Sanchez, A.R. et al. (2011) International Worm Meeting "Gene by environment interaction reveals proteostasis sub network for adult health and longevity."

Larsen, P.L., Takano, S., Sanchez, A.R.

[International Worm Meeting, 2011]

Cool core-body temperature promotes longevity in mammals, fishes, flies and worms. Molecular mechanisms underlying this cool-mediated longevity are not well understood. Mutations in the daf-2 gene increase adult lifespan. In C. elegans, wild-type adult maximal lifespan is doubled at 15 deg C compared to 25 deg C, but this pattern is aberrant in adults bearing mutations in the daf-2 gene. We investigated the gene by environment interaction using microarrays on samples from wild-type or daf-2(m577, m41, e1370, m579 or m596) adults acclimated to 15 deg C or 25 deg C. Differentially expressed (DE) genes revealed five categories: pro-longevity, pro-aging, context-specific-pro-longevity, context-specific-pro-aging, and irrelevant. DE genes lower at 25 deg C than 15 deg C included particular protein synthesis and lysosomal proteolysis genes. Adult lifespan was increased at 25 deg C and decreased lifespan at 15 deg C when treated with pharmacological lysosomal inhibitors. Lysosomal protease RNAi caused paralysis in middle-aged adults only at 15 deg C. RNAi of protein synthesis genes caused an increased lifespan in wild-type adults only at 25 deg C and vulval herniation or paralysis in middle-aged adults only at 15 deg C. Together, these results indicate that temperature-dependent regulation of the proteostatic network is adaptive for long-term healthy adult survival. Aspects of temperature regulation were retained in daf-2 adults. These mutants were hyper-responsive to temperature at the molecular level. There were 338 temperature-responsive genes in wild type, whereas the number in the mutants ranged from 902 to 3410 DE genes. Of the wild-type DE protein synthesis genes, 22 of 26 showed temperature regulation in the mutants, with four elevated at 25 deg C rather than lowered. There were 615 temperature DE genes shared in mild daf-2 alleles and 653 genes in severe daf-2 alleles. An additional 38 genes became DE in daf-2 mutants. Temperature regulation of 10 of 15 lysosomal proteases was lost in daf-2 mutants with elevated levels at 25 deg C correlating with allele severity. RNAi of protein synthesis genes in daf-2 mutants dramatically increased lifespan at 25 deg C with no increase in lifespan at 15 deg C. RNAi of a lysosomal protease increased lifespan in the daf-2 mutants at 15 deg C and decreased lifespan at 25 deg C. This suggests one consequence of the dysregulated daf-2 transcriptome results in a pro-aging liability at both temperatures. In summary, we have identified proteostatis network profiles which favor long-term healthy survival given a genotype and environmental condition.

Chen, P. et al. (2009) International Worm Meeting "The Early-Onset Torsion Dystonia Gene Product, torsinA, is a Mediator of ER Stress and Protein Homeostasis."

Roberts, N., Porter, J.C., Burdette, A.J., Berkowitz, L.A, Ricketts, J.C., Caldwell, K.A., Caldwell, G.A., Chen, P., Fox, S.A.

[International Worm Meeting, 2009]

The capacity of cells to carry out their various functions is wholly dependent upon efficient protein synthesis, processing, trafficking, and degradation. In this context, dysfunction or deficit of proteins that monitor and ensure the proper maintenance of cellular homeostasis can have serious consequences in terms of disease onset, penetrance, or progression. A single codon deletion (GAG = DE) in the gene encoding human torsinA causes a non-degenerative neurological disorder termed early-onset torsion dystonia. TorsinA, an endoplasmic reticulum (ER) resident protein, belongs to the diverse AAA+ (ATPases Associated with a variety of cellular Activities) family. Here we use a well established ER stress model in the nematode C. elegans (hsp-4::GFP) to investigate the role of torsinA in maintaining protein homeostasis in the ER. Using GFP as an ER stress reporter, we found that transgenic nematodes expressing wild type (WT) human torsinA exhibited a striking reduction in ER stress caused by tunicamycin, while animals with either DE or a mixture of WT/DE torsinA failed to do so. Interestingly, DE and WT/DE torsinA worms exhibited higher ER stress than WT torsinA animals, even in the absence of tunicamycin treatment. In addition, mutations within torsinA ER signal sequence and N-terminal hydrophobic region abolished its capacity to maintain an ER stress threshold against cellular stressors, indicating the importance of its localization to the ER for this activity. Furthermore, although torsinA possesses very low ATPase activity, the ATPase domain appeared important for ER stress suppression, as two different mutations within the ATPase domain, K108A and E171Q, resulted in loss of stress suppression. A single nucleotide polymorphism (SNP), D216H, diminishes the penetrance of dystonia from 30-40% (WT/DE) to 3% (D216H/DE) in patients. In C. elegans, coexpression of DE torsinA with D216H torsinA significantly protected worms from ER stress caused by tunicamycin, thereby recapitulating the effect on penetrance observed with patients. In order to find genetic interacting factors of torsinA, a small scale of RNAi screen for altered ER stress levels in DE torsinA worms, was carried out. Positives from the screen included glutaredoxin, H3 histones, and 13 core components and regulators of Ras signaling. Taken together, our data indicate that torsinA serves to maintain protein homeostasis in the ER, shedding new light on the pathophysiology of torsion dystonia.

McReynolds, Melanie et al. (2017) International Worm Meeting "Eukaryotic de novo NAD+ biosynthesis from tryptophan in the absence of a QPRTase homolog."

Wang, Wenqing, Holleran, Lauren, Hanna-Rose, Wendy, McReynolds, Melanie

[International Worm Meeting, 2017]

NAD+ is found in all living cells, and is an essential coenzyme, which impacts the entire metabolome. Hence, NAD+ biosynthesis has proven to be an attractive and promising therapeutic target for influencing health-span and obesity-related phenotypes as well as tumor growth. A wide range of animals and yeast synthesize NAD+ via de novo synthesis from the degradation of tryptophan (Trp), via the kyneurine pathway. The C. elegans' genome encodes all the enzymes involved in the kynurenine pathway. However, the genome lacks the critical quinolinic acid phosphoribosyltransferase (QPRTase) that converts QA into NaMN for biosynthesis of NAD+. Because C. elegans lack an apparent QPRTase homolog, it's been assumed that this species lack active NAD+ de novo synthesis. We've uncovered evidence that suggests NAD+ de novo synthesis is active and contributing to NAD+ biosynthetic capacity in C. elegans. We previously found that phenotypes that are dependent on NAD+ levels could be reversed upon supplementation with QA and other kynurenine metabolites, suggesting that the kyneurine pathway may contribute to NAD+ biosynthesis. We have directly demonstrated that isotopic label from Trp is incorporated into NAD+. Furthermore, we show that loss of kynurenine pathway activity decreases global NAD+ levels and prevents incorporation of isotopic label supplied from Trp into NAD+, supporting the presence of de novo synthesis in C. elegans. Using genetics and isotope tracing experiments, we determined that the enzyme UMPS-1, (uridine monophosphate synthetase), is required for NAD+ biosynthesis from Trp in this organism. Finally, we connected the kyneurine pathway to normal fecundity. This evidence demonstrates that NAD+ de novo synthesis is active and contributes to NAD+ biosynthetic capacity and homeostasis in C. elegans. Intriguingly, a conserved enzyme is substituting for the missing QPRTase, raising questions about the relevance of similar underground metabolic activity in higher organisms.

Fern P Finger et al. (2001) International C. elegans Meeting "Involvement of septins in development of the pharynx"

John G White, Fern P Finger

[International C. elegans Meeting, 2001]

The septins are a family of proteins often essential for cytokinesis in organisms ranging from yeast to mammals. They are also expressed in some post-mitotic cells, indicating that they function in other cellular processes . Two septins, encoded by the unc-59 and unc-61 genes, were recently identified in Caenorhabditis elegans , and have been described as having no essential function in embryogenesis . Many of the septin mutant phenotypes are common, but not restricted, to mutants affecting cell division. These include defects in the male tail, protruding vulvae, uncoordination, and gaps in the alae. A new phenotype that we found at low penetrance for the septin mutants, defects in the hermaphrodite tail, has not been previously described for cell cycle mutants, but is seen in a hypodermal fusion mutant, as are male tail and vulval defects (W. Mohler, personal communication). We now find that loss of septin function results in approximately 20% embryonic lethality. Of those larvae that hatch, up to 50% do not survive the first larval stage. Closer examination revealed that the L1 lethality was due to defects in the formation of the pharynx. The defects range from pharynges that do not appear to have properly elongated and have not attached to the buccal cavity, to pharynges that appear morphologically normal, but are detached, to those that are blocked by deposition of cuticle in the buccal cavity. L1 lethality in all cases results from the worms’ inability to feed. This phenotype has not been described before for mutations affecting the cell cycle or cell fusions, and may indicate a direct role for the septins in organogenesis of the pharynx. We are currently using pha-4::GFP::CAAX strains (gift of Susan Mango) to determine if these defects result from failures in cytokinesis, and performing indirect immunofluorescence to localize the septins in the developing pharynx. Preliminary results suggest that, in the case of the most extreme mutant phenotype, there are no obvious early failures in cell division. By analogy to studies on septin function in other organisms, potential roles for the septins in morphogenesis include polarized secretion, activation or assembly of machinery necessary for the synthesis or assembly of cuticle structures, a scaffold for assembly of other proteins (such as signaling molecules), interaction with the actin cytoskeleton, and the specification of membrane subdomains. Longtine, M. S., DeMarini, D. J., Valencik, M. L., Al-Awar, O. S., Fares, H., De Virgilio, C. & Pringle, J. R. (1996) Curr. Opin. Cell Biol. 8, 106-119. Nguyen, T. Q. & White, J. G. (2000) J. Cell Sci. 113, 3825-3837.

Wang, Wenqing et al. (2013) International Worm Meeting "New path to NAD+: In addition to salvage biosynthesis, NRK and de novo NAD+ synthesis contribute to NAD+ recycling in C. elegans."

Lynes, Matthew R., Wang, Wenqing, Hanna-Rose, Wendy

[International Worm Meeting, 2013]

NAD+ is a vital molecule in cellular redox reactions and acts as a cosubstrate for NAD+ consuming enzymes, which are critical to aging, stress response and energy metabolism homeostasis. NAD+ salvage biosynthesis is an important pathway to recycle the nicotinamide (NAM) liberated by NAD+ consumers to rebuild NAD+ reservoir. Mutation of PNC-1, a nicotinamidase in the NAD+ salvage pathway in C. elegans, results in a series of defects that are individually linked to NAD+ insufficiency or NAM accumulation[1, 2]. Specifically, NAD+ insufficiency in pnc-1 causes gonad developmental delay. Our recent mass-spectrometry data showed that there is a drastic 18-fold increase in NAM levels following pnc-1 loss of function. However, NAD+ levels in pnc-1 mutant only decrease by 30%. These results led us to hypothesize that there are other NAD+ synthesis pathways contributing to NAD+ synthesis in C. elegans. Here we tested two potential pathways that have been studied in yeast: the NRK pathway and de novo NAD+ synthesis. The NRK pathway uses nicotinamide riboside kinase (NRK) to convert nicotinamide riboside (NR) from the diet to nicotinamide mononucleotide, which can be processed into NAD+. Mutation of NRK-1 exacerbates gonad developmental delay in pnc-1. In addition, supplementing NR rescues gonad developmental delay in pnc-1 mutants, but not in pnc-1;nrk-1 double mutants. NAD+ can also be synthesized de novo from tryptophan in yeast. By BLAST, we found that C. elegans lacks a key enzyme in de novo NAD+ synthesis, quinolinic acid phosphoribosyl transferase (QPRTase). Surprisingly, supplementing quinolinic acid (QA), the substrate of QPRTase, to pnc-1 mutants rescues gonad developmental delay. Moreover, RNAi of tryptophan 2,3-dioxygenase, another enzyme in the de novo NAD+ synthesis pathway, exacerbates gonad developmental delay. In conclusion, our results suggest that both NRK and de novo NAD+ biosynthesis pathways actively contribute to NAD+ synthesis in C. elegans. Despite the fact that a QPRTase homologue is missing in C. elegans, it is possible that a novel mechanism converts QA into NAD+. We are working to identify this new mechanism. Reference: 1. Vrablik et al. Development 2009 2. Vrablik et al. Dev Biol. 2011.

McReynolds, Melanie et al. (2015) International Worm Meeting "Maintaining Global NAD+ Homeostasis Reveals Separable Functional and Compensatory Roles for NAD+ Biosynthetic Pathways in C. elegans."

Wang, Wenqing, Hanna-Rose, Wendy, McReynolds, Melanie

[International Worm Meeting, 2015]

NAD+ biosynthesis has proven to be a useful therapeutic target for influencing health-span and obseity-related phenotypes as well as tumor growth. The goal of our research is to understand how NAD+ homeostasis is maintained to support its core metabolic roles and its signaling and regulatory roles involving NAD+ consumers. NAD+ is synthesized via distinct routes including de novo synthesis from tryptophan, salvage synthesis from nicotinamide, which feeds into the Preiss-Handler pathway from nicotinic acid in C. elegans, and via the phosphorylation of nicotinamide or nicotinic acid ribosides using nicotinamide riboside kinase (NRK). We hypothesize that each pathway will have tissue-specific functions independent of the others and that they will also have compensatory roles in contributing to global NAD+ homeostasis when another pathway is compromised, creating a homeostatic network. We have previously shown, using pnc-1 nicotinamidase mutants, that salvage NAD+ biosynthesis is required for the appropriate temporal progression of reproductive development and for optimum muscle function. Here we examine functions of the NRK and the de novo pathways using genetics. We find that both pathways function to maintain muscle activity, but neither pathway has an independent role in gonad development. Using LC-MS we show that NAD+ is decreased when either pathway is blocked, as previously shown for PNC-1. This data supports the hypothesis that all pathways are required for maintenance of NAD+ levels and for muscle function but only the salvage pathway normally plays a role in gonad development. To examine homeostatic mechanisms, we investigated the function of the NRK and de novo pathways in a pnc-1 mutant background. Mutations in either pathway further exacerbate pnc-1-associated muscle defects, but not the reproductive development defect. We revealed using qRT-PCR that in the absence of salvage synthesis, key enzymes in de novo and NRK-mediated synthesis are up-regulated. We also observed reciprocal trends when the de novo or NRK pathways were blocked. Consistent with these results, supplementation with either nicotinamide riboside or de novo pathway metabolites rescues the gonad delay in pnc-1, indicating that boosting other pathways can restore gonad development when salvage synthesis is blocked, even though these pathways play no role in gonad development normally. We interpret these data as evidence that homeostatic mechanisms are indeed occurring between NAD+ biosynthetic pathways. Our work has implications for efforts to therapeutically target individual NAD+ biosynthetic pathways. .

Carissa Perez et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "A Novel 13C Isotope Labeling Strategy Identifies Insulin/IGF Receptor Mutations that Selectively Modulate de novo Fatty Acid Synthesis and Longevity"

Marc Van Gilst, Carissa Perez

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Although studies in C. elegans have identified numerous genes and pathways involved in fat storage, it is now important to determine how these factors affect in vivo lipid metabolism. We developed a 13C-isotope assay to quantify the contribution of dietary fat absorption and de novo fatty acid synthesis to triglyceride and phospholipid synthesis in C. elegans. We employed this method to define the impact of insulin/IGF signaling on fat storage and membrane synthesis. Worms harboring the long-lived daf-2(e1370) insulin receptor mutation contained significantly higher levels of synthesized fats in triglycerides and phospholipids. Elevated fat synthesis was completely dependent upon the DAF-16/FoxO transcription factor. Intriguingly, other long-lived alleles of daf-2 did not increase de novo fatty acid synthesis, suggesting that site-specific mutations in the insulin receptor can selectively modulate longevity and fat metabolism. Additionally, we have begun to employ similar labeling strategies to monitor the turnover of fatty acid molecules found in triglyceride stores as well as phospholipid membranes. These stable isotope-labeling assays permit measurement of nutrient flux in C. elegans and present a strategy for combining quantitative metabolic studies with genetic analysis.

Charles Baer (2008) Development & Evolution Meeting "Quantifying the De-canalizing Effects of Spontaneous Mutations in Rhabditid Nematodes"

Charles Baer

[Development & Evolution Meeting, 2008]

The evolution of canalization - the robustness of the phenotype to environmental or genetic perturbation - has attracted considerable recent interest. A key step toward understanding the evolution of any phenotype is to characterize the rate at which mutation introduces genetic variation for the trait (the mutational variance, VM) and the average directional effects of mutations on the trait mean (deltaM). In this study, the mutational parameters for canalization of productivity and body volume are quantified in two sets of mutation accumulation lines of nematodes in the genus Caenorhabditis and compared to the mutational parameters for the traits themselves. Four results emerge: (1) spontaneous mutations consistently de-canalize the phenotype; (2) the mutational parameters for de-canalization - VM (quantified as mutational heritability) and deltaM - are of the same order of magnitude as the same parameters for the traits themselves; (3) the mutational parameters for canalization are roughly correlated with the parameters for the traits themselves across taxa; and (4) there is no evidence that residual segregating overdominant loci contribute to the decay of canalization. These results suggest that canalization is readily evolvable, and that any evolutionary factor that causes mutations to accumulate will, on average, de-canalize the phenotype.

Carissa L. Perez et al. (2007) International Worm Meeting "Using Metabolic Tracers to Quantify Fatty Acid Synthesis, Absorption, and Expenditure in C. elegans."

Marc Van Gilst, Carissa L. Perez

[International Worm Meeting, 2007]

Although C. elegans is being recognized as a potent genetic tool to study the relationship of nutrition to growth and aging, surprisingly little is known about how worms utilize dietary molecules. Therefore, we have established a stable isotope enrichment strategy to characterize the ingestion and metabolism of dietary nutrients in developing and aging animals. Here, we present a study using 13C labeled bacteria to determine the relative abundance of dietary and de novo synthesized fatty acids in worm lipids using mass spectrometry. Quantitative analysis of 13C enrichment in feeding worms revealed that most lipid species; e.g., triglycerides, phospholipids, sphingolipids, etc., are primarily composed of fatty acids directly absorbed from the bacterial food source. Therefore, it is clear that direct incorporation of dietary fats plays a significant role in the formation of fat stores and lipid membranes. Because our labeling approach is effective at quantifying both dietary and synthesized fatty acids, we are now exploiting this strategy to identify genetic factors that impact de novo fat synthesis, dietary fat absorption, and/or fat expenditure. Thus far, we have surveyed genes known to significantly affect overall fat storage in nematodes to specifically identify factors that affect fatty acid synthesis. We found that mutations in the daf-2 gene result in a dramatic increase in the de novo synthesis of fatty acids, which likely explains the high fat phenotype of these mutants. In contrast, mutations in eat-2, which cause caloric restriction, lead to a significant decrease in de novo fat synthesis. We are continuing this approach to examine other genes known to affect overall fat storage. We expect our novel labeling strategy to generate new insights into how worms incorporate and process their dietary fats and other nutrients.

Gert de Voer et al. (2006) European Worm Meeting "Overexpression of Subunit C, the Main Component of the Storage Material in Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), Causes Disruption of Mitochondria in C. Elegans and Subsequent Death"

Gert de Voer, Gert-Jan B. van Ommen, Ronald O.B. de Keizer, Peter E.M. Taschner, Paola van der Bent, Dorien J.M. Peters

[European Worm Meeting, 2006]

Overexpression of Subunit C, the Main Component of the Storage Material in Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), Causes Disruption of Mitochondria in C. Elegans and Subsequent Death. Gert de Voer, Ronald O.B. de Keizer, Paola van der Bent, Gert-Jan B. van Ommen, Dorien J.M. Peters, and Peter E.M. Taschner . Subunit c of the mitochondrial ATP synthase (subunit c) normally is present in the F0 part of the ATP synthase complex in mitochondria. The very hydrophobic subunit c also forms the main component of the lysosomal storage material found in many forms of the hereditary neurodegenerative disorders called Neuronal Ceroid Lipofuscinosis (NCL, Batten disease). The juvenile form of NCL is caused by mutations in the CLN3 gene, which has three homologs in C. elegans. In order to obtain more insight in the etiology of juvenile NCL and how the mitochondrial subunit c protein ends up in large quantities in lysosomes, we have constructed cln-3 triple mutants, in which all three cln-3 genes have been mutated. The cln-3 triple mutants had a decreased life span and brood size, but no lysosomal storage was observed, probably due to the relatively short life span of the worm. We hypothesized that overexpression of subunit c might directly or indirectly induce lysosomal storage in C. elegans and potentially lead to a phenotype useful for genetic screens. Therefore, we identified the atp-9 gene, which encodes the C. elegans subunit c protein, and generated transgenic worms carrying an hsp-16.2 promoter-atp-9 fusion construct. Induction of subunit c overexpression by a 2-hr heat shock causes the nematodes to disintegrate, presumably as a result of disrupted mitochondria. Electron micrographs of transgenic worms show altered mitochondria after induction of subunit c overexpression, but no lysosomal storage was detected irrespective of a wildtype or cln-3 triple mutant background. Milder induction of overexpression affects the reproduction and the morphology of the worms.. This work was financially supported by the Center for Biomedical Genetics, the Batten Disease Support and Research Association, and the European Union, EU project NCL models (EU LSHM-CT-2003-503051).