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FUdR treatment improved proteostasis before the onset of reproduction. (A-B) The motility of age-synchronized temperature-sensitive unc-45(e286) (A), or unc-54(e1301) (B) animals raised in the absence (black) or presence (gray) of FUdR was examined on the first and second days of adulthood and the percent of paralyzed animals was scored. Data represent means 6 SEM of 5 independent experiments. P values compare age-matched treated and untreated animals. (**) P,0.01. (C) Confocal images of age-synchronized unc54(e1301) animals raised in the absence or presence of FUdR, and stained with anti-UNC-54 antibodies (green) and Phalloidin (red). Arrows indicate myofilaments. Scale bar is 10 mm. doi:10.1371/journal.pone.0085964.g002
Source publication
Protein homeostasis (proteostasis) networks are dynamic throughout the lifespan of an organism. During Caenorhabditis elegans adulthood, the maintenance of metastable proteins and the activation of stress responses are inversely associated with germline stem cell proliferation. Here, we employed the thymidylate synthase inhibitor 5-fluoro-2'-deoxyu...
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Context 1
... of wild type animals raised on regular plates to induce mRNA levels of hsp-70 and hsp-16.11 declined strongly on the second day of adulthood, while FUdR- treated animals maintained their ability to induce hsp-70 and hsp- 16.11 (Fig. 1C). No significant changes were detected in the basal expression levels of HS genes in FUdR-treated animals (Fig. S2B). Likewise, induction of other stress responses, such as oxidative stress response genes (gcs-1, gst-4, and sod-3), UPR ER (hsp-4) and UPR mt (hsp-6), was not detected in FUdR-treated animals, suggesting that FUdR-mediated effects on HS response activation are not due to hormesis. These data indicate that treatment with FUdR can ...
Context 2
... of both unc-45(ts) and unc-54(ts) animals, with 1.461.4% and 2.161.4% of the population being paralyzed, respectively. At the same time, the mobility of the wild type animals was unaffected (data not shown). Motility was also maintained on the second day of adulthood, with 2.362.3% and 2.261.5% of these populations being paralyzed, respectively ( Fig. 2A and ...
Context 3
... in the presence or absence of FUdR. As expected, the myofilaments of unc-54(ts) animals shifted to 25uC were disrupted and UNC-54 was completely mislocalized, compared to wild type or unc-54(ts) animals raised at 15uC. In contrast, myofilaments of FUdR-treated animals were partially maintained, and showed reduced number of myo-filaments (Fig. 2C). These observations support our finding that FUdR treatment modulated proteostasis during development, thereby increasing both stress resistance and folding ...
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The heat shock response (HSR) is essential for proteostasis and cellular health. In metazoans, aging is associated with a decline in quality control, thus increasing the risk for protein conformational disease. Here, we show that in C. elegans, the HSR declines precipitously over a 4 hr period in early adulthood coincident with the onset of reprodu...
Citations
... Day 1 of adulthood (D1) was defined as 24 h post-L4. Given the potential confounding effects of FUDR on aging (Feldman, Kosolapov, and Ben-Zvi 2014;Van Raamsdonk and Hekimi 2011), key experiments were repeated without FUDR, moving animals onto fresh plates daily until D5, every 2 days thereafter. FUDR-free experiments are noted in figure legends. ...
Despite advances in understanding molecular and cellular changes in the aging nervous system, the upstream drivers of these changes remain poorly defined. Here, we investigate the roles of non‐neural tissues in neuronal aging, using the cutaneous PVD polymodal sensory neuron in Caenorhabditis elegans as a model. We demonstrate that during normal aging, PVD neurons progressively develop excessive dendritic branching, functionally correlated with age‐related proprioceptive deficits. Our study reveals that decreased collagen expression, a common age‐related phenomenon across species, triggers this process. Specifically, loss‐of‐function in dpy‐5 or col‐120, genes encoding cuticular collagens secreted to the epidermal apical surface, induces early‐onset excessive dendritic branching and proprioceptive deficits. Adulthood‐specific overexpression of dpy‐5 or col‐120 mitigates excessive branching in aged animals without extending lifespan, highlighting their specific roles in promoting neuronal health span. Notably, collagen reduction specifically drives excessive branching in select sensory neuron subclasses but does not contribute to PVD dendritic beading, another aging‐associated neurodegenerative phenotype associated with distinct mechanosensitive dysfunction. Lastly, we identify that rig‐3, an immunoglobulin superfamily member expressed in interneurons, acts upstream of collagen genes to maintain PVD dendritic homeostasis during aging, with collagen's regulatory role requiring daf‐16/FOXO. These findings reveal that age‐related collagen reduction cues neuronal aging independently of collagen's traditional structural support function, possibly involving bi‐directional communication processes between neurons and non‐neuronal cells. Our study also offers new insights into understanding selective neuron vulnerability in aging, emphasizing the importance of multi‐tissue strategies to address the complexities of neuronal aging.
... While FUdR does not prolong the lifespan of wild-type worms [16], its introduction at the L4 larval stage extends the lifespans of fat-storing tub-1 and gas-1 mutants, which exhibit compromised mitochondrial complex functions [17,18]. Interestingly, FUdR-induced sterility in wild-type adult worms significantly enhances their proteostasis, protein folding, and stress responses [19,20,21,22]. While commonly attributed to its sterilizing capabilities, FUdR has been shown to enhance longevity and proteostasis in nematodes devoid of a germline, suggesting that its beneficial effects are not exclusively linked to reproductive inhibition [22]. ...
... Interestingly, FUdR-induced sterility in wild-type adult worms significantly enhances their proteostasis, protein folding, and stress responses [19,20,21,22]. While commonly attributed to its sterilizing capabilities, FUdR has been shown to enhance longevity and proteostasis in nematodes devoid of a germline, suggesting that its beneficial effects are not exclusively linked to reproductive inhibition [22]. It is postulated that the mechanism for FUdR proteostasis improvements stems from metabolic shifts or the activation of the DNA damage response pathway [23,24,25]. ...
... While FUdR is primarily known for inducing reproductive arrest, it also exerts secondary effects on various facets of C. elegans physiology, such as proteostasis and stress response mechanisms [16,19,20,21,22]. Our data indicated that FUdR augments the proteasome's activities, akin to the effects observed in the sterile glp-1(e2144) mutant [28]. ...
The ubiquitin-proteasome system (UPS) is critical for maintaining proteostasis, influencing stress resilience, lifespan, and thermal adaptability in organisms. In Caenorhabditis elegans, specific proteasome subunits and activators, such as RPN-6, PBS-6, and PSME-3, are associated with heat resistance, survival at cold (4°C), and enhanced longevity at moderate temperatures (15°C). Previously linked to improving proteostasis, we investigated the impact of sterility-inducing floxuridine (FUdR) on UPS functionality under proteasome dysfunction and its potential to improve cold survival. Our findings reveal that FUdR significantly enhances UPS activity and resilience during proteasome inhibition or subunit deficiency, supporting worms’ normal lifespan and adaptation to cold. Importantly, FUdR effect on UPS activity occurs independently of major proteostasis regulators and does not rely on the germ cells proliferation or spermatogenesis. Instead, FUdR activates a distinct detoxification pathway that supports UPS function, with GST-24 appearing to be one of the factors contributing to the enhanced activity of the UPS upon knockdown of the SKN-1-mediated proteasome surveillance pathway. Our study highlights FUdR unique role in the UPS modulation and its crucial contribution to enhancing survival under low-temperature stress, providing new insights into its mechanisms of action and potential therapeutic applications.
... Many longitudinal transcriptome studies have worked around this issue by inhibiting reproduction either through application of chemical inhibitors like Fluorodeoxyuridine (FUDR) (Ham et al., 2022;Li et al., 2019;Rangaraju et al., 2015), or by using strains with condition-specific induced sterility such as rrf-3 and fem mutants (Barton et al., 1987;Garigan et al., 2002;Lund et al., 2002;Murphy et al., 2003). Although these are useful methods to ensure no progeny are mixed in with the adult population when extracting RNA, the practice does move the animals away from a natural condition, affecting the transcriptome (Anderson et al., 2016;Feldman et al., 2014). A recent study also showed that the amino acid biosynthesis of E. coli exposed to FUDR is altered, which can affect gene transcription of feeding nematodes (McIntyre et al., 2021). ...
C. briggsae offers a robust system for comparative investigations of genetic pathways that affect physiological processes. One key process, reproduction, significantly impacts longevity due to its high energetic cost, which limits resources for somatic maintenance. Long-lived mutants often exhibit reproductive impairments, and studies in C. elegans have demonstrated that germline mutations and complete germline removal can promote longevity, underscoring the link between reproduction and aging. We are interested in identifying genes and biological processes affected during the reproductive and post-reproductive periods in C. briggsae. To achieve this, we conducted whole-genome transcriptome profiling on animals at various adult stages. analysis of differentially expressed (DE) genes revealed that the majority were downregulated during the reproductive period. Interestingly, this trend reversed post-reproduction, with three-quarters of the genes upregulated - a phenomenon we termed the 'reproductive shift'. A similar analysis in C. elegans also showed a downregulation bias during the reproductive period, but the reproductive shift was absent. Further examination of C. briggsae DE genes showed enrichment in processes related to the matrisome, muscle development and function during the reproductive period. Post-reproductive downregulated genes were enriched in DNA damage repair, stress response, and immune response. Additionally, terms related to fatty acid metabolism, catabolism, and transcriptional regulation exhibited complex patterns, with different biological processes being up or downregulated between the reproductive and post-reproductive stages. Overall, our transcriptomic data provides a valuable resource for cross-sectional comparative studies of reproductive and post-reproductive changes in nematodes. Additionally, the findings prompt similar studies in other animal models thereby advancing our understanding of genetic pathways affecting reproduction and aging.
... Second, staining did not happen if there was no X-gal added in the staining solution implying that it is the action of the β-gal enzyme on the X-gal in the animals that resulted in the blue end-product (Fig. S1C). Third, since we used FuDR to prevent progeny production and FuDR also affects protein homeostasis before and beyond the reproductive time period [36], we checked if FuDR had any effect on the staining efficiency. We found similar staining intensity between applying FuDR on wild type (N2) or using a temperature-sterile wild-type background mutant (spe-9(hc88)) ( Fig. S1D). ...
Accumulation of senescent cells accelerates aging and age-related diseases, whereas preventing this accumulation extends the lifespan in mice. A characteristic of senescent cells is increased staining with β-galactosidase (β-gal) ex vivo. Here, we describe a progressive accumulation of β-gal staining in the model organism C. elegans during aging. We show that distinct pharmacological and genetic interventions targeting the mitochondria and the mTORC1 to the nuclear core complex axis, the non-canonical apoptotic, and lysosomal-autophagy pathways slow the age-dependent accumulation of β-gal. We identify a novel gene, rege-1/Regnase-1/ZC3H12A/MCPIP1 , modulating β-gal staining via the transcription factor ets-4/SPDEF . We demonstrate that knocking down Regnase-1 in human cell culture prevents senescence-associated β-gal accumulation. Our data provide a screening pipeline to identify genes and drugs modulating senescence-associated lysosomal phenotypes.
... Kato et al. (2017) suggest that FUdR influences lifespan ''by interfering with fertility, which extends lifespan, and by inducing DNA base damage, which reduces lifespan''. Furthermore, FUdR was shown to influence hormetic responses (Anderson et al. 2016), proteostasis (Angeli et al. 2013;Feldman et al. 2014), the lifespan and metabolism of certain mutant strains (Aitlhadj and Stürzenbaum 2010;Davies et al. 2012;Van Raamsdonk and Hekimi 2011), as well as polyglutamine aggregation in a Huntington's disease model (Brunquell et al. 2014). Therefore, to prevent potential false-negative or falsepositive effects, its use should be avoided, if possible. ...
Several biogerontology databases exist that focus on genetic or gene expression data linked to health as well as survival, subsequent to compound treatments or genetic manipulations in animal models. However, none of these has yet collected experimental results of compound-related health changes. Since quality of life is often regarded as more valuable than length of life, we aim to fill this gap with the ‘‘Healthy Worm Database’’ (http://healthy-worm-database.eu). Literature describing health-related compound studies in the aging model Caenorhabditis elegans was screened, and data for 440 compounds collected. The database considers 189 publications describing 89 different phenotypes measured in 2995 different conditions. Besides enabling a targeted search for promising compounds for further investigations, this database also offers insights into the research field of studies on healthy aging based on a frequently used model organism. Some weaknesses of C. elegansbased aging studies, like underrepresented phenotypes, especially concerning cognitive functions, as well as the convenience-based use of young worms as the starting point for compound treatment or phenotype measurement are discussed. In conclusion, the database provides an anchor for the search for compounds affecting health, with a link to public databases, and it further highlights some potential shortcomings in current aging research.
... FUdR inhibits germline stem cell proliferation and the production of intact eggs in adults 40 . However, FUdR alone has been shown to extend lifespan by promoting stress responses in mutant animals or during stressing growth conditions [41][42][43][44][45] . Therefore, we asked if there is a synergic effect between FUdR and HGD in promoting the longevity of HGD-5 animals. ...
Metabolic diseases often share common traits, including accumulation of unfolded proteins in the endoplasmic reticulum (ER). Upon ER stress, the unfolded protein response (UPR) is activated to limit cellular damage which weakens with age. Here, we show that Caenorhabditis elegans fed a bacterial diet supplemented high glucose at day 5 of adulthood (HGD-5) extends their lifespan, whereas exposed at day 1 (HGD-1) experience shortened longevity. We observed a metabolic shift only in HGD-1, while glucose and infertility synergistically prolonged the lifespan of HGD-5, independently of DAF-16. Notably, we identified that UPR stress sensors ATF-6 and PEK-1 contributed to the longevity of HGD-5 worms, while ire-1 ablation drastically increased HGD-1 lifespan. Together, we postulate that HGD activates the otherwise quiescent UPR in aged worms to overcome ageing-related stress and restore ER homeostasis. In contrast, young animals subjected to HGD provokes unresolved ER stress, conversely leading to a detrimental stress response.
... Recently, it has also been shown that E. coli's metabolic conversion of FUdR to 5-fluorouridine monophosphate (FUMP) can impair C. elegans fecundity and highlight the role of bacteria in host health [10][11][12]. Moreover, FUdR may directly affect C. elegans hosts by improving thermal stress response, protein homeostasis, and altering lifespan in some mutant backgrounds [6,[13][14][15]. Thus, further studies analyzing the gene expression changes in OP50 E. coli exposed to FUdR may provide insight into aging and other studies. ...
Objective
Many C. elegans aging studies use the compound 5-fluro-2ʹ-deoxyuridine (FUdR) to produce a synchronous population of worms. However, the effects of FUdR on the bacterial gene expression of OP50 E. coli , the primary laboratory C. elegans food source, is not fully understood. This is particularly relevant as studies suggest that intestinal microbes can affect C. elegans physiology. Therefore, it is imperative that we understand how exposure to FUdR can affect gene expression changes in OP50 E. coli.
Results
An RNAseq dataset comprised of expression patterns of 2900 E. coli genes in the strain OP50, which were seeded on either nematode growth media (NGM) plates or on FUdR (50 µM) supplemented NGM plates, was analyzed. Analysis showed differential gene expression in genes involved in general transport, amino acid biosynthesis, transcription, iron transport, and antibiotic resistance. We specifically highlight metabolic enzymes in the l -histidine biosynthesis pathway as differentially expressed between NGM and FUdR exposed OP50. We conclude that OP50 exposed to FUdR results in differential expression of many genes, including those in amino acid biosynthetic pathways.
... The practice of using FUDR in lifespan experiments has been questioned previously due to a lack of complete comprehension for its mechanism of action and potential effects on worm development. Indeed, FUDR has been shown to alter worm proteostasis, a process critically involved in lifespan regulation (Feldman et al., 2014). The temporal administration of FUDR has been shown to impact N2 phenotypes and possibly senescence (Wang et al., 2019), and an extension of lifespan after FUDR administration to mutant C. elegans has been previously established (Aitlhadj and Stürzenbaum, 2010;Van Raamsdonk and Hekimi, 2011;Kato et al., 2016). ...
The nematode Caenorhabditis elegans is a powerful model organism for studying cell development, apoptosis, neuronal circuits, and aging. The isolate N2 is recognized by the C. elegans community as the reference wild-type strain. Interestingly, the lifespan of presumably isogenic C. elegans N2 worms—even when grown under comparable conditions—varies significantly amongst distinct laboratories. This hinders the inter-laboratory comparability of C. elegans lifespan data and raises questions regarding data interpretation and reproducibility. Here, we hypothesized slight alterations in experimental design and worm handling could explain the observed discrepancies. To test this hypothesis, we collected and assessed data from over 1000 published C. elegans N2 lifespan assays as well as corresponding methodological meta-data. We find that mean N2 lifespans range from approximately 7 days to upwards of 35 days, despite laboratories disclosing seemingly comparable experimental conditions. We further demonstrate that, in addition to temperature, the use of the chemical sterilizer 5-fluoro-2′-deoxyuridine (FUDR) may change N2 lifespan. Additionally, we observed differences in average N2 lifespan from experiments originating from distinct geographic locations, indicating a potential effect of location-specific factors on experimental outcomes. Taken as a whole, our work indicates the sum of many small, rather than a few critical, differences in experimental conditions may account for the observed variance in N2 lifespan. We also find that the absence of standardized experimental methods and the insufficient disclosure of experiment details in the peer-reviewed literature limits the inter-lab comparability of published results. We thus propose the establishment of a succinct reporting standard for C. elegans lifespan experiments to increase the reliability and reproducibility, and thus scientific value, of these studies.
... Recently, it has also been shown that E. coli's metabolic conversion of FUdR to 5-fluorouridine monophosphate (FUMP) can impair C. elegans fecundity (10). Moreover, FUdR may directly affect C. elegans hosts by improving thermal stress response, protein homeostasis, and altering lifespan in some mutant backgrounds (6,(11)(12)(13). ...
Objective: Many C. elegans aging studies use the compound, 5-fluro-2’-deoxyuridine (FUdR), to produce a synchronous population of worms in aging studies. However, it is not fully clear what effects FUdR have on the bacterial gene expression in the E. coli strain OP50, the primary laboratory C. elegans food source. This is particularly relevant as studies indicate that intestinal microbes can affect C. elegans physiology. Here, we examine how exposure to FUdR can affect gene expression changes in OP50 E. coli.
Results: RNAseq datasets were used to compare the expression patterns of E. coli genes in the strain OP50 seeded on either nematode growth media (NGM) plates or on FUdR (50µM) supplemented NGM plates. Analysis showed differential expression of genes involved in general transport, amino acid biosynthesis, transcription, iron transport, and antibiotic resistance. We specifically highlight metabolic enzymes in the L-histidine biosynthesis pathway may be regulated by FUdR exposed OP50. We conclude that OP50 exposed to FUdR results in many differentially expressed genes, including those in amino acid biosynthetic pathways.
... Kato et al. (2017) suggest that FUdR influences lifespan ''by interfering with fertility, which extends lifespan, and by inducing DNA base damage, which reduces lifespan''. Furthermore, FUdR was shown to influence hormetic responses (Anderson et al. 2016), proteostasis (Angeli et al. 2013;Feldman et al. 2014), the lifespan and metabolism of certain mutant strains (Aitlhadj and Stürzenbaum 2010;Davies et al. 2012;Van Raamsdonk and Hekimi 2011), as well as polyglutamine aggregation in a Huntington's disease model (Brunquell et al. 2014). Therefore, to prevent potential false-negative or falsepositive effects, its use should be avoided, if possible. ...
Several biogerontology databases exist that focus on genetic or gene expression data linked to health as well as survival, subsequent to compound treatments or genetic manipulations in animal models. However, none of these has yet collected experimental results of compound-related health changes. Since quality of life is often regarded as more valuable than length of life, we aim to fill this gap with the “Healthy Worm Database” ( http://healthy-worm-database.eu ). Literature describing health-related compound studies in the aging model Caenorhabditis elegans was screened, and data for 440 compounds collected. The database considers 189 publications describing 89 different phenotypes measured in 2995 different conditions. Besides enabling a targeted search for promising compounds for further investigations, this database also offers insights into the research field of studies on healthy aging based on a frequently used model organism. Some weaknesses of C. elegans -based aging studies, like underrepresented phenotypes, especially concerning cognitive functions, as well as the convenience-based use of young worms as the starting point for compound treatment or phenotype measurement are discussed. In conclusion, the database provides an anchor for the search for compounds affecting health, with a link to public databases, and it further highlights some potential shortcomings in current aging research.
