Cellular senescence and ageing
Co-workers: Hayley Lees, Thibault Teissier, Hannah Walters
Collaborators: Alison Woollard (Biochemistry, Oxford); Robert Saunders (Open University); Richard Faragher (University of Brighton); David Kipling (Cardiff University)
My lab studies the molecular basis of ageing, focusing on the process of cellular senescence. Our work aims to define molecular and genetic pathways that govern cell senescence and that underlie organismal ageing and frailty. We are following up our pathway discovery with targeted intervention using pharmacological agents to delay or inhibit senescence, or even reverse detrimental phenotypes.
While ageing in humans manifests in many different ways, with a range of age-related diseases and frailties that affect some people much more than others, it is becoming increasingly accepted that the underlying cause is the biological ageing of cells - cellular senescence. Cells undergo senescence in several different ways in response to DNA damage, activation of oncogenes or simply after they have undergone a large number of cell divisions until their telomeres have become critically short. Senescence is an important tumour suppressor mechanism to prevent damaged cells from dividing, but the many changes cells undergo as they become senescent leads to them altering their local environment, including degrading tissue structure and promoting a pro-inflammatory and pro-cancer environment. We aim to develop ways of altering the rate of onset or outcomes of cell senescence, in order to better treat age-associated diseases and frailty, by identifying the biochemical pathways and specific molecules involved.
Key molecules involved in driving and maintaining cellular senescence are the tumour suppressor proteins p16 and p53, and the p53-activated cyclin-dependent kinase inhibitor p21, while the mTORC1 kinase and its downstream effectors are antagonistic and promote cell growth and proliferation, but senescence then results through telomere attrition after multiple rounds of cell division.
Other anti-ageing genes are also involved; one of the most promising for molecular study is WRN, mutation of which leads premature ageing Werner's syndrome. That a mutation in a single gene causes highly premature cell senescence which results in the early development of many age-related diseases including atherosclerosis, diabetes, cataracts, osteoporosis and cancer provides very strong evidence that there is a central underlying - and importantly, tractable - cause of many of the diseases of later life. We have shown that WRN stabilises the genome and acts to limit DNA damage - without it, excess DNA damage signals through the p53 damage response pathway to trigger senescence.
We are using a combination of mammalian cell culture with biochemical and proteomics analyses together with fly and worm genetics to investigate and modulate pathways of senescence and ageing. Such studies have to date allowed us to identify a major DNA replication defect in human Werner syndrome, to provide proof-of principle correction of that defect in cultured Werner syndrome patient cells, to identify and characterise the Drosophila orthologue of human WRN exonuclease, and to identify novel pathways of ageing in C. elegans based on genetic interactions with the worm wrn-1 gene. Our human proteomic studies are at an earlier stage but already show significant promise in identifying biochemical targets suitable for pharmacological intervention to modulate senescence. Finally, we are addressing a major problem in replicative senescence research – that of obtaining senescent cells without oncogene activation – by developing approaches to induce senescence through physiological pathways.
We have recently established the Oxford Ageing Network to bring together ageing researchers from all fields across the University, to provide a forum for discussion, sharing of resources and productive collaboration.
- Mason, P. A., Boubriak, I. and Cox, L. S. (2013) A fluorescence-based exonuclease assay to characterise DmWRNexo, orthologue of human progeroid WRN exonuclease, and its application to other nucleases. JoVE 82. Doi: 10.3791/50722
- Mason PA, Boubriak I, Robbins T, Saunders RDC and Cox LS (2013) DmWRNexo processes replication fork and bubble substrates but is inhibited by the presence of uracil or abasic sites. AGE (Dordr). 35(3):793-806
- Bird, JLE, Jennert-Burston, KCB, Bachler, MA, Mason PA, Lowe, JE, Heo, S-J, Campisi, J, Faragher, RGA, Cox, LS (2012) Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease. Biogerontology 13 (1) 49-62
To find out more, check out our videos and podcasts on cell senescence, Werner syndrome and ageing in general at:
- http://rss.oucs.ox.ac.uk/oxitems/generatersstwo2.php?channel_name=manstud/entrepreneurship-audio (Feb 2010, 2nd of 3 speakers, location -49.31)
- Cox LS, Mason PA, Bagley MC, Steinsaltz D, Stefanovska A, Bernjal A, McClintock PVE, Phillips AC, Upton J, Latimer JE, David T. (2014) Understanding ageing: biological and social perspectives. Chapter 2 in 'New Science of Ageing' Ed Alan Walker, Policy Press (Bristol) ISBN 978 1 4731 467 7
- Kenessary, A. Zhumadilov, Z, Nurgozhin, T, Kipling, D, Yeoman, M, Cox, L, Ostler, E. and Faragher, F. (2013) Biomarkers, interventions and healthy ageing. N. Biotechnology 30(4) 373-377
- Mason, PA and Cox, LS (2012) The role of DNA exonucleases in protecting genome stability and their impact on ageing. AGE (Dordr). 2012 Dec;34(6):1317-40
- Mason, PA and Cox, LS (2010) Prospects for rejuvenation of aged tissue by telomerase reactivation. Rejuvenation Research 13, 749-754
- Cox, LS (2009) Live fast, die young: new lessons in mammalian longevity. Rejuvenation Res. 12(4): 283-288.
- Cox, LS and Mattison, JA (2009) Increasing longevity through caloric restriction or rapamycin feeding in mammals: common mechanisms for common outcomes? Aging Cell. 8(5): 607-13
- Cox, LS (2009) Cell senescence - the future of ageing? Biogerontology 10 (3) 229-233
- Cox LS and Faragher RG. (2007) From old organisms to new molecules: integrative biology and therapeutic targets in accelerated human ageing. Cell Mol Life Sci. 64:2620-264
- Cox, LS. (Ed) (2009) Molecular Themes in DNA Replication. Royal Society of Chemistry (Cambridge) ISBN 978-0-85404-164-0
- Cox, LS , Harris, DA, Pears, CJ. (2012) Thrive in Biochemistry and Molecular Biology. OUP (Oxford). ISBN: 978-0-19-964548-0
Figure 1: Young and senescent human fibroblasts (photographed at same magnification). Young healthy proliferating skin fibroblasts are elongated in shape with a narrow diameter. As they undergo replicative cell senescence after multiple rounds of cell division (with concomitant telomere shortening), the cell size increases markedly, cells become more granular, and in some cases, multiple nuclei are detected within individual cells. The ability to form cell monolayers is impaired and the cells show marked biochemical changes.
Figure 2. Major biochemical pathways of senescence. mTORC1 integrates cellular signals such as those from hormones, nutrients and stress to co-ordinate the cellular response; under favourable conditions, cells will proliferate, though over multiple cell generations, telomeres will shorten and cell senescence will result (replicative senescence, RS). In the presence of activated oncogenes, tumour suppressor genes p16 and/or p53 are activated, leading to inhibition of cyclin-dependent kinases and cell cycle arrest via the cyclin-kinas inhibitor p21; this state of arrest and 'early' senescence is reinforced by retrograde signalling and production of ROS, to establish a state of 'deep' senescence (oncogene-induced senescence, OIS). Similarly, acute or chronic DNA damage (or other cellular stresses) is signalled through p53 to activate senescence (stress-induced premature senescence, SIPS). WRN interacts physically and functionally with p53, and may act antagonistically either by preventing accumulation of DNA damage (e.g. during DNA replication and by telomere stabilisation) or by repairing damage (e.g. it is implicated in HR and BER). Like p53 and p16, WRN is also a tumour suppressor protein.
Figure 3. Correction of DNA replication defects in Werner syndrome (WS) patient cells. (A) Replicating DNA was fluorescently labelled and progression from a bidirectional origin of replication assessed using fibre spread confocal microscopy. (B) Replication forks stall at high frequency in WS, leading to formation of 4-way Holliday junctions. (C) Defective replication can be restored back to wild type control levels by expressing a small enzyme that cleaves the Holliday junctions. (see also Rodríguez-López, A.M., Jackson, D.A., Iborra, F., and Cox, L.S. (2002) Asymmetry of DNA replication fork progression in Werner's syndrome. Ageing Cell 1, 30-39; Rodríguez-López, A.M., Jackson, D.A., Nehlin, J. O., Iborra, F., Warren, A. V. and Cox, L. S. (2003) Characterisation of the interaction between WRN, the helicase/exonuclease defective in progeroid Werner's syndrome, and an essential replication factor, PCNA. Mech. Ag. Dev 124, 167-174; Rodriguez-Lopez, A. M, Whitby, M. C, Borer, C. M, Bachler, M. A. and Cox, L. S. (2007) Correction of Proliferation and Drug Sensitivity Defects in the Progeroid Werner's Syndrome by Holliday Junction Resolution. Rejuvenation Research 10 (1) 27-40))
Figure 4. Identification and characterization of Drosophila WRN exonuclease. (A) We identified the gene on chromosome 3R 91A3 at locus CG7670. (B) A piggyBac insertional mutation greatly reduces gene expression (as shown here by RT-PCR). (C) Flies with the hypomorphic pBac mutation show excess homologous recombination, as detected by emergence of clones of multiple wing hairs. (D) We cloned the cDNA for DmWRNexo, expressed it and purified the encoded protein, which shows 3'-5' exonuclease activity in a dose and time dependent manner. (E) We carried out mutagenesis of the gene, and correlated genomic instability with various mutations (Blue and red). We propose that the mutation D229V affects the fold of the protein which could impact either on DNA path through the nuclease or on protein-protein interactions. (see also Saunders RD, Boubriak I, Clancy DJ and Cox LS. (2008) Aging Cell 7 (3) 418-425; Cox, L S, Clancy, DJ, Boubriak, I and Saunders, RDC. (2007) Annals New York Acad. Sci. 1119: 274-288; Boubriak, I, Mason, PA, Clancy, DJ, Dockray, J, Saunders, RDC, Cox, LS. (2009) Biogerontology, 10(3): 267-77.Mason PA, Boubriak I, Robbins T, Saunders RDC and Cox LS (2013) Age (Dordr). 35(3):793-806; Mason, PA, Boubriak, I and Cox, LS (2013) J Vis Exp. Dec 23;(82):e50722. doi: 10.3791/50722).
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