Spurlock, B., Tullet, J., Hartman, J. and Mitra, K. (2020). Interplay of mitochondrial fission-fusion with cell cycle regulation: Possible impacts on stem cell and organismal aging. Experimental Gerontology [Online] 135:110919. Available at: https://doi.org/10.1016/j.exger.2020.110919.
Declining mitochondrial function and homeostasis is a hallmark of aging. It is appreciated that the role of mitochondria is much more complex than generating reactive oxygen species to cause aging-related tissue damage. More recent literature describes that the ability of mitochondria to undergo fission or fusion events with each other impacts aging processes. A dynamic balance of mitochondrial fission and fusion events is required to sustain critical cellular functions including cell cycle. Specifically, cell cycle regulators modulate molecular activities of the mitochondrial fission (and fusion) machinery towards regulating cell cycle progression. In this review, we discus literature leading to our understanding on how shifts in the dynamic balance of mitochondrial fission and fusion can modulate progression through, exit from, and re-entry to the cell cycle or undergo senescence. Importantly, core regulators of mitochondrial fission or fusion are emerging as crucial stem cell regulators. We discuss the implication of such regulation in stem cells in the context of aging, given that aberrations in adult stem cells promote aging. We also propose a few hypotheses that may provide direction for further understanding about the roles of mitochondrial fission-fusion dynamics in aging biology.
Benedetto, A., Bambade, T., Au, C., Tullet, J., Monkhouse, J., Dang, H., Cetnar, K., Cabreiro, F., Chan, B. and Gems, D. (2019). New Label‐Free Automated Survival Assays Reveal Unexpected Stress Resistance Patterns during C. Elegans Aging. Aging Cell [Online] 18:e12998. Available at: https://doi.org/10.1111/acel.12998.
Caenorhabditis elegans is an excellent model for high‐throughput experimental approaches but lacks an automated means to pinpoint time of death during survival assays over a short time frame, that is, easy to implement, highly scalable, robust, and versatile. Here, we describe an automated, label‐free, high‐throughput method using death‐associated fluorescence to monitor nematode population survival (dubbed LFASS for label‐free automated survival scoring), which we apply to severe stress and infection resistance assays. We demonstrate its use to define correlations between age, longevity, and severe stress resistance, and its applicability to parasitic nematodes. The use of LFASS to assess the effects of aging on susceptibility to severe stress revealed an unexpected increase in stress resistance with advancing age, which was largely autophagy‐dependent. Correlation analysis further revealed that while severe thermal stress resistance positively correlates with lifespan, severe oxidative stress resistance does not. This supports the view that temperature‐sensitive protein‐handling processes more than redox homeostasis underpin aging in C. elegans. That the ages of peak resistance to infection, severe oxidative stress, heat shock, and milder stressors differ markedly suggests that stress resistance and health span do not show a simple correspondence in C. elegans.
Lawrence, A., Nemoto-Smith, E., Deery, E., Baker, J., Schroeder, S., Brown, D., Tullet, J., Howard, M., Brown, I., Smith, A., Boshoff, H., Barry, C. and Warren, M. (2018). Construction of Fluorescent Analogs to Follow the Uptake and Distribution of Cobalamin (Vitamin B 12 ) in Bacteria, Worms, and Plants. Cell Chemical Biology [Online]. Available at: https://doi.org/10.1016/j.chembiol.2018.04.012.
Vitamin B12 is made by only certain prokaryotes yet is required by a number of eukaryotes
such as mammals, fish, birds, worms and Protista, including algae. There is still much to learn
about how this nutrient is trafficked across the domains of life. Herein, we describe ways to
make a number of different corrin analogues with fluorescent groups attached to the main
tetrapyrrole-derived ring. A further range of analogues were also constructed by attaching
similar fluorescent groups to the ribose ring of cobalamin, thereby generating a range of
complete and incomplete corrinoids to follow uptake in bacteria, worms and plants. By using
these fluorescent derivatives we were able to demonstrate that Mycobacterium tuberculosis
is able to acquire both cobyric acid and cobalamin analogues, that Caenorhabditis elegans
takes up only the complete corrinoid, and that seedlings of higher plants such as Lepidium
sativum are also able to transport B12.
Filer, D., Thompson, M., Takhaveev, V., Dobson, A., Kotronaki, I., Green, J., Heinemann, M., Tullet, J. and Alic, N. (2017). Longevity by RNA polymerase III inhibition downstream of TORC1. Nature [Online] 552:263-267. Available at: http://dx.doi.org/10.1038/nature25007.
Three distinct RNA polymerases (Pols) transcribe different classes of genes in the eukaryotic nucleus1. Pol III is the essential, evolutionarily conserved enzyme that generates short, non-coding RNAs, including transfer RNAs (tRNAs) and 5S ribosomal RNA (rRNA)2. Historical focus on transcription of protein-coding genes has left the roles of Pol III in organismal physiology relatively unexplored. The prominent regulator of Pol III activity, Target of Rapamycin kinase Complex 1 (TORC1), is an important longevity determinant3, raising the question of Pol III’s involvement in ageing. Here we show that Pol III limits lifespan downstream of TORC1. We find that a reduction in Pol III extends chronological lifespan in yeast and organismal lifespan in worms and flies. Inhibiting Pol III activity in the adult worm or fly gut is sufficient to extend lifespan, and in flies, longevity can be achieved by Pol III inhibition specifically in the intestinal stem cells (ISCs). The longevity phenotype is associated with amelioration of age-related gut pathology and functional decline, dampened protein synthesis and increased tolerance of proteostatic stress. Importantly, Pol III acts downstream of TORC1 for lifespan and limiting Pol III activity in the adult gut achieves the full longevity benefit of systemic TORC1 inhibition. Hence, Pol III is a pivotal output of this key nutrient signalling network for longevity; Pol III’s growth-promoting, anabolic activity mediates the acceleration of ageing by TORC1. The evolutionary conservation of Pol III affirms its potential as a therapeutic target.
Tullet, J., Green, J., Au, C., Benedetto, A., Thompson, M., Clarke, E., Gilliat, A., Young, A., Schmisser, K. and Gems, D. (2017). The SKN-1/Nrf2 transcription factor can protect against oxidative stress and increase lifespan in C. elegans by distinct mechanisms. Aging Cell [Online] 16:1191-1194. Available at: http://dx.doi.org/10.1111/acel.12627.
In C. elegans the skn-1 gene encodes a transcription factor that resembles mammalian Nrf2 and activates a detoxification response. skn-1 promotes resistance to oxidative stress (Oxr) and also increases lifespan, and it has been suggested that the former causes the latter, consistent with the theory that oxidative damage causes aging. Here we report that effects of SKN-1 on Oxr and longevity can be dissociated. We also establish that skn-1 expression can be activated by the DAF-16/FoxO transcription factor, another central regulator of growth, metabolism and aging. Notably, skn-1 is required for Oxr but not increased lifespan resulting from over-expression of DAF-16; concomitantly, DAF-16 over-expression rescues the short lifespan of skn-1 mutants but not their hypersensitivity to oxidative stress. These results suggest that SKN-1 promotes longevity by a mechanism other than protection against oxidative damage.
Bastow, E., Peswani, A., Tarrant, D., Pentland, D., Chen, X., Morgan, A., Staniforth, G., Tullet, J., Rowe, M., Howard, M., Tuite, M. and Gourlay, C. (2016). New links between SOD1 and metabolic dysfunction from a yeast model of amyotrophic lateral sclerosis. Journal of cell science [Online] 129:4118-4129. Available at: http://dx.doi.org/10.1242/jcs.190298.
A number of genes have been linked to familial forms of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Over 150 mutations within the gene encoding superoxide dismutase 1 (SOD1) have been implicated in ALS, but why such mutations lead to ALS-associated cellular dysfunction is unclear. In this study, we identify how ALS-linked SOD1 mutations lead to changes in the cellular health of the yeast Saccharomyces cerevisiae We find that it is not the accumulation of aggregates but the loss of Sod1 protein stability that drives cellular dysfunction. The toxic effect of Sod1 instability does not correlate with a loss of mitochondrial function or increased production of reactive oxygen species, but instead prevents acidification of the vacuole, perturbs metabolic regulation and promotes senescence. Central to the toxic gain-of-function seen with the SOD1 mutants examined was an inability to regulate amino acid biosynthesis. We also report that leucine supplementation results in an improvement in motor function in a Caenorhabditis elegans model of ALS. Our data suggest that metabolic dysfunction plays an important role in Sod1-mediated toxicity in both the yeast and worm models of ALS.
Fabris, F., Freitas, A. and Tullet, J. (2015). An Extensive Empirical Comparison of Probabilistic Hierarchical Classifiers in Datasets of Ageing-Related Genes. IEEE/ACM Transactions on Computational Biology and Bioinformatics [Online] 13:1045-1058. Available at: http://doi.org/10.1109/TCBB.2015.2505288.
This study comprehensively evaluates the performance of 5 types of probabilistic hierarchical classification methods used for predicting Gene Ontology (GO) terms related to ageing. Of those tested, a new hybrid of a Local Hierarchical Classifier (LHC) and the Predictive Clustering Tree algorithm (LHC-PCT) had the best predictive accuracy results. We also tested the impact of two types of variations in most hierarchical classification algorithms, namely: (a) changing the base algorithm (we tested Naive Bayes and Support Vector Machines), and the impact of (b) using or not the Correlation based Feature Selection (CFS) algorithm in a pre-processing step. In total, we evaluated the predictive performance of 17 variations of hierarchical classifiers across 15 datasets of ageing and longevityrelated genes. We conclude that the LHC-PCT algorithm ranks better across several tests (7 out of 12). In addition, we interpreted the models generated by the PCT algorithm to show how hierarchical classification algorithms can be used to extract biological insights out of the ageing-related datasets that we compiled.
Tullet, J. (2015). DAF-16 target identification in C. elegans: past, present and future. Biogerontology [Online] 16:221-234. Available at: http://dx.doi.org/10.1007/s10522-014-9527-y.
In C. elegans, mutations in the conserved insulin/IGF-1 signaling (IIS) pathway lead to a robust extension in lifespan, improved late life health, and protection from age-related disease. These effects are mediated by the FoxO transcription factor DAF-16 which lies downstream of the IIS kinase cascade. Identifying and functionally testing DAF-16 target genes has been a focal point of ageing research for the last 10 years. Here, I review the recent advances in identifying and understanding IIS/DAF-16 targets. These studies continue to reveal the intricate nature of the IIS/DAF-16 gene regulation network and are helping us to understand the mechanisms that control lifespan. Ageing and age related disease is an area of intense public interest, and the biochemical characterization of the genes involved will be critical for identifying drugs to improve the health of our ageing population.
Alic, N., Tullet, J., Niccoli, T., Broughton, S., Hoddinott, M., Slack, C., Gems, D. and Partridge, L. (2014). Cell-nonautonomous effects of dFOXO/DAF-16 in aging. Cell reports 6:608-16.
Drosophila melanogaster and Caenorhabditis elegans each carry a single representative of the Forkhead box O (FoxO) family of transcription factors, dFOXO and DAF-16, respectively. Both are required for lifespan extension by reduced insulin/Igf signaling, and their activation in key tissues can extend lifespan. Aging of these tissues may limit lifespan. Alternatively, FoxOs may promote longevity cell nonautonomously by signaling to themselves (FoxO to FoxO) or other factors (FoxO to other) in distal tissues. Here, we show that activation of dFOXO and DAF-16 in the gut/fat body does not require dfoxo/daf-16 elsewhere to extend lifespan. Rather, in Drosophila, activation of dFOXO in the gut/fat body or in neuroendocrine cells acts on other organs to promote healthy aging by signaling to other, as-yet-unidentified factors. Whereas FoxO-to-FoxO signaling appears to be required for metabolic homeostasis, our results pinpoint FoxO-to-other signaling as an important mechanism through which localized FoxO activity ameliorates aging.
Tullet, J., Araiz, C., Sanders, M., Au, C., Benedetto, A., Papatheodorou, I., Clark, E., Schmeisser, K., Jones, D., Schuster, E., Thornton, J. and Gems, D. (2014). DAF-16/FoxO directly regulates an atypical AMP-activated protein kinase gamma isoform to mediate the effects of insulin/IGF-1 signaling on aging in Caenorhabditis elegans. PLoS genetics [Online] 10. Available at: http://dx.doi.org/doi/10.1371/journal.pgen.1004109.
The DAF-16/FoxO transcription factor controls growth, metabolism and aging in Caenorhabditis elegans. The large number of genes that it regulates has been an obstacle to understanding its function. However, recent analysis of transcript and chromatin profiling implies that DAF-16 regulates relatively few genes directly, and that many of these encode other regulatory proteins. We have investigated the regulation by DAF-16 of genes encoding the AMP-activated protein kinase (AMPK), which has ?, ? and ? subunits. C. elegans has 5 genes encoding putative AMP-binding regulatory ? subunits, aakg-1-5. aakg-4 and aakg-5 are closely related, atypical isoforms, with orthologs throughout the Chromadorea class of nematodes. We report that ?75% of total ? subunit mRNA encodes these 2 divergent isoforms, which lack consensus AMP-binding residues, suggesting AMP-independent kinase activity. DAF-16 directly activates expression of aakg-4, reduction of which suppresses longevity in daf-2 insulin/IGF-1 receptor mutants. This implies that an increase in the activity of AMPK containing the AAKG-4 ? subunit caused by direct activation by DAF-16 slows aging in daf-2 mutants. Knock down of aakg-4 expression caused a transient decrease in activation of expression in multiple DAF-16 target genes. This, taken together with previous evidence that AMPK promotes DAF-16 activity, implies the action of these two metabolic regulators in a positive feedback loop that accelerates the induction of DAF-16 target gene expression. The AMPK ? subunit, aakb-1, also proved to be up-regulated by DAF-16, but had no effect on lifespan. These findings reveal key features of the architecture of the gene-regulatory network centered on DAF-16, and raise the possibility that activation of AMP-independent AMPK in nutritionally replete daf-2 mutant adults slows aging in C. elegans. Evidence of activation of AMPK subunits in mammals suggests that such FoxO-AMPK interactions may be evolutionarily conserved.
Riesen, M., Feyst, I., Rattanavirotkul, N., Ezcurra, M., Tullet, J., Papatheodorou, I., Ziehm, M., Au, C., Gilliat, A., Hellberg, J., Thornton, J. and Gems, D. (2014). MDL-1, a growth- and tumor-suppressor, slows aging and prevents germline hyperplasia and hypertrophy in C. elegans. Aging 6:98-117.
In C. elegans, increased lifespan in daf-2 insulin/IGF-1 receptor mutants is accompanied by up-regulation of the MDL-1 Mad basic helix-loop-helix leucine zipper transcription factor. Here we describe the role of mdl-1 in C. elegans germline proliferation and aging. The deletion allele mdl-1(tm311) shortened lifespan, and did so significantly more so in long-lived daf-2 mutants implying that mdl-1(+) contributes to effects of daf-2 on lifespan. mdl-1 mutant hermaphrodites also lay increased numbers of unfertilized oocytes. During aging, unfertilized oocytes in the uterus develop into tumors, whose development was accelerated by mdl-1(tm311). Opposite phenotypes were seen in daf-2 mutants, i.e. mdl-1 and daf-2 mutant germlines are hyperplastic and hypoplastic, respectively. Thus, MDL-1, like its mammalian orthologs, is an inhibitor of cell proliferation and growth that slows progression of an age-related pathology in C. elegans (uterine tumors). In addition, intestine-limited rescue of mdl-1 increased lifespan but not to wild type levels. Thus, mdl-1 likely acts both in the intestine and the germline to influence age-related mortality.
Schuster, E., McElwee, J., Tullet, J., Doonan, R., Matthijssens, F., Reece-Hoyes, J., Hope, I., Vanfleteren, J., Thornton, J. and Gems, D. (2010). DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO. Molecular systems biology [Online] 6. Available at: http://dx.doi.org/10.1038/msb.2010.54.
Insulin/IGF-1 signaling controls metabolism, stress resistance and aging in Caenorhabditis elegans by regulating the activity of the DAF-16/FoxO transcription factor (TF). However, the function of DAF-16 and the topology of the transcriptional network that it crowns remain unclear. Using chromatin profiling by DNA adenine methyltransferase identification (DamID), we identified 907 genes that are bound by DAF-16. These were enriched for genes showing DAF-16-dependent upregulation in long-lived daf-2 insulin/IGF-1 receptor mutants (P=1.4e(-11)). Cross-referencing DAF-16 targets with these upregulated genes (daf-2 versus daf-16; daf-2) identified 65 genes that were DAF-16 regulatory targets. These 65 were enriched for signaling genes, including known determinants of longevity, but not for genes specifying somatic maintenance functions (e.g. detoxification, repair). This suggests that DAF-16 acts within a relatively small transcriptional subnetwork activating (but not suppressing) other regulators of stress resistance and aging, rather than directly regulating terminal effectors of longevity. For most genes bound by DAF-16::DAM, transcriptional regulation by DAF-16 was not detected, perhaps reflecting transcriptionally non-functional TF 'parking sites'. This study demonstrates the efficacy of DamID for chromatin profiling in C. elegans.
Wang, J., Robida-Stubbs, S., Tullet, J., Rual, J., Vidal, M. and Blackwell, T. (2010). RNAi screening implicates a SKN-1-dependent transcriptional response in stress resistance and longevity deriving from translation inhibition. PLoS genetics [Online] 6:1-17. Available at: http://dx.doi.org/10.1371/journal.pgen.1001047.
Caenorhabditis elegans SKN-1 (ortholog of mammalian Nrf1/2/3) is critical for oxidative stress resistance and promotes longevity under reduced insulin/IGF-1-like signaling (IIS), dietary restriction (DR), and normal conditions. SKN-1 inducibly activates genes involved in detoxification, protein homeostasis, and other functions in response to stress. Here we used genome-scale RNA interference (RNAi) screening to identify mechanisms that prevent inappropriate SKN-1 target gene expression under non-stressed conditions. We identified 41 genes for which knockdown leads to activation of a SKN-1 target gene (gcs-1) through skn-1-dependent or other mechanisms. These genes correspond to multiple cellular processes, including mRNA translation. Inhibition of translation is known to increase longevity and stress resistance and may be important for DR-induced lifespan extension. One model postulates that these effects derive from reduced energy needs, but various observations suggest that specific longevity pathways are involved. Here we show that translation initiation factor RNAi robustly induces SKN-1 target gene transcription and confers skn-1-dependent oxidative stress resistance. The accompanying increases in longevity are mediated largely through the activities of SKN-1 and the transcription factor DAF-16 (FOXO), which is required for longevity that derives from reduced IIS. Our results indicate that the SKN-1 detoxification gene network monitors various metabolic and regulatory processes. Interference with one of these processes, translation initiation, leads to a transcriptional response whereby SKN-1 promotes stress resistance and functions together with DAF-16 to extend lifespan. This stress response may be beneficial for coping with situations that are associated with reduced protein synthesis.
Selman, C., Tullet, J., Wieser, D., Irvine, E., Lingard, S., Choudhury, A., Claret, M., Al-Qassab, H., Carmignac, D., Ramadani, F., Woods, A., Robinson, I., Schuster, E., Batterham, R., Kozma, S., Thomas, G., Carling, D., Okkenhaug, K., Thornton, J., Partridge, L., Gems, D. and Withers, D. (2009). Ribosomal protein S6 kinase 1 signaling regulates mammalian life span. Science (New York, N.Y.) [Online] 326:140-144. Available at: http://dx.doi.org/10.1126/science.1177221.
Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.
Tullet, J., Hertweck, M., An, J., Baker, J., Hwang, J., Liu, S., Oliveira, R., Baumeister, R. and Blackwell, T. (2008). Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans. Cell [Online] 132:1025-1038. Available at: http://dx.doi.org/10.1016/j.cell.2008.01.030.
Insulin/IGF-1-like signaling (IIS) is central to growth and metabolism and has a conserved role in aging. In C. elegans, reductions in IIS increase stress resistance and longevity, effects that require the IIS-inhibited FOXO protein DAF-16. The C. elegans transcription factor SKN-1 also defends against oxidative stress by mobilizing the conserved phase 2 detoxification response. Here we show that IIS not only opposes DAF-16 but also directly inhibits SKN-1 in parallel. The IIS kinases AKT-1, -2, and SGK-1 phosphorylate SKN-1, and reduced IIS leads to constitutive SKN-1 nuclear accumulation in the intestine and SKN-1 target gene activation. SKN-1 contributes to the increased stress tolerance and longevity resulting from reduced IIS and delays aging when expressed transgenically. Furthermore, SKN-1 that is constitutively active increases life span independently of DAF-16. Our findings indicate that the transcription network regulated by SKN-1 promotes longevity and is an important direct target of IIS.
Tullet, J., Pocock, V., Steel, J., White, R., Milligan, S. and Parker, M. (2005). Multiple signaling defects in the absence of RIP140 impair both cumulus expansion and follicle rupture. Endocrinology [Online] 146:4127-4137. Available at: http://dx.doi.org/10.1210/en.2005-0348.
The nuclear receptor corepressor RIP140 is essential in the ovary for ovulation, but is not required for follicle growth and luteinization. To identify genes that may be subject to regulation by RIP140 or play a role in ovulation, we compared ovarian gene expression profiles in untreated immature wild-type and RIP140 null mice and after treatment with pregnant mare serum gonadotropin and human chorionic gonadotropin. Many genes involved in signaling, extracellular matrix formation, cell-cell attachment, and adhesion were aberrantly regulated in the absence of RIP140, varying according to the hormone status of the mice. Notable among these was the reduced expression of a number of genes that encode components of signaling pathways and matrix proteins required for cumulus expansion, a key remodeling process necessary for ovulation. Histological analysis confirmed that cumulus expansion in RIP140 null mice is reduced, oocyte detachment from the mural cell wall is impaired, and follicles fail to rupture in response to LH. Although the expression of many genes involved in cumulus cell expansion was reduced, there was a subset of genes involved in extracellular matrix formation and cell-cell interactions that was up-regulated and may interfere with ovarian tissue remodeling. We propose that widespread gene dysregulation in ovarian tissues in the absence of RIP140 leads to the anovulatory phenotype. This helps to define an important role for RIP140 in the regulation of multiple processes leading to ovulation.
Christian, M., Tullet, J. and Parker, M. (2004). Characterization of four autonomous repression domains in the corepressor receptor interacting protein 140. The Journal of biological chemistry [Online] 279:15645-15651. Available at: http://dx.doi.org/10.1074/jbc.M313906200.
Receptor interacting protein (RIP) 140 is a corepressor that can be recruited to nuclear receptors by means of LXXLL motifs. We have characterized four distinct autonomous repression domains in RIP140, termed RD1-4, that are highly conserved in mammals and birds. RD1 at the N terminus represses transcription in the presence of trichostatin A, suggesting that it functions by a histone deacetylase (HDAC)-independent mechanism. The repressive activity of RD2 is dependent upon carboxyl-terminal binding protein recruitment to two specific binding sites. Use of specific inhibitors indicates that RD2, RD3, and RD4 are capable of functioning by HDAC-dependent and HDAC-independent mechanisms, depending upon cell type.