Morton, Katherine et al. (2021) International Worm Meeting "Refining Sugar: A neuroprotective role for high sugar diets despite lifespan and reproductive losses"

Heffernan, Nathan, Hartman, Jessica, Morton, Katherine, Meyer, Joel

[International Worm Meeting, 2021]

High sugar diets are cited as a causal factor in the ongoing increase in obesity rates as Americans consume over 75 g of refined sugar and corn syrup per day on average. Further, obesity is associated with increased risk for multiple negative health outcomes including the neurodegenerative disease Parkinson's Disease. Utilizing C. elegans to investigate the effects of two common dietary sugars, glucose and fructose, we investigated the systemic and dopaminergic neuron-specific effects of high sugar diets on bioenergetics and susceptibility to mitochondrial dysfunction. Through the use of in vivo fluorescent reporters and Seahorse XF whole worm respiratory analysis, we found that glucose-fed worms exhibited a 14.9% increase in basal oxygen consumption and alterations to dopaminergic neuronal mitochondrial dynamics and morphology. Our study also indicated that unlike exposures beginning during development, chronic adult high glucose and high fructose diets are protective from 6-hydroxydopamine-induced dopaminergic neurodegeneration, despite deleterious impacts on lifespan and reproduction. These findings support the idea that a shift to glycolytic metabolism protects from acute electron transport chain inhibition, while also supporting previously observed adverse impacts of high sugar diets.

Maaike C. W. van den Berg et al. (2006) European Worm Meeting "Sex-Dependent Resistance to the Fungal Pathogen Cryptococcus neoformans"

Maaike C. W. van den Berg, Jessica Z. Woerlee, Robin C. May, Hansong Ma

[European Worm Meeting, 2006]

Maaike C. W. van den Berg1,2, Jessica Z. Woerlee2, Hansong Ma1,2 & Robin C. May1. Cryptococcus neoformans is the causative agent of cryptococcosis, a fatal fungal disease of immunocompromised patients. Our group and others have made use of C. elegans as an alternative host for Cryptococcus, in order to investigate the molecular basis of host-pathogen interactions. Using this system, we now report that male C. elegans show greater resistance to killing by Cryptococcus than hermaphrodite animals and that this resistance can be induced in hermaphrodite animals by inappropriate activation of the male sex-determination pathway. Resistance is molecularly determined, rather than resulting from behavioural changes or reproductive differences, and requires the activity of the stress-response transcription factor DAF-16. Finally, we demonstrate that resistance to Cryptococcus neoformans correlates broadly with longevity within the Caenorhabditis genus. Our results suggest that many of the molecular determinants of longevity and immunity are the same and that differential regulation of these determinants may underlie much sex-dependent and species-dependent variation.

Ahmed S et al. (2000) European Worm Meeting "A mutator phenotype found in C. elegans checkpoint mutants"

Hodgkin JA, Ahmed S

[European Worm Meeting, 2000]

DNA damage checkpoint proteins are caretakers of the genome. These checkpoint proteins function by sensing DNA damage and inducing either CELL CYCLE ARREST, which allows for the DNA damage to be repaired, or APOPTOSIS, which eliminates the cell that has been damaged. Alteration of the p53 checkpoint protein is frequently observed in cancer cells. Since mutations in multiple genes occur in developing cancers, it seems reasonable to surmise that a DNA damage checkpoint defect might result in an elevated mutation rate that leads to cancer. However, controversial results have been obtained regarding mutation rates in mammalian cells with checkpoint defects, and no mutator phenotype has been reported for yeast checkpoint mutants. Several different C. elegans mutants with DNA damage checkpoint defects have been recently identified (1,2) - the germlines of these checkpoint mutants are hypersensitive to X-rays and to other agents that damage DNA. We observed an elevated rate of spontaneous mutation in one of these C. elegans checkpoint mutants, and another checkpoint mutant was previously shown to be a mutator (3). This increase in mutation frequency is much higher for some genes than for others. These results may explain the controversy surrounding mutation rates in human cells with checkpoint defects. 1. Ahmed S, Hodgkin J (2000). MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans. Nature 403, 159-164. 2. Gartner A, Milstein S, Ahmed S, Hodgkin J, Hengartner MO (2000). A conserved checkpoint pathway mediates DNA damage-induced apoptosis and cell cycle arrest in C. elegans. Molecular Cell, in press. 3. Hartman PS, Herman RK (1982). Radiation-sensitive mutants of Caenorhabditis elegans. Genetics 102, 159-178.

Kumar, Sandeep et al. (2021) International Worm Meeting "Enhanced functional restoration through axon regeneration by swimming exercise"

Ghosh-Roy, Anindya, Behera, Sibaram, Dey, Shirshendu, Kumar, Sandeep, Basu, Atrayee

[International Worm Meeting, 2021]

Axonal regeneration is a promising approach to overcome impaired functionality due to axonal injury. In mammals, central nervous system has poor regenerative capacity due to both extrinsic and intrinsic factors. The regenerative capacity also declines significantly with ageing. Therefore, functional axon regeneration in adulthood is challenging and needs more understanding. The pharmacological manipulations are not very successful for functional restoration whereas rehabilitation and physical activity shows improvement. As physical exercise has complex systemic effects, understanding the downstream effectors of physical exercise that is relevant for axon regeneration might be useful. Studying this using simple model organism has several advantages. Using posterior gentle touch circuit neuron (PLM) of Caenorhabditis elegans, we are studying effect of swimming exercise on functional restoration after laser assisted axotomy. We found that a single swimming exercise session of 90 minutes, which is an established paradigm of exercise in worm (Laranjeiro et al., 2017; Laranjeiro et al., 2019) improves functional recovery irrespective of age. However multiple swimming session is required for older worms (A5 stage). Anatomical correlation showed that swimming session improves regrowth initiation, regrowth length and functional connections. We found that the energy sensor kinase AMPK/AAK-2 plays an essential role mediating swimming benefits. Characterizing tissue specific requirement, we found that it has both cell autonomous (PLM neuron) and non-autonomous (muscle) requirement. Pharmacological activation of AMPK/AAK-2 showed enhanced functional restoration similar to swimming. We are studying the downstream molecules and their specific roles in various tissues for swimming mediated functional enhancement which will be helpful for better implementation of this approach. References Laranjeiro R, Harinath G, Burke D, Braeckman BP, Driscoll M (2017) Single swim sessions in C. elegans induce key features of mammalian exercise. BMC Biology 15. Laranjeiro R, Harinath G, Hewitt JE, Hartman JH, Royal MA, Meyer JN, Vanapalli SA, Driscoll M (2019) Swim exercise in Caenorhabditis elegans extends neuromuscular and gut healthspan, enhances learning ability, and protects against neurodegeneration. Proc Natl Acad Sci U S A 116:23829-23839.

Oge Arum et al. (2004) West Coast Worm Meeting "Reduction in cep-1 expression increases lifespan"

Oge Arum, Tom Johnson

[West Coast Worm Meeting, 2004]

We found that reduced expression of cep-1 (a homolog of p53) confers extension of maximal lifespan in screens of RNAi libraries. Subsequent survival assays confirmed a modest mean lifespan increase and mild resistance to heat, but no effect on resistance to oxidative, stress. cep-1 feeding-RNAi failed to increase dauer formation of N2 worms at 27 degC or of daf-2 worms at 25.5 degC. cep-1 feeding-RNAi also failed to induce nuclear-localization of DAF-16::GFP; all suggesting that CEP-1 does not participate in the daf-2 pathway. However, the daf-16 -dependence lifespan assays showed daf-16 worms on cep-1 feeding-RNAi bacteria as having lifespans similar to those of daf-16 worms on GFP feeding-RNAi bacteria. As we have often pointed out, shortening of life span by supposed 'suppression' by daf-16 mutations is not a reliable indication of direct interaction between the genes under study because of the known life-shortening effects of mutations in daf-16 and the oxidative toxicity in daf-16 hypomorphs. A cep-1 knockout has a lifespan increase slightly longer than that observed on cep-1 RNAi. A survey of all C. elegans mutants defective for response to DNA damage in the germline revealed that only cep-1 reduction causes an increased life expectancy. Thus, there does not appear to be a universal correlate of DNA damage-response mutants and increased lifespan, consistent with studies published fifteen years ago by Phil Hartman and by us. Since mammalian Sir2alpha decreases p53 expression and SIR2 overexpression in Saccharomyces cerevisiae increases replicative lifespan, we are currently studying possible cep-1 interactions with sir-2.1 in double mutants with the sir-2.1 overexpression and knockout strains. Thus, consistent with murine findings that an increase in p53 activity decreases lifespan and engenders segmental progeria, decreased expression of a C. elegans p53-like gene leads to a daf-16 -independent increase in lifespan.

Shawn Ahmed et al. (2001) International C. elegans Meeting "C. elegans DNA damage checkpoint mutants mutate"

Shawn Ahmed, James Thomas, Monika Tzoneva, Jonathan Hodgkin

[International C. elegans Meeting, 2001]

The DNA damage checkpoint is composed of a group of proteins that sense DNA damage and induce either cell cycle arrest and DNA repair (which mends the damage) or programmed cell death (which removes the damaged cell). Although many cancer cells are checkpoint-defective, efforts to show that mammalian cells with defects in the DNA damage checkpoint are mutating spontaneously have produced controversial results. In addition, it is unclear which DNA damage checkpoint protein physically binds to damaged DNA. We have been addressing both of these issues using the nematode C. elegans . Three C. elegans DNA damage checkpoint mutants have been identified to date: mrt-2 (e2663) , op241 , and rad-5 (mn159) (1,2). The rates and nature of spontaneous mutation in these mutants have been examined using a variety of tests. We find that all of these DNA damage checkpoint mutants display a spontanaeous mutator phenotype, and that most of these spontaneous mutations are deletions. Although mrt-2 (e2663) and op241 mutate at all loci tested, rad-5 (mn159) is mutating primarily at a single locus. This surprising result agrees with the observation that rad-5 (mn159) is Temperature-sensitive and is therefore unlikely to be a null allele (3). Since rad-5 (mn159) is mutating mostly at a single locus, rad-5 (mn159) may be defective in sensing a particular kind of spontaneous DNA damage that occurs at this locus. Thus, the RAD-5 checkpoint protein may be a primary 'sensor' of DNA damage, a protein whose identity has eluded the DNA damage checkpoint community for many years. 1. Gartner, A, Milstein, S, Ahmed, S, Hodgkin, J, and Hengartner, MO (2000). A conserved checkpoint pathway mediates DNA damage-induced apoptosis and cell cycle arrest in C. elegans . Molecular Cell 5: 435-443. 2. Ahmed, S, and Hodgkin, J (2000). MRT-2 checkpoint protein is required for germline immortality and telomere replication in C. elegans . Nature 403: 159-164. 3. Hartman, PS and Herman RK (1982). Radiation-sensitive mutants of C. elegans . Genetics 102: 159-178.

Wu Y-C et al. (1997) International C. elegans Meeting "TUNELING FOR CORPSES IN C. ELEGANS"

Horvitz HR, Wu Y-C, Stanfield GM

[International C. elegans Meeting, 1997]

The principal assay for cell death in C. elegans has been light microscopic observation using Nomarski optics of dying cells, which are readily distinguishable by their raised, refractile appearance. Some cell- death mutants affect the morphology or time of appearance of cell corpses as visualized by Nomarski optics, so it would be useful to have additional markers to follow the progression of the cell-death process. We have adapted the TUNEL (TdT-mediated dUTP Nick End Labeling) technique (1) for studies in C. elegans. TUNEL has been widely used to identify cell corpses in other organisms. TUNEL specifically labels DNA ends, which are more abundant in cell corpses as a consequence of DNA degradation. Thus, in C. elegans, TUNEL should provide both an additional marker for cell corpses and a tool for studying the control of DNA degradation during programmed cell death. We found that TUNEL stains only a small subset of the cell corpses visualized in wild-type embryos using Nomarski optics. However, nuc-1 embryos, which are defective in some aspects of DNA degradation (2,3,4), contain many persistent TUNEL-positive nuclei. Since ced-3, ced-4, and ced-9(gf) mutations significantly reduce TUNEL-staining in a nuc-1 background, the TUNEL-positive nuclei are likely to be those of cell corpses. We propose that DNA degradation is normally very rapid in dying cells, that only certain transient intermediates are TUNEL-reactive, and that mutations in nuc-1 may block DNA degradation from proceeding beyond the TUNEL-reactive stage(s). If so, DNA degradation may involve multiple steps, only some of which are mediated by nuc-1. We have begun to use the TUNEL technique to stain other cell-death mutants. We have found that some mutants with previously indistinguishable engulfment-defective phenotypes have distinct TUNEL-staining patterns. These results indicate that some steps in DNA degradation are dependent on engulfment gene function. (1) Gavrieli et al., J. Cell Biol. 119, 493, 1992 (2) Sulston, Philos. Trans. R. Soc. London 275, 287, 1976 (3) Hedgecock et al., Science 220, 1277, 1983 (4) Hevelone & Hartman, Biochem. Genetics 26, 447, 1988

Jonathan W. Astin et al. (2006) European Worm Meeting "Defects in C. elegans Nucleotide Excision Repair Lead to Hypermutability, Growth Arrest and Degradation of RNA Pol II"

Patricia E. Kuwabara, Nigel J. O'Neil, Jonathan W. Astin

[European Worm Meeting, 2006]

. In eukaryotes, UV-induced photolesions are repaired through the multistep nucleotide excision repair (NER) pathway. In humans, the absence of NER activity results in the disorder xeroderma pigmentosum (XP). XP patients suffer from extreme photosensitivity, a high incidence of skin cancer and neurological defects. Most studies of NER have employed the single-celled yeast or somatic cell lines. To understand how defects in NER affect the development and survival of a multicellular organism, we have characterised a UV hypersensitive and NER defective mutant, rad-3 [1].. We have established that the rad-3 gene encodes the human XPA homolog (K07G5.2), a DNA-binding protein that is essential in the early steps of NER. We also obtained a deletion mutant from the KO consortium, xpa-1(ok698). Both alleles of xpa-1 are likely to be null because they encode truncated proteins, which are missing essential domains. In the absence of UV exposure, xpa-1 worms develop normally; we have shown that xpa-1(ok698) mutants have a low rate of spontaneous mutation and are not hypersensitive to reactive oxygen species (ROS). By contrast, when xpa-1 animals are exposed to UV irradiation, they undergo an immediate growth arrest and decline in survival, and become hypermutable. Surprisingly, transcriptionally quiescent dauer stage larvae survive after receiving UV doses that are lethal to worms at other stages of development. We have successfully rescued the UV sensitivity of both xpa-1 mutants using a XPA-1::GFP construct; XPA-1::GFP is expressed in nuclei throughout development, and in most tissues.. We further show that the UV-induced growth arrest and subsequent death of xpa-1 worms is correlated with transcriptional inhibition. This inhibition is probably due to the stalling of transcription complexes on unrepaired UV-lesions, a phenomenon that leads to the proteolytic degradation of the large subunit of RNA Polymerase II [2]. In support of this hypothesis we show that the steady-state levels of Pol II decline in xpa-1 worms after UV exposure. Finally, we demonstrate that the degradation of Pol II is dependent on the activity of an Rsp-5 like E3 ubiquitin ligase, encoded by the wwp-1 gene. The UV sensitivity of the wwp-1 xpa-1 double mutant is greater than either single mutant alone. These results, together with studies in yeast, establish that the ubiquitylation and degradation of Pol II aids the survival of organisms after they are exposed to UV irradiation. [1] Hartman et al. Genetics 122, 379-85. (1989), [2] Ratner et al. J Bio Chem 273, 5184-89. (1998).

Jonathan W Astin et al. (2005) International Worm Meeting "UV irradiation induces a transcriptional block in C. elegans deficient in nucleotide excision repair."

Nigel J O'Neil, Patricia E Kuwabara, Jonathan W Astin

[International Worm Meeting, 2005]

Many pathways have been identified in eukaryotes that are responsible for the repair of damaged DNA. In humans, repair of UV-induced photolesions is dependent on the multistep nucleotide excision repair (NER) system. An absence of NER activity in humans results in the disorder xeroderma pigmentosum (XP). XP patients suffer from extreme photosensitivity, a high incidence of skin cancers and neurological defects. Most studies of NER have employed yeast or somatic cell lines; hence, we are interested in understanding how defects in NER affect the development and survival of a multicellular organism, such as C. elegans.rad-3(mn157) mutants are hypersensitive to UV because they are defective in NER [1]. We have established that the rad-3 gene encodes the human XPA homolog (K07G5.2), a DNA-binding protein that is essential in the early steps of NER. We also obtained a K07G5.2 deletion mutant from the KO consortium (ok698). Both rad-3 alleles are likely to be null because they encode truncated proteins, which are missing essential XPA domains. We have successfully rescued the UV sensitivity of both rad-3 mutants using a rad-3::gfp construct; RAD-3::GFP is expressed in nuclei throughout development, and in most tissues.UV irradiation of rad-3 mutants leads to an arrest in growth and a decrease in survival. We show that the growth arrest in rad-3 mutants after exposure to UV is due to an inhibition in cell division. In addition we find that the lifespan of rad-3 mutants is reduced after UV treatment throughout all stages of development; however lifespan is most severely reduced during transcriptionally active stages, such as the late embryo and L1. By contrast, transcriptionally quiescent dauers remain healthy. We postulate that the death of rad-3 mutants following UV exposure is due to the stalling of transcription complexes on unrepaired UV-lesions leading to the degradation of RNA pol II (AMA-1)[2]. We present three lines of evidence to support this model: 1) as discussed above, rad-3 mutants display a heightened hypersensitivity to UV during transcriptionally active stages of development 2) the ability to initiate transcription is lost and 3) AMA-1 is preferentially degraded in rad-3 mutants after UV exposure. We believe our results are the first to highlight the effects of UV damage on transcription and its consequences on the survival and development of a multicellular organism. [1] Hartman et al. Genetics 122, 379-85. (1989) [2] Ratner et al. J Bio Chem 273, 5184-89. (1998)

Stanfield GM et al. (1998) East Coast Worm Meeting "DNA DEGRADATION DURING PROGRAMMED CELL DEATHS"

Horvitz HR, Tsung N, Stanfield GM

[East Coast Worm Meeting, 1998]

One hallmark of programmed cell death is the degradation of the DNA of cell corpses by endonuclease(s). This degradation can be detected either by analyzing internucleosomal DNA fragmentation on agarose gels or by TUNEL (TdT-mediated dUTP Nick End Labeling) staining in situ (1). One gene, nuc-1, is known to be required for the elimination of cell corpse DNA in C. elegans. Mutations in nuc-1 result in the persistence of the DNA of dead, engulfed cells and also prevent the breakdown of bacterial DNA in the lumen of the gut (2,3). An endonuclease activity present in extracts from wild-type worms is greatly reduced in or absent from nuc-1(e1392am), nuc-1(n334) and nuc-1(n887) mutant extracts (4). Very few corpses are TUNEL-reactive at any stage in wild-type embryos, but embryos mutant for any of the three nuc-1 alleles have an increased number of TUNEL-staining corpses and cell corpse DNA persists in a TUNEL-positive state (Y.-C. Wu, G. Stanfield and B. Horvitz, unpublished results). These data suggest that DNA degradation is initiated in nuc-1 embryos, perhaps by an endonuclease functioning upstream of nuc-1, and that degradation proceeds more slowly in a nuc-1 mutant than in a wild-type animal. nuc-1 could encode either an endonuclease or a positive regulator of DNA degradation. To understand better the control of DNA degradation during programmed cell death, we are trying to clone the nuc-1 gene. nuc-1 lies between sma-5 and lin-2 on LGX. We are mapping nuc-1 more finely and attempting transformation rescue with cosmids from the region. We are also examining candidate genes from other organisms. Of the various endonuclease activities implicated in DNA degradation during programmed cell death, perhaps the best candidate for having a nuc-1-like activity is either mouse CAD (caspase-activated deoxyribonuclease) or mouse ICAD (inhibitor of CAD) (5). CAD activity is inhibited in living cells via association with ICAD; however, ICAD is required for functional CAD synthesis. When ICAD is cleaved by caspase-3, a CED-3-like protease, CAD is released and DNA is degraded (6). We are testing whether expression of CAD or ICAD can rescue the Nuc-1 mutant phenotype. (1) Gavrieli et al., J. Cell Biol. 119, 493, 1992 (2) Sulston, Philos. Trans. R. Soc. London 275, 287, 1976 (3) Hedgecock et al., Science 220, 1277, 1983 (4) Hevelone & Hartman, Biochem. Genetics 26, 447, 1988 (5) Enari et al., Nature 391, 43, 1998 (6) Sakahira et al., Nature 391, 96, 1998