Kawata, Konami et al. (2019) International Worm Meeting "C. elegans calcineurin modulates lifespan via SKN-1 signaling."

Blackwell, T. Keith, Kawata, Konami, Mizunuma, Masaki, Ogawa, Takafumi

[International Worm Meeting, 2019]

Much of our understanding of aging derives from studies in model organisms, including yeasts, worms and mice. However, to date the causes of aging and determinants of lifespan are not fully understood. SKN-1 is the C. elegans ortholog of the mammalian Nrf proteins, which defend against oxidative and proteotoxic stress. SKN-1 is important in several scenarios of lifespan extension, including reduced TORC1/2 or insulin/IGF-1 signaling, and dietary restriction. Much remains to be learned about how specific phosphorylation events regulate SKN-1, and that little is understood about how SKN-1 activity might be regulated by specific phosphatases. To investigate how SKN-1 activity might influenced by phosphatases, we performed RNAi against predicted C. elegans phosphatases. We first monitored expression of a transgene in which the promoter for the SKN-1 target gene gcs-1 (?-glutamyl cysteine synthetase) is fused to GFP. Among phosphatases we have tested, RNAi against calcineurin (TAX-6) upregulated gcs-1p::GFP in the anterior intestine. Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine protein phosphatase that regulates cellular Ca2+ signaling responses. We next investigated whether TAX-6 knockdown influences the localization of SKN-1. TAX-6 knockdown dramatically increased SKN-1 accumulation in intestinal nuclei. We further show that TAX-6 RNAi causes an increase of mean lifespan, reduced both brood size and body length. Lifespan extension by TAX-6 RNAi requires SKN-1 but does not depend on the DAF-16/FoxO transcription factor. However, surprisingly, TAX-6 RNAi animals show reduced resistance to thermal- and oxidative stress. We are currently investigating how calcineurin affects lifespan and oxidative stress resistance through SKN-1.

Susoy, Vladislav et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "EVOLUTIONARY DIVERSIFICATION THROUGH THE GAIN AND LOSS OF DEVELOPMENTAL PLASTICITY"

Sommer, Ralf J., Susoy, Vladislav, Ragsdale, Erik J.

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Developmental plasticity is common among plants and animals, yet its role in mediating evolutionary processes is debated. Several species of Rhabditina, including Pristionchus pacificus, can execute two alternative mouth phenotypes, one of which is associated with predatory feeding. Studies in P. pacificus have revealed that environmental factors act through a conserved genetic pathway that enables a rapid developmental response to the environment (Bento et al., 2010). In addition, a developmental switch that controls the expression of the alternative mouth phenotypes was recently identified (Ragsdale et al., 2013). In contrast, little is known about the macroevolutionary potential of the mouth plasticity. We studied the relationship between plasticity and morphological change by a macroevolutionary analysis of nematode mouthparts when accompanied by a dimorphism. To test whether plasticity facilitates or hinders morphological change, we analyzed variation in form and complexity in 90 nematode species with or without a mouth dimorphism. Our analyses revealed a two-step process of morphological diversification associated with the gain and loss of plasticity. First, acquisition of a dimorphism was accompanied by an increase in complexity, including structural innovations such as moveable teeth. Second, the fixation of a single mouth-phenotype in several nematode lineages was associated with a decrease in mouth complexity but a sharp increase in evolutionary rates when measured as change of shape and size. Thus, plasticity facilitates phenotypic diversification by fostering evolutionary novelties, whereas subsequent loss of the dimorphism enables acceleration of evolution by releasing novel morphologies from developmental constraints. 1. G. Bento, A. Ogawa, R. J. Sommer. Nature 466, 494-497 (2010). 2. E. J. Ragsdale, M. R. Muller, C. Rodelsperger, R. J. Sommer, Cell 155, 922-933 (2013).

He XY et al. (1998) J Biol Chem "A human brain L-3-hydroxyacyl-coenzyme A dehydrogenase is identical to an amyloid beta-peptide-binding protein involved in Alzheimer's disease."

He XY, Schulz H, Yang SY

[J Biol Chem, 1998]

A novel L-3-hydroxyacyl-CoA dehydrogenase from human brain has been cloned, expressed, purified, and characterized. This enzyme is a homotetramer with a molecular mass of 108 kDa. Its subunit consists of 261 amino acid residues and has structural features characteristic of short chain dehydrogenases. It was found that the amino acid sequence of this human brain enzyme is identical to that of an endoplasmic reticulum amyloid beta-peptide-binding protein (ERAB), which mediates neurotoxicity in Alzheimer's disease (Yan, S. D., Fu, J., Soto, C., Chen, X., Zhu, H., Al-Mohanna, F., Collison, K., Zhu, A., Stern, E., Saido, T., Tohyama, M., Ogawa, S., Roher, A., and Stern, D. (1997) Nature 389, 689-695). The purification of human brain short chain L-3-hydroxyacyl-CoA dehydrogenase made it possible to characterize the structural and catalytic properties of ERAB. This NAD+-dependent dehydrogenase catalyzes the reversible oxidation of L-3-hydroxyacyl-CoAs to form 3-ketoacyl-CoAs, but it does not act on the D-isomers. The catalytic rate constant of the purified enzyme was estimated to be 37 s-1 with apparent Km values of 89 and 20 microM for acetoacetyl-CoA and NADH, respectively. The activity ratio of this enzyme for substrates with chain lengths of C4, C8, and C16 was approximately 1:2:2. The human short chain L-3-hydroxyacyl-CoA dehydrogenase gene is organized into six exons and five introns and maps to chromosome Xp11.2. The amino-terminal NAD-binding region of the dehydrogenase is encoded by the first three exons, whereas the other exons code for the carboxyl-terminal substrate-binding region harboring putative catalytic residues. The results of this study lead to the conclusion that ERAB involved in neuronal dysfunction is encoded by the human short chain L-3-hydroxyacyl-CoA dehydrogenase gene.

Akira Ogawa et al. (2010) East Asia Worm Meeting "Ppa-daf-16 regulates Dauer Formation but not Buccal Cavity Dimorphism in Pristionchus pacificus"

Gabi Bartelmes, Gilberto Bento, Akira Ogawa, Christoph Dieterich, Ralf J. Sommer

[East Asia Worm Meeting, 2010]

Based on knowledge obtained from C. elegans, we are exploring how the regulatory mechanisms for dauer formation evolved, using the genetically tractable nematode Pristionchus pacificus and other nematode species. So far we and others have shown that the nuclear hormone receptor DAF-12 and its ligands represent an evolutionarily conserved mechanism for dauer formation in free-living species as well as infective larva formation in parasitic species [1, 2]. In addition to the developmental plasticity of dauer formation, P. pacificus shows plasticity in buccal cavity (BC) formation in the adult stage. One morph called eurystomatous has a wide and shallow mouth opening and is specialized for predation and fungi-feeding whereas the other morph, stenostomatous, has a narrow and deep mouth opening and is specialized for bacteria-feeding. This dimorphism is regulated by at least two environmental factors including starvation and dauer pheromone [3]. In addition multiple daf-d mutants that include daf-12 strains also show defects in the regulation of BC dimorphism, suggesting that the mechanisms for dauer formation and BC dimorphism are largely overlapping. However, we have found several other strains that show strong daf-d phenotype but normal regulation of BC dimorphism. To identify the gene responsible for these mutations, we sequenced the genome of the mutant strains using Illumina Genome Analyzer and identified mutations in the orthologue of daf-16. Our results suggest that daf-16 represents one of the evolutionarily conserved mechanisms for dauer formation and among the two regulatory mechanisms that involve DAF-12 and DAF-16, only DAF-12 steroid signaling was co-opted for the regulation of BC dimorphism. We are currently investigating how upstream regulators of these two transcription factors evolved. Reference 1. Ogawa et al. (2009). Curr Biol 19, 67-71. 2. Wang et al. (2009). PNAS 106, 9138-9143. 3. Bento et al. Nature in press.

Melanie G. Mayer et al. (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "Natural Variation in Dauer Entry and Dauer Exit in the Nematode Pristionchus pacificus"

Melanie G. Mayer, Akira Ogawa, Ralf J. Sommer

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

Evolutionary ecology investigates how the developmental response to the environment and the ecological interactions of an organism shape the evolution of new phenotypes. Under harsh environmental conditions, the diplogastrid nematode Pristionchus pacificus is able to arrest its development in the third larval stage and form dauer juveniles (Ogawa et al. 2009). The well-known association of P. pacificus with scarab beetles is exclusively in the dauer stage, indicating that the dauer stage is essential not only for enduring unfavorable conditions but also for dispersal. The supernatant prepared from P. pacificus liquid cultures contains dauer pheromone, which induces J2 larvae to enter the dauer stage. To study natural variation in dauer entry, we tested 18 P. pacificus strains with the dauer pheromone of each strain. Interestingly, these strains show a high level of natural variation regarding dauer entry, even in response to the same dauer pheromone. We will report recombinant-inbred-line experiments, which try to identify the molecular basis of natural variation in dauer entry. Furthermore, we investigated the ability of P. pacificus to exit the dauer stage after different periods of time and at different temperatures, an experiment that is still ongoing. As with dauer entry, the 10 tested strains show a wide range of natural variation regarding dauer exit. However, in nearly all of the strains, many dauers were still able to recover and reproduce after 28 weeks at 8C. Some strains display a distinct trade-off between survival (the ability to exit the dauer stage) and recovery (the ability to develop into J4 larvae) or between recovery and brood size. When incubated at higher temperatures, P. pacificus dauers seem to compensate for lower survival and recovery by increasing their brood size. These studies provide a better understanding of the ecologically most important nematode stage, the dauer stage.

Pierce SB et al. (1998) East Coast Worm Meeting "IDENTIFICATION OF A PUTATIVE DAF-2 INSULIN-LIKE LIGAND"

Ruvkun GB, Pierce SB

[East Coast Worm Meeting, 1998]

The activity of daf-2, which encodes a homolog of insulin and insulin-like growth factor I (IGF-I) receptors (1), is required for non-dauer development and normal adult life span, and is likely to be regulated by an insulin-like ligand. By searching the worm database, we determined that the C. elegans genome contains at least eight genes predicted to encode insulin-like peptides, in addition to two previously identified related genes (WBG 15(2): 47). The predicted proteins contain the invariant cysteines and, in some cases, predicted proteolytic cleavage sites that are hallmarks of the insulin superfamily. The gene most closely related to the vertebrate insulins, which is on cosmid F13B12 and was not predicted by the C. elegans Genome Project, was chosen for further analysis. The expression pattern of F13B12(ins) was determined using a GFP transgene. F13B12(ins)::GFP is expressed only in neurons, including a pair of pharyngeal neurons, several pairs of head neurons, and three tail neurons. To investigate whether F13B12(ins) can play a role in dauer formation, the entire F13B12(ins) genomic region was amplified by PCR and used to construct transgenic worms, with the goal of overexpressing F13B12(ins). We hypothesized that, if F13B12(ins) is the DAF-2 ligand, overexpression of F13B12(ins) might suppress the Daf-c phenotype of weak daf-2 mutants or mutants in the parallel daf-7 transforming growth factor-beta (TGF-beta) pathway, which synergizes with the daf-2 insulin-like signaling pathway (2) . The F13B12(ins) transgene was unable to suppress either of two non-null daf-2 alleles (e1370 and e1365). Although these are non-null alleles, there may not be enough extra ligand produced in these worms to overcome the effect of the impaired DAF-2 receptor. However, the F13B12(ins) transgene maternally suppressed the Daf-c phenotype of daf-7(e1372), which encodes a TGF-beta-like ligand (3). Mutations in the unc-64 gene, which encodes a homolog of syntaxin (4), a component of the neurosecretory apparatus, have a Daf-c phenotype at 27 degrees and extended adult life span at 20 degrees. The fact that these phenotypes are suppressed by mutations in daf-16, which also suppress daf-2 mutations, suggests that this may be the result of decreased secretion of the DAF-2 ligand (J. Thomas, personal communication). unc-64(e246); F13B12(ins) worms briefly arrest as dauers at 27 degrees and then, unlike unc-64(e246) control worms, rapidly recover and develop into fertile adults. The expression pattern of F13B12(ins)::GFP and the ability of the F13B12(ins) transgene to suppress dauer formation and promote dauer recovery in daf-7(e1372) and unc-64(e246) mutants, respectively, are consistent with a role for F13B12(ins) in the neurosecretory signaling system that regulates dauer formation. We are continuing to investigate the ability of F13B12(ins) to affect dauer formation and aging and are exploring candidate mutations in the F13B12 region. (1) Kimura, et. al., 1997, Science 277, 942-946. (2) Ogg, et al., 1997, Nature 389, 994-999. (3) Ren, et. al., 1996, Science 274, 1839-1391. (4) Ogawa, et. al., 1998, J. Biol. Chem. 273, 2192-2198.