Towards understanding the biological drivers of cellular ageing
Human ageing is associated with gradual loss of tissue and organ function, with consequent frailty and illness leading to poor quality of later life for many older people. It is thought to result from a failure to repair the body as cells stop dividing and become senescent. We will investigate the causes of senescence at the very fundamental level of the genes involved and the proteins they encode, using an experimental system in which we will turn off an ageing-associated gene in normal human cells to induce senescence, and analyse the proteins that are altered as cells enter the senescent state.
Using the powerful modern technique of proteomics, we can simultaneously measure the levels of many different proteins in the senescent cells and compare them with cells still able to divide; the technique even allows us to look at tiny chemical changes on the proteins that affect their function. This study will allow us to identify proteins or protein modifications involved in the onset of senescence, with the longer-term aim of highlighting novel drug targets that could be used to treat or prevent conditions that currently contribute to major loss of life quality for many older people.
Lynne Cox, University of Oxford
Penelope Mason, University of Oxford
Partner and Collaborator:
The Proteomics Facility, University of Oxford
Biological ageing results in decreased ability of the body’s cells to proliferate and renew damaged tissues. This failure of cell division - replicative senescence - leads to loss of tissue and organ function, and the increased frailty characteristic of the oldest old. Since many factors are thought to contribute to this loss of function, it is hard to dissect any individual causes that represent the key biological drivers of ageing.
A premature ageing syndrome, Werner’s syndrome (WS), in which patients show many normal features of ageing but in an accelerated manner, provides an excellent model for studying normal ageing in a simplified biological system, as mutation of a single gene (WRN) leads to a very wide range of ageing characteristics: loss of WRN is in itself a driver of ageing.
We will exploit this experimentally by removing WRN from human cells in culture and assessing the degree of cellular ageing in terms of cell size, shape, ability to divide and to maintain the cellular DNA (genes and regions that regulate gene activity).
A comparison of cells in which WRN is normal and those in which its activity has been blocked experimentally will be carried out to determine which proteins are elevated and which diminished or modified in ageing cells.
Proteins identified as having a causative role in driving ageing present good targets for inhibitory drugs to prevent or attenuate loss of cell function, while proteins that are shown to decrease on ageing may highlight alternative therapeutic strategies necessary to maintain function in old age.
The overall aim of this project is to identify proteins associated with, and causative in, cellular ageing.
The following are the objectives of the three experimental strategies:
Develop tools to induce cell senescence by regulated loss of WRN helicase/exonuclease: We will generate molecular tools including miRNA for regulated knockdown of WRN in stably transfected cells. This will provide isogenic cells lines that differ initially only in terms of WRN expression.
Determine senescent phenotype upon miRNA-WRN expression: We will assess key phenotypes of senescent cells following WRN knockdown to verify that the experimental treatment is robust and reproducible in causing cellular senescence in vitro.
Conduct proteomic comparison of miRNA-WRN induced senescence vs non-senescent cells: Heavy isotope amino acid labelling will be used followed by proteomic analysis of senescent and non-senescent isogenic cells to assess differences in protein levels and modification following experimentally induced senescence.
Discovery and documentation (including publication in peer-reviewed journals) of cellular and proteomic differences in senescence consequent on WRN knockdown, which will hugely increase ourunderstanding of the WRN-related ageing phenotype, and support knowledge-based research of normal ageing.
A system for looking at the senescent proteome in vitro, that can be disseminated as a validated tool to other researchers in the field.
Use of this system as a tool for high-throughput analyses of possible inhibitor molecules which may be used to inform future drug and therapy trials both for WS and associated disease symptoms, and to increase healthy ageing.
Strengthened links with the UK and international ageing research community and also the wider public (current and future end-users)
Interdisciplinary research collaborations with a mandate for future ageing research and pharma development.