Osman, Hadley C et al. MicroPubl Biol

Lucanic, Mark, Hall, David, Osman, Hadley C, Sedore, Christine A, Foulger, Anna, Jackson, E Grace, Battistoni, Elena T, Lithgow, Gordon J, Guo, Max, Driscoll, Monica, Phillips, Patrick

[MicroPubl Biol, 2021]

The Caenorhabditis Intervention Testing Program (CITP) is a multi-institutional, National Institute on Aging (NIA)-funded consortium with the goal of identifying compounds that will robustly extend lifespan with reproducible effects across genetically diverse Caenorhabditis species and strains (Lucanic et al., 2017). Compounds are prioritized for testing based on computational prediction for lifespan or healthspan effects (Coleman-Hulbert et al., 2019), predicted or known interactions with documented lifespan-regulating pathways, or previous reports for lifespan or healthspan extension in laboratory animals. In this case, diuron was of interest to CITP based on the report of a positive effect on the lifespan of C. elegans strain TJ1060 (spe-9;fer-15) (Lucanic et al., 2018; personal communication). Diuron (1-(3,4-dichlorophenyl)-3,3-dimethylurea) is an herbicide that inhibits photosynthesis by binding photosystem II (Haynes et al., 2000). Diuron has been previously tested in nematode species for toxicity (Neury-Ormanni et al., 2019) and reproductive effects (Mugova et al., 2018), and in both cases showed detrimental effects at high doses only.

McVeigh P et al. (2008) Int J Parasitol "Neuropeptide-like protein diversity in phylum Nematoda."

Mousley A, Alexander-Bowman S, Maule AG, Marks NJ, Veal E, McVeigh P

[Int J Parasitol, 2008]

This study reports the identification of nematode neuropeptide-like protein (nlp) sequelogs from the GenBank expressed sequence tag (EST) database, using BLAST (Basic Local Alignment Search Tool) search methodology. Search strings derived from peptides encoded by the 45 known Caenorhabditis elegans nlp genes were used to identify more than 1000 ESTs encoding a total of 26 multi-species nlp sequelogs. The remaining 18 nlps (nlp-4, -16, -24 through -36, -39, -41 and -45) were identified only in C. elegans, while the sole EST representative of nlp-23 was from Caenorhabditis remanei. Several ESTs encoding putative antibacterial peptides similar to those encoded by the C. elegans genes nlp-24-33 were observed in several parasite species. A novel gene (nlp-46) was identified, encoding a single, amidated dodecapeptide (NIA[I/T]GR[G/A]DG[F/L]RPG) in eight species. Secretory signal peptides were identified in at least one species representing each nlp sequelog, confirming that all 46 nematode nlp genes encode secretory peptides. A random sub-set of C. elegans NLPs was tested physiologically in Ascaris suum ovijector and body wall muscle bioassays. None of the peptides tested were able to modulate ovijector activity, while only three displayed measurable myoactivity on somatic body wall muscle. AFAAGWNRamide (from nlp-23) and AVNPFLDSIamide (nlp-3) both produced a relaxation of body wall muscle, while AIPFNGGMYamide (nlp-10) induced a transient contraction. Numerical analyses of nlp-encoding ESTs demonstrate that nlp-3, -13, -14, -15 and -18 are amongst the most highly represented transcripts in the dataset. Using available bioinformatics resources, this study delineates the nlp complement of phylum Nematoda, providing a rich source of neuropeptide ligands for deorphanisation of nematode neuropeptide receptors.

Coleman-Hulbert, Anna L. et al. MicroPubl Biol

Guo, Max, Driscoll, Monica, Coleman-Hulbert, Anna L., Lithgow, Gordon J., Phillips, Patrick C., Johnson, Erik, Sedore, Christine A., Banse, Stephen A.

[MicroPubl Biol, 2019]

The CaenorhabditisIntervention Testing Program (CITP) is a National Institutes of Aging (NIA)-funded multi-institutional research consortium that investigates chemical interventions for their potential to extend lifespan and healthspan across a genetically diverse panel of Caenorhabditisnematodes (Lucanic et al.,2017a). To date, the CITP has tested more than 20 compounds, including many of those previously surveyed in mice by the NIA-funded Interventions Testing Program (ITP) (Miller et al.,2007; Nadon et al.,2008). With compounds tested across multiple strains, species, labs, and concentrations, the CITP produces a large body of work before a compound traverses the full testing workflow. The intensive effort required poses an important challenge for the CITP as to how to systematically identify and prioritize compounds to test. Indeed, the selection of chemical interventions that might hold the greatest potential for efficacy is a challenge to the aging field in general. One approach to chemical intervention prioritization is to use computational approaches that rank candidate compounds based on their likelihood to confer longevity benefits. For example, Ziehm et al.(2017) developed a ranking algorithm that combined information on genetic effects on aging, drug-target orthology relationships and sequence conservation, 3D protein structures, drug binding, and bioavailability. Here, we present the results of a test for lifespan effects of imatinib mesylate (a tyrosine kinase inhibitor; commercially known as Gleevec). Imatinib mesylate was the highest scoring drug-like compound with known mammalian targets ranked for likelihood to modulate aging in the invertebrates C. elegansand D. melanogaster (Ziehmet al.,2017). Imatinib was also highly ranked bya separate computational approach that predicted compound interventions for human aging (Fuentealba et al.,2019). We assayed lifespan in response to imatinib mesylate exposure in three Caenorhabditisspecies in triplicate using our previously published workflow (Lucanic et al.,2017a; b). In brief, imatinib mesylate (Toronto Research Chemicals) was dissolved in water and diluted appropriately such that 125 l of solution could be added to 35 mm diameter NGM plates containing 51 m FUdR in order to generate the following final imatinib mesylate concentrations: 0.01 M, 0.1 M, 1.0 M, 10 M, 50 M, 100 M, 500 M, and 1 mM. Worms were age-synchronized by timed egg-lays on standard NGM plates, then transferred at a density of 50 animals per 35 mm plate (control or imatinib mesylate) in triplicate when they reached adulthood. Animals were maintained at 20 C and moved to fresh experimental plates on the first, second, and fifth day of adulthood, then once weekly afterward, and fed on OP50-1 lawns for the duration of the experiments. Thrice weekly, we observed animals for spontaneous movement or movement after gentle perturbation with a 0.2 mm diameter platinum wire. Death was scored as a lack of movement. Our results indicate that imatinib mesylate does not extend lifespan in any of the Caenorhabditisspecies at the concentrations tested here; in fact, at some concentrations, this compound reduced nematode lifespan, although this effect was not consistent among species (Fig. 1). However, as there was only a single biological replicate, this conclusion should be considered as preliminary. Interventions may be ineffective due to a range of causes including permeability barriers, compound stability in vivo, and metabolism by the bacterial food source. While we did not observe any lifespan-extending properties of imatinib mesylate in this study, it is important to note that the ranking methodology used to prioritize imatinib mesylate (Ziehm et al.,2017) predicts whether compounds have aging-modulating effects rather than lifespan-extending properties per se. Notably, Ziehm et al.(2017) identified two aging-associated likely targets of imatinib, ABL1 and MAPK14. InC. elegans,pmk-1(MAPK14 ortholog) deletion mutants exhibit decreased survival compared to wild-type (Parket al.,2018) indicating that inhibition by imatinib mesylate could likewise result in decreased lifespan. In contrast, abl-1(ABL1 ortholog) mutants are resistant to endoplasmic reticulum stress induced by tunicamycin (Judy et al.,2013). Imatinib mesylate may target abl-1as it phenocopies abl-1mutants with respect to cell death phenotypes (Deng et al.,2004). In future studies, it will be important to further characterize whether compounds highly ranked in methodologies, such as the one discussed here, possess health or longevity-promoting effects as well as putative aging-modulating effects.

Morshead, Mackenzie L et al. MicroPubl Biol

Morshead, Mackenzie L, Guo, Max, Plummer, W Todd, Jones, E Grace, Garrett, Theo, Sedore, Christine A, Lithgow, Gordon, Phillips, Patrick C, Driscoll, Monica, Lucanic, Mark, Hall, David

[MicroPubl Biol, 2020]

The Caenorhabditis Intervention Testing Program (CITP) is a multi-institutional, National Institute on Aging (NIA)-funded consortium. The goal of the program is to identify chemical compounds that extend lifespan robustly and reproducibly across genetically diverse Caenorhabditis strains (Lucanic et al. 2017). The CITP test compounds are selected if they are consistently highly ranked via computational prediction for lifespan or healthspan effects (Coleman-Hulbert et al. 2019), if they are predicted or known to interact with known lifespan-regulating pathways, or if they have previously been reported as extending lifespan or healthspan in laboratory animals. Obeticholic acid is an analog of the natural bile acid chenode oxycholic acid, which acts as an agonist of the farnesoid X receptor (FXR) (Neuschwander-Teri et al. 2015), a nuclear receptor (NR) closely involved with hepatic triglyceride homeostasis. Obeticholic acid is most commonly used to treat the autoimmune liver disease, primary biliary cholangitis. The most likely homolog of FXR in C. elegans is DAF-12, which can bind and be activated by human bile acids (Held et al. 2006; Zhi et al. 2011). DAF-12 modulation is of particular interest because it is closely linked to dauer formation, lifespan extension, and metabolism homeostasis (Antebi 2015). We assayed lifespan in response to different concentrations of obeticholic acid exposure in three Caenorhabditis species using the flatbed scanner-based Automated Lifespan Machine (ALM) workflow previously published (Banse et al. 2019). To summarize, the worms were age synchronized by egg-lays on standard 60 mm diameter Nematode Growth Media (NGM) plates with lawns of Escherichia coli OP50-1, and transferred to compound-treated 38 mm NGM plates containing 51 M 5-Fluoro-2-deoxyuridine (FUdR) at a density of 50 worms per plate on day one of adulthood. For treatment plates, we used standardized protocols (Caenorhabditis Intervention Testing Program 2020); in short, obeticholic acid (Apexbio Technology) was dissolved in dimethyl sulfoxide (DMSO) and diluted appropriately such that addition of 7.5 l of stock solution and 125 l water for 35 mm diameter plates, and 17.5 l of stock solution and 232.5 l water for 50 mm diameter scanner plates would generate 50, 100, and 150 M final obeticholic acid concentrations. For control plates, DMSO was added instead of stock solution using the same method. The worms were maintained at 20C and transferred to new treatment plates again on day two of adulthood. One week after age-synchronization (day five of adulthood for C. elegans and C. briggsae, day four for C. tropicalis), the worms were transferred to compound-treated scanner plates and loaded onto the ALM. At this point, automated survival monitoring began, and the scanner data was collected and analyzed using Lifespan Machine software (https://github.com/nstroustrup/lifespan; Stroustrup et al.. 2013). Our results indicate that obeticholic acid does not have a consistent beneficial effect on lifespan in any of the C. elegans or C. briggsae strains tested at the concentrations used. Although we did see some significant differences from the control for some of the concentrations in the C. tropicalis strains, overall the difference was not robust. We actually saw a significant decrease in lifespan in C. tropicalis JU1630, a weakly significant increase in C. tropicalis QG834 at some concentrations, and a relatively significant increase in C. tropicalis JU1373, but with only a 5.7-7.9% change in mean survival from the control (Fig. 1). In summary, our results do not indicate a robust effect of obeticholic acid on Caenorhabditis lifespan. This conclusion is based upon two biological replicates at each concentration performed in one lab, resulting in an average of 104 individuals measured per strain and concentration, and should be considered preliminary. The effect on lifespan in this study may pertain to a lack of physiological relevance of obeticholic acid to Caenorhabditis. Obeticholic acid was of interest to the CITP because of its effect on the mammalian NR FXR. Although DAF-12 has been identified as a potential Caenorhabditis homolog of FXR and other bile acids have been shown to bind with DAF-12 (Zhi et al. 2011), it is possible that obeticholic acid was not able to bind with high affinity to the receptor, therefore eliciting little to no effect on lifespan. Alternatively, obeticholic acid may be rapidly metabolized in Caenorhabditis.

Coleman-Hulbert, Anna L. et al. MicroPubl Biol

Johnson, Erik, Banse, Stephen A., Guo, Max, Phillips, Patrick C., Sedore, Christine A., Driscoll, Monica, Coleman-Hulbert, Anna L., Lithgow, Gordon J.

[MicroPubl Biol, 2020]

The Caenorhabditis Intervention Testing Program (CITP) is a multi-institutional, National Institutes of Aging (NIA)-funded consortium charged with identifying chemical compounds that robustly extend lifespan in a genetically diverse panel of Caenorhabditis strains. Compounds are prioritized for screening if they are highly ranked via computational prediction for lifespan or healthspan effects (Coleman-Hulbert et al. 2019), if they are predicted to engage known lifespan regulating pathways, or if they have previously been reported as extending lifespan or healthspan in model systems (Lucanic et al. 2017). -guanidinopropionic acid (-GPA) is a creatine analog (Shields and Whitehair 1973), commonly used as a dietary supplement, and has been shown to extend lifespan in Drosophila under stress via 5′ AMP-activated protein kinase (AMPK) activity (Yang et al. 2015). The AMPK pathway is conserved in nematodes and humans (Apfeld et al. 2004) and is involved in multiple pathways affecting stress response and metabolism (Wang et al. 2012). We assayed lifespan in response to -GPA exposure in three Caenorhabditis species using our previously published workflow (Lucanic et al. 2017). In brief, worms were age-synchronized by timed egg-lays on standard 60 mm diameter Nematode Growth Media (NGM) plates and transferred at a density of 50 individuals per 35 mm treated plate in triplicate when they reached adulthood (for control plates, there were six replicates of 50 animals each). -GPA (Sigma-Aldrich) was dissolved in water and diluted appropriately such that addition of 125 l of solution to 35 mm diameter plates containing NGM with lawns of E. coli OP50-1 and 51 m FUdR would generate the following final -GPA concentrations: 0.1 M, 1 M, 10 M, 50 M, 100 M, 500 M, 1 mM, 10 mM and 300 mM. Worms were maintained at 20 C and moved to fresh plates on the first, second, and fourth (C. tropicalis) or fifth (C. elegans and C. briggsae) day of adulthood, then once weekly afterward. Due to our previous experience with compound interventions that potentially alter bacterial viability through transient pH changes upon plate treatment (Banse et al. 2019), we investigated the effects of -GPA on our assay plates. At the above listed concentrations, -GPA-treated plates had a pH of 6.5 and the bacterial lawns survived treatment when tested by replica plating. Thrice weekly, we observed animals for spontaneous movement or movement after gentle perturbation with a 0.2 mm diameter platinum wire. Death was scored as a lack of movement. Our results indicate that -GPA does not extend lifespan in the three nematode species at the concentrations tested here; in fact, in only one instance was an effect detected and, in that case, the compound reduced lifespan (Fig. 1). This conclusion is based on one biological replicate per concentration and, as such, could be considered preliminary. While interventions may be ineffective due to a range of causes, including permeability barriers, compound stability in vivo, and metabolism by the bacterial food source, we believe that the lack of response in this study was due to a lack of physiological relevance in C. elegans. In Drosophila and mammalian models, -GPA reduces the level of intracellular phosphocreatine that can be used by creatine kinases to regenerate ATP (Oudman et al. 2013; Yang et al. 2015) resulting in a decreased cellular ATP/AMP ratio, which activates AMPK and ultimately increases lifespan and stress-resistance. Our a priori expectation for lifespan extension in C. elegans was built on the observations that: (1) creatine is reported as detectible in C. elegans (Atherton et al. 2008; Jones et al. 2012; Wan et al. 2017); (2) C. elegans has a creatine-like kinase, ARGK-1, whose activity modulates AMPK signaling (McQuary et al. 2016); (3) modulation of AMPK (Apfeld et al. 2004; Greer et al. 2007) and ARGK-1 (McQuary et al. 2016) in C. elegans can affect lifespan and stress resistance; and (4) regulation of lifespan by insulin signaling is partially dependent on AMPK signaling in C. elegans (Tullet et al. 2014). As such, we were surprised to find no changes in lifespan upon treatment with a creatine analog. One possible explanation is that, despite the similarity between ARGK-1 and mammalian creatine kinase, the enzymes substrates differ. Biochemical analysis suggests that ARGK-1 uses arginine instead of creatine as a substrate to recharge ADP (Fraga et al. 2015). Additionally, it has been postulated that the biochemical characterization of C. elegans metabolites may have misidentified creatine, and that creatine is not relevant to C. elegans physiology (Witting et al. 2018). Given these caveats, it may not be surprising that -GPA does not alter Caenorhabditis lifespan.