Investigating the role of the primary cilium in muscle regeneration

Skeletal muscle is an important tissue, not only for athletes or young people, but for everyone during their everyday life. As we get older our muscle mass and strength tends to diminish and to support continued physical health and a good quality of life it is important to keep muscle healthy. This tissue has an amazing ability to repair itself after injury and muscle can rebuild itself, for example after long-term immobilization by doing exercise. This regenerative capacity depends on a population of stem cells that is present in muscle. These stem cells are called satellite cells (SC) and they reside in a specialised location, called a 'niche', within the muscle.
The SC are normally quiescent (inactive), but in response to signals from the body they become activated and begin to multiply. The descendents of SCs will differentiate into more muscle, but also re-enter the niche in order to maintain the pool of stem cells throughout life. After SCs are activated they migrate along muscle fibres towards sites of injury, which they then repair.
As we get older we lose some of our SCs and the remaining ones become less efficient at generating more muscle. So it is important to understand how to keep these cells healthy. We have found that SCs have a cellular protrusion called a primary cilium, which acts as an 'antenna' for important signals. One such signal is the Sonic hedgehog protein (Shh), which we know needs a primary cilium to be sensed by the cells. We have created a unique line of mice, engineered to carry mutant SCs, which lack a primary cilium. We showed that this leads to much less efficient muscle regeneration, with thinner muscle fibres, after injury. Our finding suggests that activating the Shh pathway may have potential therapeutic benefits for muscle diseases, or help sustain muscle function in the elderly.
First we need to investigate in more detail how the primary cilium and Shh affect SC function - and this is what this project is about. We will examine the ability of the mutant SCs to become activated, to divide, to migrate, to differentiate, to re-enter their niche. It is also possible that SCs die if they don't have a cilium and we will test if muscle repair can be improved by injecting a drug that activates the Sonic hedgehog signalling pathway. All the methods we need to do these experiments are established in our laboratories. In addition to the detailed characterization of the repair mechanism we will examine the molecular changes that happen in SCs that do not have a primary cilium, specifically we will ask which genes are expressed differently as a result. This will give additional important insights and may reveal potential novel therapeutic targets in muscle disease, or help to maintain a healthy tissue in elderly and frail people.

To investigate effects of primary cilia-loss on SC function, Ta3-floxed mice were crossed with Pax-7CreERT2 mice, leading to tamoxifen-inducible Cre recombinase-mediated deletion of Ta3 in Pax7-expressing postnatal muscle SCs. Pilot data show that Ta3-loss significantly impairs muscle repair after injury with a myotoxin (cardiotoxin). Underlying mechanisms will be investigated using:
-IN VITRO assays of primary SCs in culture to examine proliferation/differentiation/ migration/self-renewal and apoptosis (Obj 1a).
-EX VIVO analysis of single muscle fibres will use time-lapse microscopy and immunostaining to determine effects on proliferation/differentiation/ migration/self-renewal and apoptosis (Obj 1b).
Both assays will also be performed in the presence of a Hedgehog (Hh) pathway agonists (purmorphamine), which may overcome the requirement for a cilium/Ta3 (Obj 3). In addition to pharmacological activation of Hh-signaling, we will use a Ta3 expressing retrovirus to restore function in isolated SCs.
-IN VIVO injury by cardiotoxin injection followed by repeat injury after one cycle of regeneration will determine whether Ta3-loss leads to a diminished stem cell pool (Obj 1c). In vivo rescue experiments will examine the effects of systemic delivery of the Hh pathway agonist, purmorphamine, and of microRNA: miR-133 (Obj 3)
-The impact of Ta3 loss on the stem cell niche will be investigated with a focus on basement membrane components and receptors (Obj 2).
-Global molecular changes resulting from Ta3-loss will be analysed by transciptomics (Obj 4).
The project is underpinned by our finding that muscle regeneration is affected in the absence of Ta3 function. It benefits from complementary expertise in myogenic signalling and mouse genetics, from existing mouse lines, established cell and molecular biology approaches and expert collaborators, who support different aspect of the project.

OVERVIEW: A key component of our proposal is the hypothesis that reduced function or loss-of-function of primary cilia is responsible for specific muscle defects. This could be relevant for increased skeletal muscle weakness and/or the diminished ability to repair muscle injury in disease states or in older people. As such, our research has the potential to benefit a range of clinical researchers and health professionals, students, patient and family groups. Furthermore, the increased understanding of mechanisms that underlie muscle regeneration, will underpin the development of new treatments and interventions to maintain muscle strength. This would have significant economic benefits by allowing people affected by muscle weakness and frailty to fully integrate in society and contribute to the economy. It will also potentially increase the 'healthspan' during ageing and therefore benefit the NHS, as it will reduce the financial burden to the health services.

CLINICAL RESEARCH - MYOLOGY: In the short term, our research will impact academic and clinical researchers interested in skeletal muscle disorders and in ciliopathies. In particular, clinical researchers interested in the causes of progressive muscle weakness, which is also associated with some ciliopathies, could benefit from our studies. For example, if we find that Shh pathway activation benefits muscle repair, this will likely stimulate further clinical research studies to assess the beneficial role of Shh agonists in patients and/or the elderly who may be at risk of increased muscle weakness.

CLINICAL RESEARCH - HUMAN GENETICS: A potential longer-term effect is the development of improved screening methods and diagnostic tools to detect impaired cilia function in skeletal muscle that could lead to preventative treatment regimes. Genetic counselors would benefit from a more detailed understanding of the critical mechanisms underpinning effective skeletal muscle regeneration, which can be passed on to patients and families by health professionals.

CLINICAL RESEARCH - CANCER: Shh signaling is a major regulator of cell differentiation, cell proliferation. Aberrant activation of the pathway is observed in human cancers, including, basal cell carcinoma, malignant gliomas, medulloblastoma, leukemias, breast, lung, pancreas, and prostate cancer. Important targets for cancer therapeutics include the Smoothened co-recpetor and the Gli transcription factors, whose processing is regulated within the cilium. Increasing our understanding of the biology of the Shh pathway, and in particular the role of the cilium, can therefore be translated to cancer studies and to the development of better targeted cancer therapeutics.

SOCIETAL IMPACT - EDUCATION: Applicants provide several lectures on muscle development, disease and stem cell biology to Undergraduate and Masters students at the University of East Anglia. These lectures benefit from the use of topical examples from our research.

SOCIETAL IMPACT - IMPROVING UNDERSTANDING: Both Münsterberg and Mayer pass on knowledge to the wider public by participating in University Open Days and by seeking opportunities to give general science talks (e.g. Pint of Science, Science Café, Teacher Training Network). These will include accessible presentations on topics such as muscle diseases, for example muscular dystrophy, or the role of muscle stem cells in muscle repair.