The role of p38 MAPK in the adult muscle stem cell fate and regeneration in ageing

In part due to advances in modern medicine, many of us are living longer, and a greater proportion of the population of the Western world is over 60 than ever before. However, there is reduced benefit to these extra years of life if they are overshadowed by poor health; we want to increase 'healthspan' not 'lifespan'. What determines healthy ageing? What does it mean to die 'of old age' rather than some disease? We do not understand these processes yet. One strong candidate for the maintenance and repair of the body is a specialized cell called the adult stem cell. These differ from the embryonic stem cells that are so often in the press. Whereas embryonic stem cells have the ability to differentiate into any lineage (and become any tissue), the adult stem cell is committed to repairing and regenerating the tissue in which it resides. It achieves this by i, activating in response to cues, such as injury ii, proliferating, providing a pool of cells that will repair the damage and iii, through one of these cell divisions, replacing itself for future use. In the elderly, the ability of adult stem cells to perform these functions in markedly reduced, leading to gradual tissue deterioration. A good exemplar of this is skeletal muscle. After the age of 50, skeletal muscle mass declines by ~10% per decade; muscle also becomes weaker and contains more fat. On the positive side, studies in animals have determined that the decline in muscle adult stem cell (called the 'satellite' cell) behavior in ageing is not due to defects within the cell itself but is rather because the cell does not receive the correct signals from its environment. The overall aim of this project is to examine the theory that a key signaling molecule, called p38 MAPK, has a key role in interpreting these signals from the environment and dictating the way in which satellite cells subsequently behave; it therefore acts as a 'gateway'. p38 regulates many of the important events in muscle satellite cell activation, ranging from stopping cells dividing to changing the genes that are activated and therefore the proteins that the cell can make. If p38 is activated too soon, adult stem cells will not have sufficient opportunity to form enough cells for repair and replacement. We will address the following specific questions: 1) Does increased p38 activity restrict the 'choices' of muscle satellite cells? What would happen if p38 activity were suppressed? We will answer this by generating mice that lack p38 in adult muscle stem cells (knockout mice). We expect that they may produce increased numbers of precursor cells. 2) What are the first stages by which p38 regulates gene expression? DNA does not exist as a naked strand but is rather wound around groups of proteins called histones (the whole unit is called chromatin). Histones can be modified by the addition of methyl groups; it is now believed that these modifications ultimately change the shape of chromatin, either making it compact and inaccessible to molecules that will encourage gene expression (transcription factors), or 'opening' the chromatin so that transcription factors can function. We will examine two fundamental histone modifications, which regulate each of these events. 3) What are the signals that control p38 activation? Some of these will come from outside the cell, and some from inside. We will increase or decrease the activity of candidate regulators and determine the consequences for p38 activity and satellite cell function. 4) Most important, what changes occur in p38 activity during ageing? What happens if we prevent p38 activation in muscle satellite cells from old animals? We will test this using the knockout mice made in point 1. We predict that the normal decrease in satellite cell activation and proliferation that occurs in old mice will be ameliorated in the p38 knockout mice. This may prevent part of the decline in muscle mass that occurs in ageing.

Continued health depends on the ability of adult stem cells to maintain tissue integrity by generating precursor progeny with the potential to both differentiate and self-renew. Newly activated stem cells must undergo a period of proliferation, transit amplification, to provide sufficient cells for regeneration. In ageing, this is significantly impaired. Skeletal muscle adult stem cells (satellite cells) are an excellent exemplar; after the age of 50, muscle mass declines by 10% per decade, due to reduced proliferation of satellite cells. This is in large part due inappropriate extrinsic signalling to satellite cells, rather than intrinsic defects. p38 MAPK regulates the progression of transit amplifying cells to irreversible differentiation via phosphorylation of multiple targets to induce cell cycle exit and expression of myogenic genes. The aim of this project is to examine the role of p38 MAPK as a 'gateway' that integrates environmental cues into epigenetic changes to dictate gene expression and adult stem cell behavior. Four related hypotheses will be addressed: 1) p38 activation restricts lineage choice to differentiation at the expense of amplification in muscle precursor and stem cell pools, investigated by generation of conditional p38 knockout mice. 2) p38 has a crucial role in terminal differentiation genes by epigenetic mechanisms, via regulation of chromatin activation (TrxG) and suppression (PcG), determined by genome-wide ChIP-Seq (Illumina sequencing) for H3K4me3 and H3K27me3 in primary satellite cells. 3) p38 is activated in a strictly spatially and temporally regulated manner to ensure sufficient proliferation of progenitors in young animals. Candidate signals will be manipulated and subsequent p38 activity assessed. 4) During ageing, mechanisms that suppress premature p38 activation are impaired. Aged control and p38 knockout mice will be investigated with the expectation that decreased muscle mass will be ameliorated in the knockout mice.

Who might benefit from this research? A key aspect of this study is to manipulate the opportunity for adult stem cells to proliferate and thus determine whether this could ameliorate the age-dependent decline in tissue regenerative capacity. The key beneficiary is therefore the ageing population; realistically this means anyone over 50, after which for example, muscle mass begins to decline by 10% per decade. A minority who might also benefit from this study are athletes, as optimizing muscle response to exercise could improve performance. A further group, outside the scope of this study, comprises cancer patients whose adult stem cells exhibit uncontrolled expansion i.e. the opposite problem to ageing. Since cancer and tissue degeneration are both problems of the elderly, this suggests that knowledge of the mechanisms that regulate adult stem cell behavior is crucial to achieve the goal of 'healthy ageing', specifically increased 'healthspan' rather than 'lifespan'. A third group of beneficiaries potentially include the pharmaceutical industry and healthcare professionals; if mechanisms are identified that could be manipulated without major intervention, then pharmacological and clinical strategies could be developed to improve muscle biology in the elderly, generating wealth for the pharmaceutical industry and easing the burden on the healthcare system. How will they benefit from this research? We hope to make one of the first observations in vivo that changing signaling pathway activity will inhibit the decline in muscle mass and ability to repair that occurs during ageing, and to demonstrate that this has occurred by manipulating the opportunity for the progenitor cell population to expand. If such benefits are observed, then we will have revealed a target for strategic intervention. The major benefit ultimately would be an improvement and enhancement in quality of life in the elderly. This would have significant economic impact in terms of reduced healthcare costs and therefore the nations health and wealth. In terms of timescale, it is unlikely that these benefits would accrue for several years; however, demonstration of the principle that muscle stem cell performance could be improved in vivo would be a significant advance. What will be done to ensure that they benefit from this research? We actively communicate with local and national stem cell, muscle and 'ageing' communities. For example we attend the local Cambridge Wellcome Trust Stem Cell Club, which attracts a very wide audience with ample opportunity for informal interaction; slightly further afield, we have started to attend the excellent and active 'muscle' group meetings at King's College, London. I am a science member of the Muscular Dystrophy Foundation. Also, I was invited to speak at the joint BBSRC/Sports UK meeting (Oct 09). We run a Schools' Day project annually called 'How muscles mend', in which we introduce concepts of stem cell-mediated repair to GCSE and A level students. Our main method of communication is, of course, publications. However, these are not very accessible to the general public, and therefore we plan to add a section to our website explaining our science and findings in 'non-jargon' terms. Babraham has an excellent public relations office, which coordinate events with the public, including for example, open days for local residents and interest groups. At present, we do not have industrial collaboration for this project. However, before the onset of this proposal, we visited Unilever Research, coordinated by Dr Jonathan Powell at Unilever, for an excellent exchange of information with their group leaders who work in the area of ageing research (they have specific muscle ageing/frailty/insulin resistance/genetics of human ageing groups). Their focus is human biology, rather than animal models, and it would be most beneficial to translate the findings of this study into their research systems.