et al. (2011) International Worm Meeting "The Caenorhabditis elegans WRN-1 RecQ helicase participates in both DNA damage signaling and DNA Repair."

Park, H, Park, S, Ahn, Byungchan, Kim, E, Hyun, M

[International Worm Meeting, 2011]

RecQ helicases play essential roles in maintenance of genomic stability from E. coli to humans. The RecQ helicases interact with proteins involved in DNA metabolic pathways such DNA repair, recombination, and replication. We found that C. elegans wrn-1 mutant is more sensitive to CPT than N2 and that the wrn-1 mutant failed to arrest the S-phase since DNA synthesis was continued following CPT treatment. In addition, more DNA strand breaks accumulate in the wrn-1 mutant. These data suggest that WRN-1 is involved in DSB processing and/or DNA damage signaling. WRN-1 colocalized with CeRPA in nucleus in germ line cells following CPT, but CeRPA foci formation was independent of WRN-1. In addition, WRN-1 foci formation was not affected in atm-1 and atl-1 mutants and WRN-1 was required for phoshorylation of CHK-1 induced by DSBs. WRN-1 also colocalized with RAD51. These results suggest that WRN-1 functions downstream of RPA and upstream of ATM-1/ATL-1 and CHK-1 in the DSB checkpoint pathway and is in involved in a DNA repair pathway with RAD51.

Dana L. Miller et al. (2007) International Worm Meeting "Adaptation to hydrogen sulfide increases thermotolerance and lifespan."

Mark B. Roth, Dana L. Miller

[International Worm Meeting, 2007]

Hydrogen sulfide (H₂S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H₂S to begin to elucidate the molecular mechanisms of H₂S action. We show that nematodes exposed to H₂S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H₂S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H₂S. Increased thermotolerance and lifespan is not observed in the sir-2.1(ok434) deletion mutant exposed to H₂S. However, mutants in the insulin signaling pathway (both daf-2 and daf-16), animals with mitochondrial dysfunction (isp-1 and clk-1) and a genetic model of caloric restriction (eat-2) all exhibit H₂S-induced increased thermotolerance. These data suggest that H₂S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H₂S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H₂S.

Oppenheimer AJ et al. (1995) International C. elegans Meeting "G-PROTEIN SIGNALING IN C. ELEGANS"

Kaplan JM, Oppenheimer AJ

[International C. elegans Meeting, 1995]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate behavioral phenotypes dependent on G-protein signaling, we targeted expression of mammalian G-proteins to a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Ectopic expression of several G-proteins under the control of the not-3 promoter results in behavioral defects. Expression of constitutively active rat G-alpha-i1 causes uncoordinated forward and backward locomotion. Expression of wild-type rat G-alpha-s impairs backward locomotion, while constitutively active rat G-alpha-s causes defective backward locomotion and lethargy. In order to identify components of the G-alpha-s signaling pathway, we have initiated a screen for mutations that suppress the behavioral effects of activated G-alpha-s expression. In a screen of approximately 7000 haploid genomes, we have identified 11 independent mutations that improve backward locomotion. Several of these mutations result in a hyperactive phenotype in the absence of activated G-alpha-s expression. Mapping and complementation tests will determine whether the suppressors are allelic to other hyperactive mutants isolated by D. Elkes and J. Kaplan. By cloning the suppressor genes, we hope to identify ion channels regulated by G-alpha-s as well as other components of the G-alpha-s signaling pathway. We also plan to determine whether the behavioral effects of G-alpha-s expression are mediated by known second messenger systems. We are first testing the role of cAMP-dependent protein kinase (PKA), because it is activated by G-alpha-s in other systems and has been shown to phosphorylate ion channels. If G-alpha-s signals through PKA, increasing the level of PKA activity should mimic the effect of activated G-alpha-s expression, and inhibiting PKA should suppress the effect of activated G- alpha-s expression.

Oppenheimer AJ et al. (1996) East Coast Worm Meeting "G-alpha-s signaling in C. elegans"

Kaplan JM, Oppenheimer AJ

[East Coast Worm Meeting, 1996]

We are interested in determining how G-proteins modulate the activity of ion channels in the C. elegans nervous system. To generate phenotypes dependent on G-protein signaling, we expressed mammalian G-proteins under the control of the glr-1 promoter, which drives expression in a set of neurons including those controlling foraging and locomotion (RMD, AVA, AVB, AVD, and PVC). Expression of wild-type rat G-alpha-s impairs backward locomotion, while consitutively active rat G-alpha-s causes paralysis and neuronal degeneration in a subset of G-alpha-s-expressing cells, primarily the PVC and AVD neurons. Our model for the neurodegeneration is that activated G-alpha-s is misregulating an ion channel in AVD and PVC, causing osmotic imbalance and consequent swelling. Degenerating neurons appear enlarged and vacuolated, similar to those in worms carrying gain-of-function mutations in deg-1 and other degenerins. We tested whether activated G-alpha-s is killing cells by misregulating known ion channels. Loss-of-function mutations in deg-1, mec-6, unc-36, unc-2, and egl-19 were unable to suppress the degenerations, suggesting that G-alpha-s is not acting through these degenerins or calcium channels. Mutations in ced-3 and ced-4 do not prevent G-alpha-s-induced degenerations, indicating that G-alpha-s is not killing through apoptosis. To identify the components of the signaling pathway downstream of G-alpha-s, we screened for mutations that suppress the paralysis caused by activated G-alpha-s. In a screen of 7,760 haploid genomes, we identified 8 mutations that suppress paralysis and neurodegeneration and 8 mutations that suppress only paralysis. Five of the degeneration suppressors map to chromosome III and may be allelic. These mutations are semidominant, and several cause a hyperactive phenotype in the absence of activated G-alpha-s. Because hyperactivity also results from mutations in the Go signaling pathway, our suppressor mutations may identify a gene invovled in both the Gs and Go signaling pathways. We are continuing to map and characterize the suppressor genes.

Costi D. Sifri et al. (2007) International Worm Meeting "Immune evasion by staphylococcal exopolysaccharide during C. elegans infection."

Jakob Begun, Dietrich Mack, Costi D. Sifri, Jessica M. Gaiani, Frederick M. Ausubel, Holger Rohde, Stephen B. Calderwood

[International Worm Meeting, 2007]

We have previously shown that C. elegans can serve as a model host for the study of S. aureus pathogenesis and host innate immunity. Here we characterize nematode killing by S. epidermidis. C. elegans fed S. epidermidis survive 3-5 days, which is ~1 day longer than their lifespan on S. aureus. Inactivation of the ica operon (the biofilm exopolysaccharide [PIA] biosynthetic operon and Yersinia hms homolog) in S. epidermidis strain M10 greatly reduces nematode killing compared to the isogenic parental strain 9142 and a plasmid complemented mutant. In S. aureus, overproduction of PIA due to derepression of ica augments nematocidal activity. FITC-WGA labeling confirmed the presence of PIA within the digestive tracts of worms fed 9142 but not M10. Attachment of bacteria or bacterial matrix to the nematode cuticle was not observed. Nematodes briefly exposed to 9142 could not be rescued by transfer to M10, and worms fed dilutions of 9142 in M10 at ratios as low as 1:10e4, respectively, still die. Pulse/chase experiments using 9142, M10, and a second PIA-deficient strain, ATCC 12228, showed that durable colonization requires an intact ica operon. Disruption of N2 worms fed 1:100 9142/M10 mixtures for 20h confirmed significant enrichment of 9142 within the digestive tract of N2 but not sek-1 animals. Moreover, sek-1 and pmk-1 animals were found to be equally sensitive to M10- and 9142-mediated killing, in contrast to wild-type N2 worms. Several conclusions can be drawn from these studies. (a) S. epidermidis can infect and kill worms. (b) Disruption of PIA biosynthesis abrogates S. epidermidis virulence, while overproduction of PIA in S. aureus augments virulence. (c) PIA-producing S. epidermidis has a competitive advantage over PIA-deficient S. epidermidis within the nematode digestive tract. (d) Genetic analysis suggests that PIA may protect luminal staphylococci from SEK-1 p38 MAPK-regulated immune responses.

Scholer, Lone V. et al. (2011) International Worm Meeting "Characterization of pathogenesis of S. aureus and involvement of checkpoint response in C. elegans."

Hansen, Frederik D, Scholer, Lone V., Fuursted, Kurt, Noerregaard, Steffen, Olsen, Anders

[International Worm Meeting, 2011]

Staphylococcus aureus (S. aureus) is a frequent colonizer of humans contributing to normal bacterial flora in nose, skin and mucosa. In addition, it is an important human pathogen causing various diseases ranging from superficial skin infections to life-threatening infections. Methicillin resistant S. aureus (MRSA) strains are rapidly spreading in the community (CA-MRSA) and is consequently becoming a more serious health threat. The capacity of S. aureus to cause this spectrum of human diseases reflects an ability to adapt to distinct microenvironments in the human body and suggests that the pathogenesis of S. aureus infection is a complex process involving a diverse array of virulence determinants that are coordinately expressed at different stages of infection. Studies have shown that virulence factors are very differently distributed among strains and are not always regulated in the same way, reflecting the ability of S. aureus to adapt and survive in different environmental niches and resist antibiotic treatment. C. elegans has previously been established as a model host for the study of S. aureus pathogenesis and CA-MRSA strains are capable of killing C. elegans[1]. We have screened thirty clinical MRSA isolates and found that in terms of pathogenicity they can be divided into separate classes. Using these classes we wish to identify new virulence markers and novel immunity pathways activated in response to S. aureus infection. We are particularly interested in evaluate virulence determinants during colonization versus infection and establishing their clinical relevance in humans. Inactivation of some S-M checkpoint proteins increase stress resistance and lifespan of C. elegans[2]. Since there may exist a potential correlation between stress response and immune function we are studying the role of checkpoint proteins to S. aureus exposure. We have identified several novel genes with potential checkpoint function which increase lifespan and thermotolerance. We find that that mutation of one of these, the transmembrane protein NDG-4, also confers resistance towards S. aureus infection. Epistasis analysis reveals that increased tolerance towards S. aureus infection is partially dependent on insulin signaling but that another unidentified component is involved as well. 1. Sifri, C.D., et al. 2003. 71(4): p. 2208-17. 2. Olsen, A., M.C. Vantipalli, and G.J. Lithgow. Science, 2006. 312(5778): p. 1381-5.

Jafa Armagost et al. (2007) International Worm Meeting "Investigating Potential Bacterial Sources of Dopaminergic Neurotoxicity in C. elegans."

Kim A. Caldwell, Guy A. Caldwell, Tyler Hodges, Jeana Blalock, Julie B. Olson, Jafa Armagost

[International Worm Meeting, 2007]

Parkinsons disease (PD) involves the progressive loss of dopamine (DA) neurons from the substantia nigra pars compacta (SNpc). Genetic forms of PD account for only 5-10% of known cases and environmental factors appear pivotal to sporadic causality. Evidence from both environmental and genetic studies of PD indicates that overloading the ubiquitin-proteasome system is a causative risk factor for PD. Furthermore, when proteasome inhibitors were injected directly into the brains of rats, PD-like symptoms resulted (McNaught et al., 2004; Ann Neurol. 56:149-162). Many proteasome inhibitors are isolated from bacterial strains within the order Actinomycetales, which are well known for the production of secondary metabolites. To determine if exposure to Actinomycetes could cause DA neurodegeneration in C. elegans, we exposed worms to three different species of Streptomyces (S. venezuelae, S. griseus, and S. coelicolor). When worms were grown on either S. griseus or S. coelicolor no DA neurodegeneration was observed. However worms exposed to S. venezuelae displayed DA neurodegeneration that increased over time. The causual factor is excreted by S. venezuelae, as conditioned media is sufficient for the neurodegenerative effect. This factor does not enter DA neurons through the DA transporter because degeneration is observed in dat-1 mutant animals. Further characterization of the S. venezuelae activity shows that DA degeneration does not result from generalized stress response or the unfolded protein response but may occur as a result of proteasome inhibition. Initial efforts to purify the factor have revealed that it is lipophilic and does not fit the molecular profile of known proteasome inhibitors.