Role of motor unit remodelling in age-related loss of skeletal muscle fibres.

As we age our muscles become smaller and weaker and the reduced muscle function eventually contributes to frailty and poor quality of life. These changes occur partly because we lose a large proportion of the muscle cells (called muscle fibres), but also the muscle that we retain is weak. It is currently unknown how muscle fibres are lost during ageing, but when these fibres die, the motor nerves that normally control their activity are also lost. A fundamental question that has not yet been answered is whether the processes of ageing lead to a loss of proper functioning of nerves that causes loss of the muscle fibres activated by the nerve, or vice versa - that the primary changes in ageing are a loss of muscle that then causes loss of the nerve. This is a crucially important point since effective interventions to preserve muscle, or reverse the age-related changes in muscle function, will need to be targeted at the primary site of changes to be effective.

We believe that, as we age, the way that nerves respond to damage changes such that eventually they become very slow to respond to everyday trauma experienced during activity and this leads to degeneration of the muscle fibres that the specific nerve branch supplies. Understanding whether the muscle fibres supplied by damaged nerve branches show the initial degenerative changes in ageing that eventually leads to loss of muscle fibres is crucial to finding ways of preventing muscle loss in ageing. Microscopes that detect very sensitive changes in nerve and muscle tissue have been developed that now allow us to examine the chronological changes that occur with ageing in individual motor nerves and the muscle fibres that they activate. This approach will be used to examine individual nerves and muscle of ageing mice, since these animals show a similar pattern of ageing and weakness to humans.

The project will therefore use this form of microscopy (intra vital confocal microscopy) to determine whether all of the muscle fibres from old mice show age-related changes in several degenerative pathways, or whether only fibres where the specific nerve branch is disrupted show these changes. Data from this study should determine whether degeneration of the nerve is likely to be the key initial change during ageing. Finally we will undertake a study to attempt to identify factors that are released from muscle of young mice that stimulate rapid re-growth of nerve branches when these are damaged. Such substances are likely to be missing or modified in muscles from old mice, but replacement of them may potentially provide a means of helping preserve muscle during ageing.

Completion of these studies will provide crucial information on the mechanisms underlying muscle weakness during ageing and provide indicators of the type of therapeutic interventions that may help preserve peripheral nerve and muscle function during ageing.

The causes of age-related loss of muscle fibres are unknown, but it is associated with remodelling and loss of motor units. Studies will use novel state-of-the-art approaches to determine how motor unit remodelling (stimulated by crush of the common peroneal nerve and subsequent re-innervation in mice) influences activation of 3 key degenerative pathways (increased hydrogen peroxide, increased pro-inflammatory cytokines, activation of FOXO-dependent atrophic processes) in single muscle fibres in vivo and whether these processes contribute to age-related loss of muscle fibres and weakness.

Intra vital imaging will be used to study the structure of peripheral axons and NMJ in superficial single muscle fibres from thy1-CFP (cyan fluorescent protein) mice expressing CFP in all neurons. Activation of the 3 key degenerative pathways will be examined in the same single muscle fibres transfected with GFP/YFP-based probes using AAV vectors. Studies will examine whether transient denervation of muscle fibres in adult mice leads to activation of these 3 key degenerative pathways in fibres and whether this is confined to fibres with compromised innervation or also occurs in innervated fibres. Studies in old mice will determine whether activation of the 3 key pathways occurs in all fibres or only in those fibres with compromised innervation and will also examine the effect of nerve crush and regeneration on the axons and NMJ of muscles of old mice.

In parallel studies, neuromuscular transmission in single fibres will be examined to determine whether changes in NMJ structure in old mice prevent neuromuscular transmission and the relationship of this to activation of degenerative pathways in the fibre. Finally, we will determine whether degenerative pathways activated in denervated muscle generate muscle-derived axonal sprouting factors, and muscle mRNA will be analysed to identify potential muscle-derived axonal sprouting factors that are attenuated by ageing.

Researchers in the area of geriatrics, gerontology and frailty will be direct beneficiaries upon completion of this research in addition to those studying other neuromuscular disorders associated with denervation and muscle atrophy such as motor neurone disease (ALS), diabetes and incontinence.

The pharmaceutical and personal care products sectors of industry will also benefit from this research. Data generated throughout the project may provide a resource to guide identification of pharmaceutical or non-pharmaceutical interventions to reduce age-related loss of muscle mass and function. With the increasing older population there is increased demand for anti-ageing products. Anti-ageing products have enormous economic potential for the pharmaceutical and personal care products sector and there is therefore potential for EU and UK economic benefit and improved quality of life as a consequence of the development of anti-ageing products. Understanding the mechanisms by which loss of muscle mass occurs with increasing age is also relevant in the agriculture and animal husbandry industry, since meat yields decline in older farm animal species.

In the longer term the impact of this research will be on the health and quality of life of older people. Thus, local and national charities and policy-makers may benefit from regular contact with the research group throughout the project to disseminate information to promote and guide healthy ageing. An example of the approach used to facilitate this will be through attendance at the Institute of Ageing and Chronic Disease annual 'lay' Open Day which is advertised as a "Meet the Scientists" day and held at the World Museum, Liverpool. In 2013 it was attended by 1500 people over 2 days and included interested parties such and local and national charities and local MPs. This will provide a forum for briefing such groups in both general terms and with specific information related to this project. In addition, local schoolchildren and teachers are invited to attend the Open Day where we hold specific interactive discussions to inform both schoolchildren and teachers. This will demonstrate the specific need for this research, inform about outcomes and provide an educational forum for increasing awareness of muscuskeletal biology. This approach also helps identify areas which school outreach parties can focus on when small groups of active research staff (at all career levels), including the applicants and postdoctoral scientist funded on this project visit local schools. Full details of these activities are provided in the Pathways to Impact section.

The timescale of development of appropriate interventions deriving from the current work may be several years. However, if successful, the prevention of age-related loss of skeletal muscle mass and function would lead to an improvement in quality of life for elderly individuals with a major economic impact on UK health and social care costs.