ADAMTS5 in healthy muscle and muscular dystrophy Adam Thomas Piers ORCID ID – 0000-003-4914-3451 Doctor of Philosophy November 2016 Faculty of Veterinary Science The University of Melbourne This thesis is submitted in total fulfillment of the requirements of the degree. i ii Abstract A Disintegrin and Metalloproteinase with Thrombospondin motifs 5 (ADAMTS5) is an extracellular matrix (ECM) protease that has been shown to exacerbate cartilage destruction in a murine arthritis model. Its function in skeletal muscle remains relatively unknown, aside from in vitro work highlighting its importance in myoblast fusion (1), and embryonic neuromuscular development (2). Microarray analysis of adult muscle from the mdx mouse model of muscular dystrophy showed that Adamts5 gene expression was upregulated compared to wild type mice (3). Significantly, this finding was supported by work showing that ADAMTS5 protein levels were elevated in the serum of both mdx mice and Duchenne muscular dystrophy (DMD) patients (4). This study went further to demonstrate that ADAMTS5 serum levels were amenable to anti- sense oligonucleotide exon skipping treatment, thus demonstrating that ADAMTS5 may be a therapy-responsive biomarker for future pre-clinical DMD trials. The first part of this thesis investigated the role of ADAMTS5 in adult skeletal muscle by comparing wild type (WT) and Adamts5-/- knockout (KO) mice. The proteolytic cleavage of a known ADAMTS5 proteoglycan target, versican, was utilised as a readout of ADAMTS activity. Versican cleavage was detected using a neo-epitope antibody specific for the newly created C- terminal DPEAAE sequence of the cleaved versican product. It was demonstrated that versican cleavage was significantly reduced in KO muscle compared to WT muscle. The lack of upregulation in the gene expression of other known ADAMTS versicanases in KO mice demonstrated that ADAMTS5 was the major versican processing ADAMTS member in skeletal muscle. However, despite these differences in proteolytic activity, no differences were observed in postnatal muscle growth or function between WT and KO mice. Thus demonstrating that ADAMTS5 is dispensable for postnatal muscle development and function. Positive immuno-staining for ADAMTS-cleaved versican fragments at the neuromuscular junction and around endothelial cells suggested that ADAMTS proteinases may play a role in these tissues. Previous work has shown that the proteolytic processing of ECM proteins is important for angiogenesis (5), while Adamts5 has been detected in the developing murine neuromuscular junction (2). To investigate the role of ADAMTS5 in angiogenesis and neuromuscular changes, WT and KO mice were exercised. Endurance exercise induces skeletal muscle adaptations and remodelling of the ECM, including the upregulation of various ECM proteins and proteases (6-8). This thesis showed that exercise significantly upregulated the gene expression of ADAMTS5 in muscle, but not the activity as measured by DPEAAE immuno- blotting. The most significant result was that the typical exercise-induced adaptations observed in WT muscle, namely oxidative fibre type switching and angiogenesis, were inhibited in KO iii muscle. No differences were observed in the gene expression of the known oxidative pathway regulators calcineurin, PGC-1a, or VEGF-α. These results suggest that ADAMTS5 is involved in how muscle adapts to the demands of endurance exercise, possibly via ECM remodelling at the neuromuscular junction and/or endothelial cells. To investigate whether the genetic ablation of ADAMTS5 ameliorated the pathology of mdx mice, KO mice were crossed with mdx mice to create mdx wt and mdx ko littermates. The gene expression and activity of ADAMTS5 was shown to be increased in mdx wt mice compared to WT mice, which led to the hypothesis that elevated ADAMTS proteoglycan proteolysis may exacerbate the mdx pathology. Previous work has suggested that ECM cleavage products act as damage-associated molecular patterns (DAMPs) to induce pathological inflammatory responses (9, 10). The pathology of 12 week old mdx wt and mdx ko mice were compared based on measures of muscle damage and function. No differences were observed in the levels of tibialis anterior (TA) muscle necrosis, diaphragm fibrosis, or the gene expression of inflammatory factors in the TA muscles of mdx wt and mdx ko mice. No improvement was observed in the force producing capacity of mdx ko TA muscles compared to mdx wt. However, mdx ko mice did display a significant improvement in their resistance to fatigue compared to mdx wt mice. The oxidative profile of mdx wt and mdx ko mice were not different when assessed based on myosin heavy chain isoform expression and gene expression analysis of oxidative markers. Overall, these results demonstrate that the genetic ablation of ADAMTS5 is not a viable therapy for the treatment of DMD. This thesis demonstrated that fibroblasts were responsible for producing ADAMTS5 in the ECM surrounding skeletal muscle. To investigate how the genetic ablation of ADAMTS5 affected the ECM proteome (matrisome), fibroblasts were isolated from WT and KO mice and grown in tissue culture. Gene expression analysis of key ECM proteins demonstrated that KO fibroblast cultures were capable of producing a normal matrix. Proteins were then extracted from WT and KO fibroblasts using a sequential extraction protocol to allow label-free quantitative proteomic analysis to be performed. ECM protein abundance differences were evident in the amount of fibromodulin and fibrillin-1 between the groups, but no major differences were observed in the overall matrisome composition. Principal component analysis revealed variability within the WT samples, while DPEAAE immuno-blotting showed no difference in the processing of versican between WT and KO fibroblasts. These results suggest that ADAMTS5 is less active in vitro in cultured fibroblasts than in skeletal muscle. Future proteomic analysis using skeletal muscle from WT and KO mice may provide more information regarding the role of ADAMTS5 in the matrisome. iv Declaration This is to certity that: (i) the thesis comprises only my original work towards the PhD except where indicated; (ii) due acknowledgement has been made in the text to all other material used; and (iii) the thesis is fewer than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices. Adam Thomas Piers v Preface All work presented in this thesis was completed during the PhD candidature of Adam Piers. The following work was performed in collaboration with others: The initial fibroblast isolation steps were performed with the help of Christopher Kintakas. Dr Constanza Angelucci provided training in sequential protein extractions and mass spectrometry sample preparation. Protein samples were run on the Orbitrap mass analyser by Dr Nicholas Williamson and Dr Ching-Seng Ang at Bio21. Training for the physiological apparatus was provided by Professor Gordon Lynch and Tim Naim at The University of Melbourne. Dr Peter Houweling aided in the setup of the physiological equipment and during some experiments. Dr Jason White kindly helped during the setup of the exercise cages and during the initial computer analysis. Finally, I am grateful to Dr Chantal Coles for training me in the dark art of Western blotting. vi Acknowledgements This has been a hugely challenging and rewarding journey, and one that I could not have undertaken without the incredible people that have been by my side here in Melbourne and across the Nullarbor. I would like to start by thanking my two supervisors Jason and Shireen. ADAMTS5 has been a hard beast for all of us to tame, but the two of you have always been there pushing me to finish. Thank you for affording me the freedom to explore my ideas, but also knowing when to reign me in when I’ve inevitably drifted off on a tangential line of enquiry. Your support has taken me across the world presenting my work and meeting some of the greatest minds in our field. I think I’m finally beginning to understand what a scientist is. To the past and present members of our lab, I also thank you for your support. I want to single out Chantal and Chris as we have formed quite the TS5 team over the past couple of years. I have loved our deep Friday chats, epic lab sessions, conference trips and of course the onion farmer shenanigans. You are both very dear friends to me and I look forward to many more adventures together out of the lab. I also want to extend my thanks to Keryn in Lebron country, Liam in Memphis, Connie in Tassie, and Pete, Alex, and Marta here in Melbourne, who were all there for me as friends and as constant sources of inspiration. Finally, I want to acknowledge the support of Boris Struk at Muscular Dystrophy Australia, who has been a vital supporter of my research. Living away from my family over the course of my PhD has been hard, but has also led to me forming the most incredible connection with my friends. In particular Willow, Tahlia, Glynn and Lauren. I have lived with all of you at different stages over the past 5 years, and I want to thank you for your immense support and warmth. Big thanks to Henrik, Akin and the TSML crew for your wise words and love. I am also deeply indebted to the Spiller family; Marisa, Bruno, Dan, and Caity, for taking me in and supporting me like a member of your family. I have always felt extremely loved and welcome in your home, and will never forget what you did for me. To my amazing family, you have given so much from afar. My trips home to the West always filled me with such love and energy, especially at the end. I will never forget my time sitting in the front room of 105 writing my chapters as I looked out on the street listening to The Boss. Thank you Mum, Dad, Hannah, Holly, Kuks, Wallace, Kaia, Bess and Bonnie for always being on the end of the phone when I needed you and for telling me that you were proud of what I was trying to accomplish.