The role of cell senescence in the age-related dysregulation of proteostasis in tendon

Tendons connect muscles to bone and are essential for movement. They stretch and recoil as we walk and run, improving the efficiency of our movements. Even taking a few steps without the Achilles tendon connecting our calf muscle to our heel would be very difficult. Unfortunately tendons are regularly injured, not only in athletes, but also during normal every day activities, particularly as we get older. Injuries are painful and can be highly limiting, preventing usual activity and decreasing quality of life. They cost the economy millions of pounds every year, not only in treatment costs, but also due to the indirect costs of both work absence and reduced health due to an inability to exercise. Horses also often suffer from tendon injuries, and provide an excellent model in which to study tendon ageing. The risk and characteristics of tendon injury are very similar between humans and horses, and tendons can easily be obtained from young and old horses that are put to sleep for reasons other than tendon injury.

Tendon injuries in both humans and horses are difficult to treat, due to a poor understanding of the underlying causes of the age-related decrease in tendon function that leads to increased risk of injury. In other tissues within the body, a common cause of age-related diseases is disruption of the balance between the synthesis and breakdown of proteins that make up the tissue. This process is known as proteostasis, and must be carefully regulated to maintain tissue health. As a tissue ages, its resident cells often lose their ability to grow and divide, and also begin to produce molecules than can cause protein breakdown. These cells are referred to as senescent cells, and cell senescence can disrupt the balance between protein synthesis and breakdown within a tissue. This can reduce the tissue's ability to function properly, which may eventually lead to injury.

In this study, we will investigate the relationship between proteostasis and cell senescence in tendon with ageing. First, we will establish how proteostasis alters with ageing in different regions of the horse forelimb superficial digital flexor tendon (SDFT). We will then identify the location of the senescent cells within the SDFT from aged horses, and develop methods to isolate these cells from tendons so we can study them in the laboratory. Using these isolated cells, we will establish how protein synthesis and breakdown changes when the cells become senescent. Finally, we will determine if reversal of senescence by treating the cells with small molecules can restore the balance between protein synthesis and breakdown.

The results of this study will allow us to understand, for the first time, the link between cell senescence and proteostasis in tendon. By establishing the effect of reversing tendon cell senescence on protein synthesis and breakdown, we may discover new therapies that can be used to prevent and treat age-related tendon injuries in the future, both in humans and horses.

This project will establish if age-related dysregulation of proteostasis in the tendon interfascicular matrix (IFM) is driven by cell senescence in this region. The IFM binds tendon fascicles, and in previous BBSRC-funded work we have shown that the IFM is highly cellular and more metabolically active than the fascicles it surrounds. With ageing, we have demonstrated a decline in cell activity and altered mechanical properties in the IFM which indicate a loss of proteostasis, and our pilot data show expression of senescent markers localised to the IFM of ageing tendon.
We therefore hypothesise that: with ageing, proteostasis in tendon is impaired in the metabolically active IFM. We further hypothesise that dysregulated turnover in ageing is linked to tendon cell senescence which initiates in the tendon IFM.
We will assess how proteostasis is altered in tendon compartments with ageing by estimating the turnover of individual proteins within the fascicles and IFM from tendons with a wide age range by measuring the rate of protein deamidation, using a combination of laser capture microdissection and tandem mass spectrometry. We will establish the location of senescent cells within tendon by integrating existing single cell RNA sequencing data with co-detection of RNA and protein in tissue sections. We will then isolate the senescent cell populations using cell sorting techniques, characterise their senescence associate secretory phenotype and establish how senescence affects synthesis and turnover of extracellular matrix proteins. Finally, we will determine if reversal of senescence using small molecule modulators can restore proteostasis.
This project will answer fundamental questions regarding the role of proteostasis and cellular senescence in tendon ageing. Ultimately, this work may inform new regenerative medicine strategies aimed at addressing age-related functional impairment in tendon, by developing therapeutics to limit senescence and maintain proteostasis.