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The Role of Nedd4l in the Regulation of Muscle Stem Cell Function

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The Role of Nedd4l in the Regulation of Muscle Stem Cell Function The Role of Nedd4L in the Regulation of Muscle Stem Cell Function Claudia Yvette Dominici Department of Human Genetics, McGill University, Montreal December 2016 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science © Claudia Dominici, 2016 Abstract Muscle wasting diseases exist as a spectrum of diseases in which muscle function is impaired. Adult stem cells are the drivers of regeneration in damaged tissue. In patients with muscle degenerative diseases, the balance between the processes of muscle stem cell (MuSC) self-renewal and differentiation is perturbed; thus creating an environment that is not conducive to tissue homeostasis. Therefore, in order to assist in developing effective cell-based therapies for muscle wasting diseases, we must investigate the molecular mechanisms that are crucial for maintaining the critical balance that promotes normal MuSC function. E3 ubiquitin ligases target proteins for degradation through the proteasome, and they are known to be critical regulators of cell function. Interestingly, our data indicate that in muscle stem cells, Nedd4L (Neural Precursor Cell Expressed, Developmentally Down-Regulated 4-Like) is the only E3 ubiquitin ligase that is highly up regulated during a specific window following MuSC activation. Given this unique time frame, we hypothesize that Nedd4L is involved in a specific set of cellular functions that determine whether an activated satellite cell will self-renew or differentiate. In order to elucidate the function of Nedd4L in the regulation of MuSCs, I have utilized a series of in vitro and in vivo analyses. C2C12 mouse myoblasts were used to generate stable cell lines overexpressing Nedd4L and a mutated Nedd4L to assess the effect of Nedd4L on their proliferation and differentiation. Additionally, we deleted Nedd4L in MuSCs using the Cre/LoxP system to study the effect of the loss of Nedd4L on MuSC number and regenerative capacity of the whole muscle. Through these experiments, I have begun to characterize the role of Nedd4L in the MuSC context. ! Abstract Maladies musculaires perdre existent comme un spectre de maladies dans lesquelles la fonction musculaire est altérée. Les cellules souches adultes sont les moteurs de la régénération dans les tissus endommagés. Chez les patients atteints de maladies dégénératives musculaires, l'équilibre entre les processus de cellules souches musculaires (MuSC) auto-renouvellement et la différenciation est perturbée; créant ainsi un environnement qui ne favorise pas l'homéostasie tissulaire. Par conséquent, afin d'aider à développer des thérapies à base de cellules efficaces pour les maladies musculaires perdre, nous devons étudier les mécanismes moléculaires qui sont cruciales pour le maintien de l'équilibre critique qui favorise la fonction MuSC normale. E3 ubiquitine ligases ciblent les protéines de la dégradation par le protéasome, et ils sont connus pour être des régulateurs critiques de la fonction cellulaire. Fait intéressant, nos données indiquent que dans les cellules souches musculaires, NEDD4L (Neural Precursor cellulaire exprimée, Developmentally Down-Regulated 4-Like) est le seul E3 ubiquitine ligase qui est très réglementé en cours d'une fenêtre spécifique après activation Musc. Compte tenu de ce laps de temps unique, nous émettons l'hypothèse que Nedd4L est impliqué dans un ensemble spécifique de fonctions cellulaires qui déterminent si une cellule satellite activé sera auto-renouvellement ou de se différencier. Afin d'élucider la fonction de Nedd4L dans la régulation de muscs, je l'ai utilisé une série d'in vitro et in vivo analyse. Myoblastes de souris C2C12 ont été utilisés pour générer des lignées cellulaires stables surexprimant Nedd4L et un Nedd4L muté pour évaluer l'effet de Nedd4L sur leur prolifération et leur différenciation. De plus, nous avons supprimé Nedd4L dans muscs en utilisant le système Cre / LoxP pour étudier l'effet de la perte de Nedd4L sur le numéro Musc et la capacité de régénération du muscle entier. Grâce à ces expériences, je commence à caractériser le rôle des NEDD4L dans le contexte MuSC. " Acknowledgements I would first like to thank my thesis supervisor, Dr. Vahab Soleimani of the Department of Human Genetics at McGill University, for his support and guidance through this important stage in my career, and without whom this thesis and all of my academic successes in the last few years would not have been possible. I would also like to thank all former and present lab members who taught me many techniques along the way and provided good company throughout my research. I would specifically like to thank Dr. Zenghui Wu for his patience in training me when I initially came to the lab. I would also like to thank Mr. Jianhong Liu for making sure things were running smoothly and for his assistance with multiple rounds of fiber isolation. I would like to also acknowledge my supervisory committee members, Drs. Marc Fabian and Colin Crist, for providing valuable guidance and useful feedback on my thesis research. Finally, I must say thank you to my parents, Madeleine and Peter, for supporting me on my path to pursue science from a young age; to my brother Matthew, for always encouraging me not to be afraid of taking a challenge; to Kyran, my partner in crime, for always giving me a reason to smile; and to Cooper, my furry friend who has followed me to 5 cities. # Table of Contents Title page………………………………………………………………………………………….1 Abstract……………………………………………………………………………………………2 Abstract (French) …………………………………………………………………………………3 Acknowledgements……………………………………………………………………………….4 List of Abbreviations……………………………………………………………………………..7 List of Figures……………………………………………………………………………………9 1.0 Introduction…………………………………………………………….…………………….10 1.1 Stem cells…………………………………………………………………………….10 1.2 Muscle stem cells (satellite cells)……………………………...…………………….11 1.3 The satellite cell niche………………….……………………...…………………….11 1.4 Satellite cells in regeneration……………………………...…………………………12 1.5 Muscle wasting disease……………………………...……………………………….13 1.6 Cellular protein removal and the Ubiquitin-Proteasome System (UPS)…………….14 1.7 E3 Ubiquitin ligases………………………………………………………………….17 1.7.1 Nedd4L…………………………………………………………………..20 1.8 Objectives…………………………………………………………………………....27 2.0 The role of Nedd4L in C2C12 myoblasts……………………………………………………35 2.1 Introduction……………………………………………………………………..……35 2.2 Materials and Methods………………………………………………………………38 2.3 Results………………………………………………………………………………..43 2.4 Discussion……………………………………………………………………………46 $ 3.0 in vivo characterization of a conditional Nedd4L knockout mouse …………………………50 3.1 Introduction…………………………………………………………………………..50 3.2 Materials and Methods……………………………………………………………….52 3.3 Results……..…………………………………………………………………………55 3.4 Discussion. …………………………………………………………………………..58 4.0 Summary and Future Directions…………………………………………………………….64 References………………………………………………………………………….…………….67 % List of Abbreviations ATP: Adenosine triphosphate DMD: Duchenne Muscular Dystrophy DNA: Deoxyribonucleic acid ECM: Extracellular matrix FACS: Fluorescence-activated cell sorting HECT: Homologous to the E6-AP Carboxyl Terminus HERC: HECT and RLD domain Containing E3 Ubiquitin Protein Ligase 3 IGF-1R: Insulin growth factor 1R kDa: Kilo Dalton MPC: Myogenic precursor cells mTOR: mechanistic target of rapamycin MuSC: Muscle stem cell N4-KO: Nedd4 knockout N4L-cKO: Nedd4L conditional knockout N4L-KO: Nedd4L knockout P13K: Phosphoinositide-3-Kinase PBS: Phosphate buffered saline PD: Parkinson’s disease PFA: Paraformaldehyde PLA: Proximity ligation assay PY motif: (ppxy) proline, proline, x, tyrosine & RBR: RING between RING RING: Really interesting new gene RNA: Ribonucleic acid UPS: Ubiquitin proteasome system ' List of Figures Figure 1. Progression of embryonic stem cells…………………………………………………..29 Figure 2. Myogenic lineage……………………………………………………………………...30 Figure 3. General mechanism of the E1, E2, E3 ubiquitination pathway………………………..31 Figure 4. 2D structures of Nedd4 family of E3 ubiquitin ligases………………………………..33 Figure 5. Microarray gene expression profile of Nedd4 family members in various stages of satellite cell activation………………………………………………………………………...…34 Figure 6. Aim 1: in vitro characterization of Nedd4L in C2C12 myoblasts…………………….49 Figure 7. Aim 2: in vivo time course expression analysis of Nedd4L and Nedd4 protein…...….61 Figure 8. Aim 2: in vivo characterization of Nedd4L using a conditional knockout mouse model……………………………………………………………………………………………..62 ( 1.0 Introduction Muscle stem cells function under the control of tightly regulated molecular pathways, many of which are not well understood. The progression of a muscle stem cell through the myogenic lineage necessitates the removal of certain proteins along the way – this is an essential process that involves protein ubiquitination by the E3 ubiquitin ligase Nedd4L, which will be the focus of this thesis. 1.1: Stem cells When tissue is damaged through disease or injury, there is an immediate need for repair. Stem cells are the drivers of regeneration in damaged tissue, and are classified based on how differentiated they are. Totipotent stem cells are the most naïve and can differentiate to give rise to entire embryo. Pluripotent stem cells, which can give rise to all individual tissue types, arise from totipotent stem cells. As pluripotent stem cells differentiate, they give rise to multipotent stem cells that are primed to carry out a specific differentiation program. Adult multipotent stem cells remain undifferentiated
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