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.

Amy Walker et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Genetic analysis of Spinal Muscular Atrophy"

Amy Walker, Jevede Harris, Anne Hart

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Infantile spinal muscular atrophy (SMA) affects roughly 1/6,000 live births with severe cases resulting in early death typically due to respiratory complications. Loss of the SMN1 gene causes SMA and results in loss of anterior spinal cord motorneurons with consequent muscular atrophy. Patient motorneuron loss and clinical severity is dependent on residual function of a related gene, SMN2. The SMN1 gene encodes the SMN protein which likely involved in small nuclear RNA processing, pre-mRNA splicing and, potentially, RNA transport. The ubiquitous expression of SMN in all cells stands in contrast to the specificity of SMA defects. It is unclear why specific motorneurons are affected in SMA and there is no effective treatment for SMA patients. Given the strong homology between human, Drosophila, and C. elegans SMN proteins, we hope to understand the pathological mechanisms underlying SMA using genetic techniques available in model organisms. Loss of function alleles for SMN genes exist in C. elegans and Drosophila. In either species, these alleles result in developmental delays and lethality. In collaboration with the Artavanis-Tsakonas and Van Vactor laboratories, we have initiated genetic studies in C. elegans and Drosophila to identify genetic modifiers of SMN defects. Genes that suppress or enhance the severity of SMN loss of function defects in one organism will be tested in the other for modulation of SMN severity. Unbiased, genome-wide screens in model organisms should reveal pathways that are critical for SMN pathology in all species.

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.

Dimitriadi M et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Genetic and pharmacological analysis of synaptic function in smn-1 animals"

Hart A, Dimitriadi M, Sleigh J, Kalloo G, Harris J, Barsby T, Satterlee J, Van Vactor D, Artavanis-Tsakonas S, Walker A, Chang H, Sen A

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

Spinal Muscular Atrophy (SMA) is caused by depletion of Survival Motor Neuron (SMN) protein, but the molecular and cellular pathways critical for SMA pathology remain unclear. SMN functions in spliceosome assembly, but SMN likely also acts in additional cellular processes including axonal mRNA transport. Loss of the C. elegans SMN ortholog, smn-1, causes neuromuscular defects that are followed by larval lethality. To identify genetic pathways critical to SMN neuromuscular function in C. elegans, we undertook an RNAi genome-wide screen using the loss-of-function smn-1(ok355) allele and identified several modifier genes that act in pathways not previously implicated in SMN loss-of-function pathology. Interestingly, Drosophila orthologs of at least two of these genes modified Smn loss-of-function defects in the fly neuromuscular junction, demonstrating that conserved genes modulate SMN neuromuscular defects across species. To identify additional conserved modifier genes, C. elegans orthologs of previously described Drosophila and human SMN/SMA modifier genes were tested using C. elegans assays for function as cross-species modifiers. We found that 14 of these altered smn-1 larval lethality and/or neuromuscular defects, suggesting a conserved functional relationship with SMN. Ongoing studies addressing the importance of these genes/pathways in SMN loss of function pathology will be described. As loss of neuromuscular function is an early event in SMA pathology, we also tested whether smn-1 loss impacts synaptic signaling. We found that smn-1 animals are resistant to inhibitors of cholinesterase (Ric), but sensitive to levamisole activation of post-synaptic receptors (nonLev). We conclude that smn-1 loss likely causes pre-synaptic neuromuscular defects. Our ongoing studies focus on genetic interactions of smn-1 with known players of the synaptic vesicle cycle and on assays that will further define smn-1 pre-synaptic defects. Delineation of smn-1 defects in C. elegans should yield insights into the neuromuscular role of SMN and reveal previously unsuspected modifiers of synaptic function.

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.

Dimitriadi, M. et al. (2009) International Worm Meeting "Conserved genes and cellular pathways modulate Survival of Motor Neuron (SMN) loss of function defects."

Sleigh, J.N., van Vactor, D., Artavanis-Tsakonas, S., Harris, J., Hart, A.C., Barsby, T., Dimitriadi, M., Kalloo, G., Chang, C-HC., Satterlee, J.S., Walker, A.K., Sen, A.

[International Worm Meeting, 2009]

Spinal Muscular Atrophy (SMA) is caused by diminished function of the Survival of Motor Neuron (SMN) protein, but the neuromuscular pathways critical for SMA pathology remain unclear. SMN functions in spliceosome assembly, but SMN likely also acts in additional cellular processes including axonal mRNA transport. There is only one genetic modifier of SMA in humans; in female patients, SMA severity is dependent on the level of Plastin 3 (PLS3), an actin-bundling protein. The mechanisms underlying this interaction remain unclear. Identifying the genetic and molecular pathways pertinent to SMA should help us to understand SMA pathological mechanisms and accelerate therapy development. Here, we have used invertebrate models to identify genetic modifiers of SMA. Drosophila and C. elegans each have one SMN gene; diminished SMN function in these invertebrate models caused lethality and neuromuscular defects. We found that RNAi knockdown of PLS3 orthologs enhanced SMN loss of function defects in C. elegans and Drosophila validating these models. To identify additional conserved genes that modulate SMN loss of function defects across species, we used two strategies. First, C. elegans orthologs of previously described, candidate Drosophila SMA modifier genes were tested using C. elegans assays for function as cross-species SMN modifiers. We found that 13 of the 40 genes crossed invertebrate species and modified larval lethality and/or neuromuscular defects caused by loss of C. elegans smn-1 function. Among the cross-species modifiers were atf-6 and atn-1, which have been implicated in Amyotrophic Lateral Sclerosis (ALS) and plastin-associated pathways, respectively. Second, we undertook an independent C. elegans genome-wide RNAi screen for SMA modifiers and recovered one known and four previously unknown modifier genes. Thus far, two of these new genes modulate neuromuscular defects in Drosophila and suggest roles for SMN in pathways not previously implicated in neurodegeneration. Our ongoing studies address the importance of these pathways in SMN loss of function pathology. Overall, our results demonstrate that conserved genes and cellular pathways act as genetic modifiers of SMN loss of function and suggest mechanisms and potential SMA therapeutic targets.

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.