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 in the various tissue types. In some tissue contexts such as muscle, when stem cells are activated due to trauma or injury, they exit the quiescent G0 phase of the cell cycle to begin the regenerative process1 . In skeletal muscle tissue, these multipotent stem cells are referred to as muscle stem cells (MuSCs), or satellite cells (Figure 1). MuSCs are the sole cell type in the muscle responsible for repairing and regenerating damaged muscle tissue throughout the life of an organism.
The embryonic origin of muscle stem cells is the dermomyotome, which is an epithelial structure that forms on the dorsal part of the somites of developing embryos2 . The dermomyotome consists of Multipotent stem cells, which can give rise to many adult cell fates
such as fibroblasts, brown fat, and all skeletal muscle. The fate of theses Multipotent stem cells lies with the identity of the surrounding tissues3 .
1.2: Muscle stem cells (satellite cells)
Alexander Mauro first discovered satellite cells in 1967. Using electron microscopy, he examined cross-sections from frog muscles where he noted the presence of a population of mononucleated cells intimately associated with the muscle fibre, situated between the sarcolemma and basal lamina. He postulated that these cells might be the answer to “the vexing problem of skeletal muscle regeneration.”4 Mauro did indeed find the source of muscle’s incredible ability to regenerate, which lies almost entirely with the satellite cell5-7 . Moving now to the modern era, the sophisticated molecular mechanisms that govern satellite cell behaviour remain a vexing problem to scientists. While much has been discovered about the function of satellite cells, the intricacies of the complex pathways that govern their behaviour remain elusive.
1.3 The satellite cell niche
Under normal and healthy conditions, satellite cells remain mitotically quiescent and rest in their niche between the sarcolemma and the basal lamina of individual muscle fibres8 . A healthy niche provides the essential components that are needed to maintain a healthy satellite cell pool. The major niche components can be roughly broken down into three parts: the host muscle fibre, which provides mechanical and chemical signals9 ; the basal lamina, which provides the necessary extracellular matrix (ECM) components for anchoring10 ; and the local
vasculature and associated interstitial cells that bring essential nutrients to the satellite cell11 .
Together, these factors create a microenvironment that facilitates normal satellite cell function.
However, it is important to note that much of the complexity of niche-stem cell interactions remains unknown, which in turn is a significant hindrance for research aimed at expanding satellite cells ex vivo for downstream engraftment into patients with degenerative muscle diseases. Without the proper “soup” surrounding the satellite cell, canonical signalling is perturbed, thus making it extremely difficult to maintain satellite cell stemness and achieve homing back to the niche12 . While there are a host of essential extrinsic factors from the niche that direct satellite cell behaviour, there is also a substantial repertoire of intrinsic factors that play an equally important role in maintaining a healthy satellite cell pool8 .
1.4: Satellite cells in muscle regeneration
Upon muscle trauma or injury, there is a need for muscle regeneration, and quiescent satellite cells will be instantaneously activated. Then, under a tightly orchestrated series of events, satellite cells will proliferate, differentiate, and fuse together to form new muscle fibres that will contribute to mature muscle (Figure 2, reviewed in Yin et al., 20138 ).
A satellite cell’s progression through the myogenic lineage is demarcated by the sequential induction of a well-defined set of transcription factors that activate genes needed throughout the progressing stages. Under normal non-disease conditions, quiescent satellite cells express the transcription factor paired box 7 (Pax7), which is required for the specification of adult muscle stem cells13 . Following activation, Pax7-expressing quiescent satellite cells give rise to myogenic precursor cells (MPCs) that express Myf5 and MyoD14 . These MPCs may either self-renew, return to quiescence15, 16 , or continue along the myogenic lineage and begin
! proliferation. Proliferating myoblasts that do not return to quiescence give rise to myocytes, which begin to differentiate as they exit cell cycle. This process is accompanied by the expression of Myogenin and Mef2 transcription factors17 . Finally, Myf6 (also called MRF4) is expressed as a terminal marker of differentiation18 . Myocytes will now begin to fuse into multi- nucleated myotubes, which will eventually fuse with mature muscle fibre thus completing the process of muscle regeneration19, 20 . Satellite cells must maintain a critical balance between self- renewal and differentiation so that the muscle stem cell pool can be replenished following rounds of muscle regeneration21, 22 . In muscle wasting diseases, this balance is perturbed and normal muscle regeneration is not carried out effectively, resulting in muscle wasting.
1.5: Muscle wasting diseases
There is a spectrum of diseases in which muscle wasting occurs as a symptom of an imbalance in the processes of muscle stem cell self-renewal and differentiation. Perturbations in theses processes create an environment that is not conducive to sustained tissue homeostasis23 .
The most severe disease on the spectrum of muscle wasting diseases is Duchenne Muscular
Dystrophy (DMD). Patients with this disease also experience impaired muscle regeneration as a result of failing to replenish the muscle stem cell pool. A recent study in the United States concluded that DMD affects 1 in every 3,500 males24 . DMD is an x-linked recessive disease that is the result of a mutation in the gene encoding dystrophin, a protein that is required for normal muscle structure and function. The mutated protein results in the formation of fragile muscle fibres due to lack of structural integrity. This subsequently signals satellite cells to activate and regenerate the muscle. Satellite cells are continuously activated in an attempt to regenerate the unremittingly deteriorating muscle. Eventually, the satellite cell pool is depleted when the rate of
" self-renewal is insufficient and muscle can no longer regenerate25, 26 . The patient resultantly succumbs to death by the third decade of life. In order for muscle regeneration to operate normally following muscle damage, there must be a sufficient pool of satellite cells.
The imbalance of muscle stem cell proliferation and differentiation is also a characteristic phenotype of age-related muscle wasting disorders such as sarcopenia17, 21 . The aging satelite cell population looses the ability to maintain quiescence and subsequently is unable to self-renew, resulting in impaired muscle regeneration and eventually severe muscle frailty leading to sarcopenia. Aging stem cells also tend to take on aternative differentiated states, and progress down adipogenic or fibroblastic lineages27, 28 . Additionally, cell senescence seems to be a favorable mechanism in the aging satellite cell29 .
In an effort to develop effective cell-based therapies for these muscle-wasting diseases, we investigate the mechanisms that are involved in maintaining the critical balance between satellite cell quiescence, self-renewal, and differentiation.
1.6: Cellular protein removal and the Ubiquitin-Proteasome System (UPS)
The processes of proliferation and differentiation of any cell type require the orchestrated execution of many cellular processes. An essential component is efficient protein turnover.
Proteins needed for proper cell function must be synthesized and subsequently degraded at the correct times and ratios to ensure cellular homeostasis30 . Identifying the full protein complement
(aka. The proteome) of any cell type can shed major light on how that cell functions. By knowing what proteins are expressed in a cell, one can generate hypotheses about what cellular pathways that cell utilizes, which transcription factors are active, what histone variants are present, and so on. However, one drawback to proteomics studies is that they often only offer a
# snapshot of the proteomic profile without accounting for the opposing processes of protein synthesis and protein degradation31 . These two processes work together to establish a dynamic equilibrium of proteins that are needed for proper cell function, and serve to regulate protein levels in the cell.
Protein synthesis is a time- and energy-consuming process that begins with transcription of RNA from DNA, followed by translation of RNA into amino acid peptides, and finally proper protein folding. These are extremely complex processes that are influenced by cell type, extrinsic signalling, and countless other factors. For the purposes of this thesis however, the focus will be on protein removal. Cells are equipped with mechanisms for removing proteins which can be broken down into two major pathways: Lysosomal proteolysis and the ubiquitin-proteasome system (UPS).
Lysosomal proteolysis
Lysosomes consist of several digestive enzymes encapsulated by a membrane. During the cellular process of autophagy, intracellular proteins are surrounded by autophagosomes (ie.
Vesicles) that eventually fuse membranes with the lysosomes. Proteins are engulfed by the lysosome and proteases along with other enzyme degrade the protein to its amino acid constituents32 . Under conditions that lead to cell starvation, the lysosome will begin to degrade cytoplasmic organelles in a process called macroautphagy33 in an attempt to conserve energy.
Initially, protein degradation was attributed entirely to the lysosomes34, 35 . However, with time it became clear that there must be another mechanism in place that would account for some of the emerging characteristics of protein degradation: substrate specificity (see, for example Poole et
$ al., 1977 36 ) and the dependence on ATP (see, for example Etlinger et al, 197737 ). Eventually, the ubiquitin-proteasome system was identified and accounted for these inconsistences.
The ubiquitin-proteasome system (UPS)
Protein degradation through the UPS begins with ubiquitination of target proteins.
Ubiquitin is an 8.5 kDa protein that exists, as the name would suggest, ubiquitously throughout almost all tissue types. Ubiquitin is covalently bonded to lysine residues of target proteins as a reversible post-translational modification38 . Ubiquitination of proteins can occur as a single instance (monoubiquitination), or as repeated instances (polyubiquitination). The C-terminus of the ubiquitin protein forms a covalent bond with the epsilon amino group on the lysine residues of target proteins through the cascading actions of the E1, E2, E3 enzymes that will be described in detail shortly. In monoubiquitination, a single ubiquitin moiety is covalently bound to the target. A subgroup of monoubiquitination is multiubiquitination, in which a single ubiquitin molecule is bonded to several different lysine residues on target proteins. Polyubiquitination occurs when multiple ubiquitin molecules are bound to each other at a single lysine residue on target proteins, creating a ubiquitin chain. Ubiquitination serves in a broad range of cellular functions, some of which are proteasome-independent39 . For example, mono and multi- ubiquitination has been shown to be a necessary modification of proteins that enter vesicles in the endosomal pathway40 . Polyubiquitination canonically is viewed by the cell as a signal to transport that protein to the ATP-dependant 26S proteasome for degradation41, 42 . UPS-mediated protein degradation regulates a vast range of critical cellular functions, including but not limited to antigen presentation43 and cell cycle44 . It therefore comes as no surprise that deregulated proteasome function can be linked to muscular atrophy45 and several other diseases46-48 .
% 1.7: E3 ubiquitin ligases: crucial components of the UPS
All ubiquitination of proteins is dependent on the E1, E2 and E3 enzymes. The E1 enzyme activates free-floating ubiquitin molecules by forming a thiol-ester bond with a C- terminal Glycine residue on the ubiquitin molecule. The resulting reaction primes the ubiquitin molecule for nucleophilic attack of the E2 enzyme. The E2 enzyme now forms a thiol-ester bond with the ubiquitin molecule in a trans-esterification reaction. The E2 substrate serves as an intermediate in the cascade and holds the ubiquitin molecule until an E3 ubiquitin ligase catalyzes the transfer of the ubiquitin molecule directly to the target protein or ubiquitin molecule where it forms a covalent bond with lysine residues 38, 39 (See figure 3 for a general schematic).
The human genome codes for 40 different E2 substrates, and 600 different E3 enzymes49,
50 . Additionally, a single E2 can react with several E3 enzymes, which adds to the complexity and creates a massive amount of potential interacting partners. To date, some of these interactions have been validated51-53 , but no large-scale study has elucidated all of the potential interactors.
E3 ubiquitin ligases target proteins for degradation through the proteasome and are critical regulators of cell function. The E3 enzymes can be categorized into 3 major groups based on how the ubiquitin molecule is transferred: RING (Really Interesting New Gene) family, RBR
(RING-between-RING), and the HECT (homology to E6AP C terminus) family.
Really Interesting New Gene (RING) family of E3 ubiquitin ligases
The RING family E3 ubiquitin ligases will simultaneously bind the E2 substrate and the target protein to the RING domain and catalyze the transfer of the ubiquitin molecule54, 55 . The
& RING domain has an evolutionarily conserved structure that requires two Zinc ions (Zn2+) at its core for stability56 , and binds to the N-terminal helix of E2 substrates. While several targets of
RING family ubiquitin ligases have been discovered57-59 , the mechanism of what confers substrate specificity for many of them remains elusive60 . RING family ubiquitin ligases may exist as monomers55 , and have recently been discovered to exist in hetero- and homodimers as well61 . Interestingly, in some specific instances the multi-subunit E3s have been shown to delegate substrate recognition to individual components in the complex. For example, in SCF
(Skp1-Cullin-F-box) E3s, the Skp1 component specifically recognizes and recruits F-box containing proteins51 .
An important member of the SCF family is atrogin-1, which has been shown to play a role in muscle atrophy. Mice lacking atrogin-1 have protective effects against denervation- induced atrophy62 . Additionally, atrogin-1 is upregulated in various forms of skeletal muscle atrophy across different model organisms62-64 . It was found that atrogin-1 targets several factors that are needed for normal muscle development such as MyoD and myogenin65, 66 .
RING family E3 ubiquitin ligases have been shown to regulate a plethora of cellular functions67 , and therefore they remain an active area of research that will undoubtedly continue to provide novel insights into the function of RING-mediated ubiquitination.
RING-between-RING (RBR) family
The RBR family of E3 ubiquitin ligases consist of two RING domains (RING1 and
RING1) separated by a conserved bridge called the in-between-RING (IBR) domain. RING2 contains a catalytic cysteine residue that mediates ubiquitin transfer from the corresponding E2 substrate, while the RING1 domain binds the E2 substrate itself68 . RBR E3 ubiquitin ligases are
' a relatively new discovery, so the understanding of how they work mechanistically is in its infancy. However, the field remains of great interest as notable RBR ligases are being discovered. For example, Parkin is an RBR E3 ubiquitin ligase that is often mutated in early- onset Parkinson’s Disease (PD)69 , although its precise mechanistic role in the pathophysiology of PD is yet to be determined.
HECT family of E3 ubiquitin ligases
The third and final category of E3 ubiquitin ligases is the HECT family. These ubiquitin ligases differ from RING and RBR family ubiquitin ligases in that they transfer ubiquitin in a two-step process. In the first step, the ubiquitin that is bound to a cysteine residue on the E2 enzyme is transferred to an active cysteine residue in the HECT domain through a transthioesterification reaction. Then, a lysine residue on the target protein attacks the newly formed HECT-Ubiquitin thioester bond, resulting in the transfer of the ubiquitin molecule to the target protein substrate. Alternatively, the lysine residues in ubiquitin molecules may act as the attacking lysine, resulting in the formation of a polyubiquitin chain on the target protein70 . There are approximately 30 HECT E3 ubiquitin ligases, each of which are involved in crucial cellular processes such as protein trafficking and signalling pathways that dictate cell proliferation and differentiation71 . The HECT domain is located at the C-terminus and it bi-lobed, containing N- and C-lobes towards the N- and C-terminal ends, respectively. The N-lobe interacts with the E2 enzyme, and the C-lobe contains the activated cysteine residue that binds ubiquitin72 . Protein crystallography studies have determines that different HECT domain E3 ligases have varying distances between the lobes, indicating that the bridging region acts as a hinge that allows them
( to come together during the ubiquitin transfer between the E2 substrate and the HECT domain73-
75 .
The E3 ubiquitin ligases that make up the HECT family can be further divided into three groups which are characterized by the contents of their N-terminal domains: Nedd4 family,
HERC family, and “other” E3 ubiquitin ligases.
1.7.1 The Nedd4 family of E3 ubiquitin ligases
There are nine members of the human Nedd4 family of E3 ubiquitin ligases: Nedd4,
Nedd4L, Itch, Smurf1, Smurf2, WWP1, WWP2, Nedl1, and Nedl2. Each member of the Nedd4 family has a characteristic modular domain structure that comprises of an N-terminal C2 domain,
2-4 WW domains, and a C-terminal HECT domain (Figure 4). The C2 domain is involved in cellular membrane interactions and binds to membrane phospholipids to regulate calcium channel function76 . The WW domains are a very important part of the Nedd4 family of ubiquitin ligases, as they confer substrate specificity. They are responsible for protein:protein interactions and contain two highly evolutionarily conserved tryptophan residues which bind to proline-rich
PPXY sequences (PY motifs), or LPSY motifs of target substrates77 . While PY and LPSY motifs are the main recognition signal for the WW domains, some recent research has revealed that the
WW3 domain also binds to a non-canonical VL***PSR motif found in the fibroblast growth factor receptor (FGFR)78 . The Nedd4 ubiquitin ligase is highly evolutionarily conserved, and has a close relative in Sachharomyces cerevisiae, Rsp5 which is the only Nedd4-type E3 ligase found in the organism79 .
Of the Nedd4 family members, Nedd4 and Nedd4L are the most closely related80 .
Therefore, it is not surprising that they share the same E2 substrate called UbcH5b81 . Despite
! their structural homology and shared affinity for the same E2 substrate, these two ligases seem to serve non-overlapping functions and do not share the same suite of target proteins. The most striking example of this can be seen with the fate of their respective mouse knockout models.
Nedd4 knockout (N4-KO) mice do not survive passed embryonic day 18 and are less than half the size of their wildtype littermates82 . Autopsy of N4-KO mice reveals major heart defects and abnormalities in vasculature development83 . The proposed mechanism for this phenotype is through the lack of Nedd4 targeting of thrombospondin-1 (Tsp1), an inhibitor of angiogenesis. A recent study showed that Tsp1 was found in extremely elevated levels in the N4-KO knockout embryos and fibroblast culture83 .
Unlike N4-KO mice, Nedd4L knockouts (N4L-KO) develop normally in utero. However, most die at birth from collapsed lungs, and those that survive birth do not live pass 3 weeks84 .
The inability to inflate their lungs is due to the targeting of the epithelial sodium channel (ENaC) by Nedd4L. The ENaC maintains sodium homeostasis across the lung epithelia, thus Nedd4L- mediated ubiquitination and subsequent removal of the sodium channel results is required to maintain optimal ENaC activity. Therefore, removal of Nedd4L would lead to increased ENaC activity and ultimately premature clearance of fetal lung fluid and failure to inflate the lungs84 .
These findings suggest that Nedd4 and Nedd4L do not have compensatory mechanisms when the function of one or the other is ablated. Nedd4 and Nedd4L have also been implicated in various other important cellular processes.
Functions of Nedd4
Nedd4 plays an important role in regulating several growth factor receptors. One study shows that ubiquitination of the epidermal growth factor receptor (EGFR) controls EGFR levels
! in a ligand-independent manner85 . Nedd4 also targets IGF-1R, a receptor tightly connected to cell growth and proliferation86 . From these two examples, it can be deduced that Nedd4 is able to positively regulate cell proliferation. Further support that Nedd4 promotes proper cell proliferation can be seen with T-cells. In Nedd4 deficient mice, T-cells do not proliferate properly. Under normal conditions, receptor signalling and subsequent proliferation is promoted when Nedd4 ubiquitinates cbl-b, a protein that would otherwise interfere with routine T-cell signalling87 .
Nedd4 is also involved in regulating the neuromuscular junction. While Nedd4 is not expressed in motor neurons, it is highly expressed in skeletal muscle. When Nedd4 is knocked out, the resulting size and number of muscle fibres and motor neurons decreases. The precise mechanism by which Nedd4 regulates this has yet to be elucidates, but it is clear that Nedd4 is essential for proper development of the neuromuscular junction88
Functions of Nedd4L
Nedd4L has been implicated in several highly variable functions. It is best characterized in its role of ubiquitinating and regulating ENaC function. The WW3 and WW4 domains of
Nedd4L target PY motifs in the β and γ subunits of the ENaC channel89 . Subsequent ubiquitination of the ENaC protein results in degradation through the proteasome. This interaction also highlights the importance of PY motif recognition for this E3 ligase, as it was found that mutating the PY sequence leads to Liddle’s syndrome, which causes severe hypertension due to improper regulation of the ENaC by Nedd4L77 .
An interesting new role of Nedd4L was found when it was discovered that it targets the serine-threonine kinase Sgk1, as evidenced by the fact that Sgk1 protein levels dropped with
!! Nedd4L overexpression, and were stabilized in the presence of Nedd4L siRNA90 .
Ubiquitination-mediated degradation of this kinase provides an interesting new non- phosphorylation based mechanism for the regulation of these signalling proteins. Interestingly, there appears to be a feedback loops between Sgk1 and Nedd4L. Sgk1 has been shown to regulate Nedd4L activity through phosphorylation. Sgk1 targets serine residues of the Nedd4L protein for phosphorylation, which happens to be within the WW domain region. The phosphorylation in turn reduces the affinity of Nedd4L for PY motifs in target proteins, and inhibits Nedd4L-mediated protein degradation91 .
To add to the repertoire of signalling pathways that Nedd4L is involved in, a recent study shows that Nedd4L regulates the P13K-AKT signalling axis92 . P13K phosphorylates AKT, which in turn sends the activated AKT to the plasma membrane where it carries out one of several possible downstream functions such as activating CREB and mTOR signalling. Due to its role in cell cycle, the P13K-AKT signalling axis is implicated in cancer progression when perturbed93 .
Nedd4L also regulates the TGF β signalling pathway by targeting Smad2/3 for degradation. TGF β binds to its receptor on the cell membrane to activate a phosphorylation cascade, which results in the phosphorylation of Smad2/3. Activated Smad2/3 is then recognized by the WW2 domain of Nedd4L, and is marked for degradation by the proteasome94 .
Given that Nedd4L is involved in a spectrum of cell signalling pathways, it is not surprising that it also has implications in various forms of cancer. Nedd4L seems to function mainly as a tumour suppressor, and when it is downregulated, leads to uncontrolled cell proliferation. In the context of colorectal cancer, Nedd4L is the only E3 ubiquitin ligase that is significantly downregulated compared to unaffected individuals.
!" This trend suggests that the presence of Nedd4L is associated with a non-proliferative state.
Regulation of Nedd4 and Nedd4L
Nedd4 and Nedd4L have a suite of adaptor and accessory proteins that can regulate ubiquitination of target proteins. In S. cerevisiae, the Nedd4 family homologue Rsp5 is regulated by Bsd2p which controls trafficking of vacuolar enzymes and the Smf1p metal ion transporter95 .
This led researchers to investigate the human homologues of Bsd2P: Ndfip1 and Ndfip2. The PY motifs in Ndfip1 and Ndfip2 facilitate binding to the WW domains in the Nedd4 family of E3 ubiquitin ligases. Once bound, they inhibit the interaction of Nedd4L with target protein through
WW domain occupation and sequestration to other locations in the cell96 .
14-3-3 proteins are ubiquitously expressed in eukaryotes and serve a wide range of functions, which include directing cellular scaffolding and eliciting conformational change of bound phosphorylated proteins97 . As previously mentioned, Sgk1 phosphorylates Nedd4L and inactivates it. Once phosphorylated, Nedd4L recruits the 14-3-3 protein, which elicits an inhibitory effect by occupying the WW domains and prevents binding of target proteins98 .
While there are examples of phosphorylation being inhibitory to Nedd4 family members, it is important to note that there are possible exceptions in which phosphorylation is required to activate them. For example, Itch is phosphorylated by JNK1, which is required to disrupt an auto-inhibitory bond between the WW and HECT domain of Itch99 .
E3 ubiquitin ligase autoregulation is a relatively new discovery, and as such the present evidence lacks depth. However, it has been shown in a few specific cases that self-regulation may be a feature of the Nedd4 family of E3 ubiquitin ligases. One study found that Nedd4L is capable of forming a weak bond between its WW domains and a PY motif located within its
HECT domain, thus inhibiting auto-ubiquitination and subsequent degradation. However, the
!# bond to substrate proteins drastically outcompetes the WW domain-HECT domain bond. Once the substrate has been ubiquitinated Nedd4L is capable of self-ubiquitinating, leading to its downstream degradation100 .
Relevance of Nedd4 and Nedd4L in muscle
The explanation of Nedd4 and Nedd4L function provided above indicates that these two
E3 ubiquitin ligases serve as important components of the machinery that drives cellular processes. They have implications in a wide variety of functions including the regulation of key signalling pathways that determine cell growth, proliferation, differentiation, and quiescence.
However, there has been a limited amount of investigation into the role of these ligases in muscle stem cell function. One study suggests that Nedd4 is important for the development of the neuromuscular junction and muscle innervation88 . An additional study suggests that Nedd4 regulates Pax7 protein levels in MuSCs101 . Beyond this however, very little is known about the role of the Nedd4 family of E3 ubiquitin ligases in satellite cells. This is what prompted our lab to delve into our previously generated gene expression data of muscle stem cells at various stages of activation to observe the levels of Nedd4 family ubiquitin ligases.
Interestingly, the data indicated that both Nedd4 and Nedd4L are expressed in satellite cells. However, their expression patterns are quite different. Our data provides gene expression levels for quiescent, proliferating, and differentiating primary mouse myoblasts. Nedd4 RNA is expressed at uniform levels throughout these phases of satellite cell progression through the myogenic lineage. Nedd4L, however, exhibits a much more dynamic expression pattern (Figure
5). Our data show that Nedd4L is very highly expressed in quiescence. Then, following activation through proliferation in cell culture conditions, Nedd4L transcript levels decrease.
!$ Upon differentiation, Nedd4L transcript levels increase once again, however not to the levels seen in quiescence.
Given this unique temporally-regulated expression signature, we hypothesize that
Nedd4L may be involved in a specific set of cellular functions that determine whether an activated satellite cell will self-renew and return to quiescence, or progress through the stem cell lineage and differentiate, eventually giving rise to mature adult muscle.
As previously mentioned, once a quiescent satellite cell is activated, a series of tightly regulated processes begin to occur which will drive the cell towards self-renewal, differentiation, or a return to quiescence. The complexities of the molecular mechanisms that are in place during these fundamental stages of myogenesis are not well understood.
Given that Nedd4L exhibits a dynamic expression pattern, we suggest that it is an important factor in the fate of an activated satellite cell. In order to elucidate the effects of
Nedd4L in satellite cells, I have utilized a series of molecular biology techniques that have been divided into two major research aims. First, I have observed the effects of Nedd4L overexpression on myoblast proliferation and differentiation in vitro. Second, a Nedd4L conditional knockout mouse was created to observe the effects on satellite cell self-renewal and muscle regeneration.
!% 1.8 Objectives
Aim 1: Assess the role of Nedd4L in cellular proliferation and differentiation
Our preliminary data suggest that Nedd4L is acting in a specific window to elicit effects on satellite cell fate decisions following activation. Once a satellite cell is activated, it must decide whether to differentiate and contribute to mature muscle fibre, or proliferate and return to quiescence. I hypothesize that Nedd4L is acting as a switch to promote cell fate decisions by removing proteins that would inhibit progression down a certain pathway. Through removal of these proteins, Nedd4L is either promoting the satellite cell differentiation program, or a return to quiescence.
In order to determine if Nedd4L regulates cellular proliferation and differentiation,
C2C12 mouse myogenic cells will be used to generate stable cell lines overexpressing Nedd4L and a catalytically inactive Nedd4L for comparison. Proliferation, differentiation, and cell cycle will be analyzed using standard molecular biology techniques.
Aim 2: Investigate the role of Nedd4L in vivo in the muscle stem cell context
While there is a broad rage of research published on the function of Nedd4L in various cell types, tissues, and diseases, there is little known about its role in muscle function. Aim 2 will characterize Nedd4L in the satellite cell context. First, protein expression of Nedd4L will be assessed using standard immunofluorescence techniques and compared to pre-existing RNA expression data. Given that Nedd4L RNA expression has an interesting temporal pattern, I hypothesize that Nedd4L protein will also exhibit a dynamic protein expression pattern at different time points following activation.
!& The literature suggests that Nedd4L acts as a tumour suppressor, and when it is downregulated, uncontrollable cell proliferation occurs. I hypothesize that in the satellite cell context, down regulation of Nedd4L will lead to increased satellite cell proliferation. Further, I hypothesize that the increased ability to proliferate will result in enhanced ability to replenish the satellite cell pool following injury and subsequent regeneration. To shed more light on whether
Nedd4L plays a part in satellite cell proliferation and/or replenishment of the stem cell pool in vivo, a mouse model will be created that has a conditional knockout of Nedd4L exclusively in
Pax7-expressing satellite cells. The knockout model will be used to assess the regenerative capacity of the muscle by introducing injury and allowing 3 weeks for regeneration. The muscle will then be analyzed for replenishment of the satellite pool. Additionally, individual muscle fibres will be assessed for total satellite cell number and expression of myogenic transcription factors.
!'
Figure 1. Stem cell progression from embryonic to adult
MSC: Muscle stem cell. NSC: Neuronal stem cell. HSC: Hematopoietic stem cell. SSC: Skin stem cell
!(
Figure 2. Myogenic lineage
A quiescent satellite cell remains dormant until activated due to injury or trauma to the muscle.
The activated satellite cell can take on one of two pathways: Proliferate and self-renew, or differentiate into myotubes and fuse with the mature muscle fibre.
" A
B
C
" Figure 3. Protein degradation through the UPS
A) Ub: ubiquitin. The E2 substrate removes a ubiquitin molecule from the E1 donor in the first step. Then, the E3 ubiquitin ligase binds to the E2 substrate and the ubiquitin molecule is transferred once more. Finally, the ubiquitinated E3 ubiquitin ligase transfers the ubiquitin molecule to its recognized target protein.
B) The target protein gets ubiquitinated several times, resulting in a branched ubiquitin chain.
C) The cell recognizes the polyubiquitin chain and the target protein is shuttled to the 26S proteasome for degradation.
"!
Figure 4. 2D structures of Nedd4 family of E3 ubiquitin ligases
Schematics depicting the modular domains of various Nedd4 family members across three species. Note that they all share an N-terminal C2 domain, intermediate WW domains, and C- terminal HECT domain.
""
Figure 5. Microarray gene expression of Nedd4 family members in various stages of satellite cell activation
Quiescence represents satellite cells that were not cultured prior to microarray analysis. Growth represents satellite cells that were cultured in myoblast growth media. Differentiation represents myoblasts that were grown in differentiation media for 3 and 5 days, respectively. The red dashed line indicates a threshold for no significant expression.
"# 2.0 Assess the role of Nedd4L in C2C12 mouse myogenic cells
2.1 Introduction
Satellite cells impart skeletal muscle with the robust ability to regenerate following trauma or injury. In muscle wasting diseases, the muscle regeneration machinery malfunctions, causing perturbations in the balance of satellite cell self-renewal, differentiation and tissue homeostasis. Once a quiescent satellite is activated, it can differentiate and contribute to muscle regeneration, or it can proliferate and expand the satellite cell population. By understanding the mechanisms that drive these processes, and more importantly what mechanisms control the cell fate decision, the balance of stem cell proliferation and differentiation can be manipulated and controlled to sustain normal muscle regeneration.
Nedd4L has already been shown to target cell-signaling pathways that affect the processes of proliferation and differentiation. For example, Nedd4L targets Smad2/3 to inhibit
TGFβ signal transduction in mouse embryonic stem cells (mESCs)94 . TGFβ signaling is responsible for directly an incredibly broad range of cellular processes such as growth and differentiation is a context-dependent manner102 . Additionally, Nedd4L was shown to affect Wnt signaling by targeting disheveled for degradation thus inhibiting signal transduction103 . The study noted effects on axis development in Xenopus laevis, however Wnt signaling is also tightly involved with cell fate104 and proliferation105 . Also of note is the fact that Nedd4L plays a tumor repressor role in various contexts106-108 . These studies would suggest that Nedd4L is required to maintain a non-proliferative state.
To investigate whether Nedd4L plays a role in myogenic cell processes, C2C12s were used as an in vitro model. C2C12s are an immortalized mouse myoblast cell line that originated
"$ from an adult dystrophic mouse109 . In addition to proliferating efficiently in culture, C2C12s can also differentiate and form multinucleated myotubes under low serum conditions, and as such serve as a suitable in vitro model of myoblast cellular mechanisms.
A Nedd4L mammalian expression vector was obtained, in addition to a mutated
Nedd4L expression vector (ΔNedd4L) in which a point mutation in the HECT domain renders it catalytically inactive (Addgene plamids 27000 and 27001, respectively). The point mutation was generated using site-directed mutagenesis to achieve a C962A mutation, thus removing the cysteine residue required for ubiquitin transfer in the HECT domain of Nedd4L94 . These two vectors, as well as an empty vector were used to generate C2C12 cell lines that stably overexpress the respective proteins.
The proliferative capacity of these C2C12 stable cell lines was assessed using a BrdU assay. Bromodeoxyuridine (5-bromo-2’-deoxyuridine; BrdU) is a synthetic analogue of thymidine, and is widely used to detect proliferating cells. As cells replicate their DNA before undergoing mitosis, they will take up BrdU molecules in place of thymidine, which can subsequently be detected with an anti-BrdU antibody. The BrdU incorporation of Nedd4L- overexpressing (N4L-OE) C2C12s will be compared to that of ΔNedd4L-overexpressing
(ΔN4L-OE) C2C12s and empty vector (EV) C2C12s.
As mentioned in the introduction, myosin heavy chain (MyHC) is a marker of late differentiation in myoblasts. MyHC is expressed in muscle filaments and serves as the motor subunit of the myosin complex. MyHC expression in C2C12s is not activated until they are terminally differentiating; therefore it is used as a marker for this stage. The ability of N4L-OE and ΔN4L-OE C2C12s to differentiate will be assessed based on MyHC expression.
"% The stable cell lines will also provide valuable information on how Nedd4L affects cell cycle. When cells proliferate, there is a series of tightly regulated processes that allow for
DNA replication and division into two daughter cells. The cell cycle includes a G1 phase, during which the cell to grows in size prior to DNA replication in the S phase, followed by a G2 phase which importantly serves as a checkpoint before mitosis occurs and the cell divides. There are several checkpoints at various stages throughout cell division, and if there is a perturbation the cell will cease entry into mitosis.
Cell cycle analysis allows for quantitation of the proportion of cells that are in the various stages of cell cycle. Cell cycle analysis can be achieved using flow cytometry, which allows a univariate analysis of DNA content. In this procedure, cells are treated with the fluorescent molecule, Propidium Idodide (PI) which intercalates between DNA bases110 . As the
cell progresses from G1 to S, the DNA content changes and can be measured by the PI signal. If
Nedd4L has an effect on satellite cell proliferation, that effect should manifest in an abnormal cell cycle results.
Taken together, these experiments will provide insight as to whether Nedd4L affects the muscle stem cell’s ability to proliferate or differentiate, and will lay the groundwork for future justification of in vivo analysis of Nedd4L.
"& 2.2 Materials and Methods
Cell culture
Cell proliferation: C2C12 mouse myoblasts were grown in Dulbecco’s modified eagle’s medium supplemented with 10% FBS and 1% penicillin/streptomycin, which will be referred to as growth media.
Cell differentiation: To achieve differentiation of C2C12 mouse myoblasts, cells were grown to
90% confluence in regular growth media. To begin differentiation, cells were then kept in
Dulbecco’s modified eagle’s medium supplemented with 5% horse serum and 1% penicillin/streptomycin, which will be referred to as differentiation media.
Cell passaging: To maintain optimal growth conditions, C2C12s were passaged at 70% confluence. Media was aspirated and cells were washed with 1X phosphate buffered solution
(PBS). Cells were then trypsinized with 0.25% trypsin and incubated at 370C for 1 minute before being resuspended in 10 mL of growth media and passaged to a new culture dish at a ratio of
1:10.
Generating stable cell lines
Lipofectamine2000 (ThermoFisher Scientific) was used according to the manufacture’s protocol to transfect C2C12 mouse myoblasts with a Nedd4L expression vector or a mutant ΔNedd4L expression vector in which the HECT domain is catalytically inactive (Addgene). Briefly, C2C12 cells were cultured to 70% confluence in a 6-well culture dish using growth media (see cell culture above). 3 μg of DNA was mixed with 150 μL of Opti-MEM medium, and in a separate tube 12 μL of lipofectamine reagent was diluted with 138 μL of Opti-MEM medium. The two solutions were mixed, vortexed for 10 seconds and then incubated at room temperature for 5
"' minutes. 250 μL of the final DNA-lipid solution was added per well. After 24 hours of incubation at 370C, the media was changed. 48 hours after transfection, the cells that successfully took up the DNA were selected for with hygromycin (200 μg/mL). After 5 days of selection, colonies of resistant cells formed and were expanded and kept in maintenance media to ensure wildtype C212s did not reemerge and dominate the culture. The maintenance media was
Dulbecco’s modified eagle medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 100 μg/mL hygromycin)
Cell cycle analysis
C2C12 cells were grown to 80% confluence and then washed with 1X PBS and trypsinized. The trypsinization was then blocked with FBS by adding growth media to the cells. Cells were then transferred to a 15 mL falcon tube and spun down at 1500 RPM for 5 minutes. The cell pellet was washed once with 1X PBS and pelleted again. The resulting pellet was resuspended in 300
μL of PBS and transferred to a 1.5 mL tube. 700 μL of ice cold 70% ethanol was then added drop-wise to the tubes while gently vortexing to fix the cells. Cells were incubated in ethanol solution for one hour at 4°C. Fixed cells were then pelleted at 6000 RPM for 5 minutes 4°C. The supernatant was decanted and the cells were once again washed in 1mL of cold 1X PBS, spun down, and resuspended in 300 μL of PBS. 5 μL of 10 mg/ml RNase A was added to the cells, followed by a 60 minute incubation at 37°C. Propidium Iodide (PI) was then added to a final concentration of 10 μg/ml, and incubated with the cells for 10 minutes at room temperature in the dark. Samples were then sent to the Flow Cytometry Facility at the Lady Davis Institute for
Medical Research to be analyzed on the BD LSR Fortessa Analyzer for relative DNA content and subsequent cell cycle analysis.
"( BrdU proliferation assay
To assess the proliferative capacity of the stable C2C12 cell lines, bromodeoxyuridine (BrdU) was used. Briefly, stable cell lines were seeded onto 4-well chamber slides and grown for 2 days to roughly 60% confluence. The Brdu labeling reagent (Thermo Fisher Scientific) was added at a ratio of 1:100 with full growth media and allowed to incubate with the cells for 24 hours at 37°C.
The cells were then washed with 1X PBS and fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After three washes with 1X PBS for 5 minutes, the cells were permeabilized with 0.2% Triton X-100, 0.1M glycine in 1X PBS for 15 minutes at room temperature. Blocking for one hour at room temperature with 2% BSA, 5% horse serum and
0.1% Triton X-100 followed. Anti-BrdU antibody (Abcam) was added to the cells at a concentration of 1:200 diluted in blocking buffer and incubated at 4°C for 16 hours. Following 3 washes with 1X PBS for 5 minutes, rat anti-goat secondary (AlexaFluor 488, Life Technologies) was used at a concentration of 1:200 for 45 minutes of incubation in the dark at room temperature. The cells were washed twice for 10 minutes each with 1X PBS and then mounted with ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific). Cells that incorporated BrdU during proliferation were visualized using the 488nm channel on the EVOS cell imaging system (ThermoFisher Scientific). Each group was repeated in triplicate. The number of BrdU positive cells was quantified in 3 fields of view for each replicate, yielding a total of 9 values per group. The values from each group were averaged and statistics were performed as described below in Statistical Analysis.
Differentiation assay
To assess whether the stable C2C12 cells lines could differentiate, they were seeded onto 4-well chamber slides. Once the cells reached 90% confluence, they were washed with 1X PBS and
# supplemented with 5% horse serum in DMEM. After 72 hours of incubation at 37°C and 5%
CO2, the cells were washed with 1X PBS and fixed with 4% PFA. Following permeabilization and blocking as described previously (see BrdU proliferation assay), cells were incubated with anti-myosin heavy chain (MyHC) primary mouse monoclonal antibody (Developmental Studies
Hybridoma Bank, DSHB) at a concentration of 2:3 antibody:blocking buffer. Following washes and incubation with a rat anti-mouse secondary antibody (AlexaFluor 568, Life Technologies,
1:200), the cells were washed again and mounted with ProLong Gold Antifade Mountant with
DAPI (ThermoFisher Scientific). Cells expressing the marker of terminal differentiation MyHC were visualized using the 568nm channel on the EVOS cell imaging system (ThermoFisher
Scientific).
Western Blots
Whole cell lysate was obtained by first trypsinizing cells in 0.25% trypsin. Cells were pelleted at
1200 xg for 5 minutes, and then lysed in RIPA buffer (25mM Tris-HCl pH 7.6, 150 mM NaCl,
1mM EDTA pH 8.0, 0.01% SDS, 1% Triton X-100), plus 1x protease inhibitor cocktail (Sigma) for 20 minutes on ice, with light vortexing every 5 minutes. Samples were then centrifuged for
30 minutes at 21000 g to separate the protein fraction. The resulting protein fractions were quantified using the Bradford assay (BioRad), and equal amounts were loaded for Western Blot analysis. 40-80 μg of each sample was loaded and proteins were separated on an 8% SDS-
PAGE. The gel was then transferred onto a PVDF membrane (BioRad), followed by blocking with 5% BSA in TBST for one hour. Nedd4L was blotted for using anti-Nedd4L primary antibody (1:1000 in 5% BSA in TBST; Cell Signaling Technologies), and tubulin was blotted for as a loading control (1:20 in 5% BSA in TBST, mouse monoclonal anti-tubulin, DSHB). Both primaries were incubated overnight at 40C, and then HRP-linked anti-rabbit or anti-mouse
# secondary antibodies were used to detect target proteins. Protein bands were detected used ECL reagents (BioRad). Bands were visualized on scientific autoradiography paper (Progene).
Statistics
The student’s paired t-test was used to determine statistical significance, and p values less than
0.05 were accepted as significant. At least three biological replicates were used.
#! 2.3 Results
C2C12s overexpressing Nedd4L lack ability to proliferate normally
Proliferation is an essential part of satellite cell lineage. It is an especially important process in terms of stem cell pool replenishment. In the context of muscle wasting diseases, the muscle stem cell pool is constantly being activated and eventually becomes exhausted due to diminished self-renewal. It is therefore important to understand what processes drive satellite cell proliferation so they can be manipulated to reverse the loss of the stem cell pool.
Generating a C2C12 cell line that stably overexpresses Nedd4L can provide insight as to how Nedd4L affects the proliferative capacity of myogenic cells. The BrdU assay was employed to determine how N4L-OE C2C12s compared to ΔN4L-OE and EV C2C12s. BrdU incorporation was severely restricted in N4L-OE C2C12s, as only 15% were positive for BrdU signal (Figure 6A). Conversely, BrdU incorporation between ΔN4L-OE and EV C2C12s was comparable (91% and 92% incorporation of BrdU, respectively; Figure 6C). N4L-OE C2C12s incorporate significantly less BrdU than the control groups (p=0.0001 when compared to the EV control), which implies that they have an impeded ability to proliferate.
Total cell number was quantified as an additional indicator of proliferative capacity.
As expected, N4L-OE C2C12s had significantly less cells per field of view than the control counterparts (p=0.0003 when compared to the EV control; Figure 6C).
Nedd4L overexpressing C2C12s are able to differentiate normally
Differentiation of proliferating satellite cells is an essential step in the muscle regeneration process. However, it has been shown that this process is perturbed during aging111 .
In sarcopenia, an age-associated disease characterized by rapid and severe muscle wasting,
#" muscle is burdened with an unresponsive satellite cell pool23 . The impaired ability of the satellite cell pool to respond to cues for regeneration leads to muscle weakness and frailty23, 111 .
Additionally, satellite cells in aged individuals tend to take on alternative differentiation pathways, and progress along adipogenic or fibroblastic lineages resulting in fatty infiltration and muscle fibrosis27, 28, 112 .
In order to test the ability of the C2C12 stable cell lines to differentiate, they were subjected to low serum conditions (5% horse serum in place of 10% fetal bovine serum used in growth media). Wildtype C2C12s begin expressing myosin heavy chain by 48-72 hours in low- serum culture. As the myogenic cells differentiate, they fuse to form multinucleated myotubes, a characteristic feature of terminally differentiated myoblasts. N4L-OE C2C12s displayed both of these hallmarks of differentiation (Figure 6B), as did the EV C2C12 controls. Interestingly, the
ΔN4L-OE C2C12s were incapable of differentiation, and did not express MyHC or form multinucleated myotubes. Rather, they formed undifferentiated colonies (Figure 6B). These findings suggest that the point mutation of the cysteine residue in the HECT domain creates a dominant negative effect, which will serve as an avenue for further investigation but will not be touched upon here.
Wildtype C2C12s do not express Nedd4L in proliferating conditions, but do express Nedd4L during differentiation
Overexpressing Nedd4L in C2C12s has shed some light as to how it may effect proliferation and differentiation in myogenic cells. To further these findings, it was necessary to observe endogenous levels of Nedd4L in C2C12 myoblasts. Whole cell lysate was analyzed for the presence of Nedd4L in C2C12s that were grown in growth media (GM), or differentiation
## media (DM) using Western Blot. The results indicate that Nedd4L is not expressed endogenously during proliferation, however protein expression is turned on during differentiation (Figure 6D).
These findings corroborate the previous experiments in the sections above, which suggest that
Nedd4L expression is associated with a non-proliferative state, as it impedes proliferation and promotes differentiation.
Nedd4L overexpressing C2C12s have impeded ability to progress to S-phase and instead accumulate in G2 phase
The previous experiments revealed that Nedd4L overexpression results in impaired proliferation. Therefore, to corroborate these findings, we interrogated the progression of the stable cell lines through the cell cycle using flow cytometry. N4L-OE C2C12s were compared to
EV C2C12s for the proportions of cells in G1, S, or G2/M phase. While both cell lines had
roughly the same proportion of cells in G1 (approximately 60%), there was a notable difference in the proportion of cells in G2 and S phase. The N4L-OE C2C12s had significantly more cells
in the G2 phase (p=0.002, Figure 6E), and resultantly less cells in the S phase (p=0.01, Figure
6E). These data indicate that cells overexpressing Nedd4L were unable to pass the G2 checkpoint and progress to S phase. This accumulation of cells in the G2 phase implies that Nedd4L overexpressing cells are unable to go through mitosis as efficiently as their EV C2C12 counterparts.
#$ 2.4 Discussion
Preliminary gene expression data suggested that Nedd4L might play an important role in the function of satellite cells. As mentioned in the introduction, RNA expression patterns show that Nedd4L transcript is produced at very high levels in quiescence, and then reduces upon activation, followed by a slight increase during differentiation (Figure 5). This dynamic expression pattern suggests that Nedd4L is acting in an important window of time to help guide the satellite cell in deciding whether to proliferate or differentiate. To investigate this possible role for Nedd4L, C2C12 cell lines that overexpress Nedd4L protein, as well as a mutated
Nedd4L and an empty vector control.
The proliferative capacity of the N4L-OE C2C12s was assessed using a BrdU incorporation assay. The results of that experiment clearly showed that when Nedd4L is overexpressed, myogenic cells are not able to proliferate as efficiently as their control group counterparts (Figure 6A,C). This phenomenon is consistent with a trend that is observed in the literature, which suggests that Nedd4L expression acts as a tumor suppressor107, 108, 113 . For example, Nedd4L expression is significantly down regulated in malignant glioma, and is inversely correlated with tumor size. Additionally, there is a significant correlation between decreasing Nedd4L expression and worse overall survival in patients with malignant glioma107 .
Additionally, one study found that Nedd4L RNA and protein levels were significantly down- regulated in colorectal cancer tumors at all stages of progression compared to the healthy surrounding mucosa. Following in vitro experiments, the authors suggest it is through the inhibition of Wnt signaling at the β-catenin level that Nedd4L controls cell growth108 . These studies and several others suggest that Nedd4L expression plays a key role in regulating cellular
#% proliferation in various tissue contexts, and the in vitro results presented here for C2C12 cells suggests this may also be the case in myogenic cells.
In addition to proliferation, differentiation of C2C12s overexpressing Nedd4L was examined. Myogenic cells that are unable to differentiate properly into mature adult muscle pose a serious threat to normal muscle regeneration. These conditions have been observed in aging muscle27, 111 , and it is therefore important to investigate whether Nedd4L plays a role in the differentiation of muscle stem cells. To date, there is no information on whether Nedd4L is involved in satellite cell differentiation. This is investigated here using C2C12s as an in vitro model of muscle stem cell differentiation.
After 3 days of differentiating culture conditions, C2C12s that over express Nedd4L show the typical hallmarks of normal muscle stem cell differentiation. They express the differentiation marker MyHC. Additionally, individual cells fuse to form multinucleated myotubes (Figure 6B). These effects are also seen in the EV C2C12 cells. From these observations it is clear that Nedd4L does not interfere with normal C2C12 myoblast differentiation. Furthermore, Nedd4L is not expressed endogenously in proliferating wildtype
C2C12s. However, Nedd4L protein expression is turned on in differentiating C2C12 myoblasts
(Figure 6D), further supporting the characterization that Nedd4L inhibits proliferation and promotes differentiation.
The striking observation that Nedd4L overexpression almost completely inhibits proliferation prompted the need for cell cycle analysis in the stable cell lines. This would provide more information about what stage of the cell cycle Nedd4L overexpression is affecting.
Interestingly, the results revealed that Nedd4L overexpression in C2C12 cells causes an
accumulation of cells in G2 phase that are unable to progress through mitosis (Figure 6E). Many
#& cancer cells present with aberrant cell cycles and fail to control their division as a result. Given
that overexpression of Nedd4L halts proliferation through inhibiting progression past G2 phase, it follows that a reduction in Nedd4L protein expression would tilt the balance the other way and result in uncontrolled proliferation. It is, however, important to note that these effects have only been observed in C212 myoblasts, which only serve as an in vitro model of actual satellite cell behavior.
The data collected in this aim provide promising evidence that Nedd4L may be involved as a key regulator in the proliferation and differentiation of satellite cells. Therefore, to gain further insights into the role of Nedd4L in vivo, we move to the second research aim, which will investigate the effects of Nedd4L on satellite cell function and muscle regeneration in a conditional knockout mouse model.
#'
Brdu Brdu DAPI MHC DAPI Overlay
EV EV EV
Brdu Brdu DAPI MHC DAPI Overlay
ΔNedd4L
ΔNedd4L
Brdu Brdu DAPI MHC DAPI Overlay
Nedd4L
Nedd4L
1.0 0.8 Cell Cycle Analysis 0.6 0.4 p=0.0001 * GM 3DM 0.2 0.6 Proportion of Brdu postive cells cells postive of Brdu Proportion EV ΔNedd4L Nedd4L α-Nedd4L p=0.01 100 0.4 p=0.002 ** EV * Nedd4L 80 p=0.0003 α-β-tubulin * 0.2 60 cells of total Proportion G1 G2 S Total number of cells number of cells Total 20
EV ΔNedd4L Nedd4L
Figure 6 – in vitro characterization of Nedd4L in C2C12 myoblasts
BrdU proliferation assay of C2C12 stable cell lines B) MHC staining in differentiating C2C12
stable cell lines C) Quantification of BrdU incorporation in C2C12 stable cell lines, as well as
total number of cells D) Nedd4L expression in wildtype C2C12 mouse myoblasts in growth
media (GM) and 3 days of differentiation media (3DM). Tubulin shown as loading control. E)
Cell cycle analysis of empty vector-overexpressing C2C12s (EV) compared to Nedd4L-
overexpressing C2C12s (Nedd4L).
#( 3.0 In vivo characterization of a conditional Nedd4L knockout mouse
3.1 Introduction
Regeneration of all tissues begins with a resident stem cell. In muscle, the satellite cells are the sole cell type responsible for regenerating injured muscle114 . Upon injury, a healthy satellite cell pool is able to instantaneously respond to muscle injury and regenerate the muscle with great efficiency. Activated satellite cells will enter the cell cycle and being to proliferate, forming clumps of satellite cells within the niche along muscle fibers. The proliferating satellite cells then differentiate and fuse to form myotubes that will become incorporated into the mature muscle5, 115, 116 . Muscle wasting is presented when regeneration is no longer possible.
Muscle wasting occurs as a symptom in a wide range of diseases, including muscular dystrophies, age-related sarcopenia, and cancer-related cachexia. One of the underlying causes of muscle wasting in these diseases is the inability of the satellite cell pool to counteract muscle loss due to atrophy. Under these conditions, satellite cells tend to lose their regenerative capacity by becoming more pro-differentiation or switching to a senescent fate23, 29 . Therefore, understanding the mechanisms that direct the satellite cell’s role in muscle regeneration is essential in the development of effective therapies targeted at restoring proper muscle regeneration in muscle wasting diseases.
Thus far, the data shown in Aim 1 suggest that Nedd4L is involved in regulating myogenic cell function in C2C12 cell lines. However, the role of Nedd4L in the satellite cell context in vivo has yet to be investigated in the literature. It was essential to first validate the protein expression of Nedd4L in satellite cells at different time points and compare it with the
RNAseq data. Since Nedd4L functions by targeting other proteins for degradation, obtaining information on when the protein is expressed is more informative from a functional point of view
$ than gene expression data in this case. Therefore, we have isolated individual muscle fibers from the Extensor Digitorum Longus (EDL) of wildtype mice and cultured them ex vivo for a range of time points from zero hours (ie. Quiescent satellite cells) to 72 hours (activated satellite cells), and used standard immunofluorescence techniques to elucidate Nedd4L protein levels during this time frame.
To shed further light on the role of Nedd4L in satellite cells, we have created a mouse model that bares a Nedd4L knockout exclusively in the satellite cells. From this model we hope to ascertain whether removing Nedd4L would affect the muscle stem cell’s ability to regenerate damaged muscle. We will explore this through the well validated method of cardiotoxin (CTX) injection into the Tibialis Anterior (TA) intramuscularly muscle followed by allowing 3 weeks for muscle regeneration prior to sacrifice. Additionally, to fortify the data acquired in Aim 1, which suggests that Nedd4L inhibits proliferation, the EDL muscle will be analyzed for the number of myofibre-associated satellite cells.
$ 3.2 Materials and Methods
Generating the Nedd4L conditional knockout mouse
To generate the Nedd4L conditional knockout mouse, a mouse in which exon 15 of the Nedd4L allele is flanked with loxP sites was obtained form Dr. Hiroshi Kawabe (Max Planck Institute,
Gottingen, Germany). This mouse (Nedd4Lf/f ) was crossed with a Pax7Cre mouse to restrict the knockout to satellite cells through a non-inducible Cre driver under the control of the Pax7 promoter (Figure 8A). The genotype of the pups was confirmed with PCR. The excision of exon
15 of the floxed Nedd4L allele induces a frame shift mutation, thus ablating expression of all downstream residues.
Fiber isolation
Mice were sacrificed and their EDL muscles were dissected using standard dissection techniques. Following incubation with collagenase/dispase (Roche) at 37°C, the whole muscle was triturated with a 1 mL pipette tip to dissociate individual fibers from the whole muscle as described previously117 . To mimic activating conditions, fibers were cultured in fiber growth media (DMEM plus 20% FBS, 1% chick embryo extract, 2.5 ng/mL bFGF, 1% penicillin/streptomycin) at 370C. For quiescent satellite cell analysis, fibers were fixed immediately following dissociation using 4% paraformaldehyde (PFA) prepared fresh in 1X
PBS.
Fiber Immunofluorescence
Once fibers were fixed, they were permeabilized with 0.2% Triton X-100, 0.125M glycine in
PBS for 15 minutes at room temperature. Blocking followed for one hour at room temperature with 2% BSA, 5% horse serum and 0.1% Triton X-100. Pax7, Myogenin and myosin heavy
$! chain were detected using mouse monoclonal antibodies (Developmental Studies Hybridoma
Bank) at a ratio of 2:3 diluted in blocking buffer and incubated at 4°C for 16 hours. Nedd4L was detected using Rabbit polyclonal anti-Nedd4L antibody (Cell Signaling Technology) at a ratio of
1:400 diluted in blocking buffer and incubated at 4°C for 16 hours. Following 3 washes with 1X
PBS for 10 minutes, the appropriate secondary antibody (AlexaFluor anti-mouse or anti-rabbit
488nm or 568nm) was used at a concentration of 1:400 for 45 minutes of incubation in the dark at room temperature. The fibers were washed three times for 10 minutes each with 1X PBS.
Finally, the fibers were transferred to a microscope slide outlined using a PAP pen, and mounted with ProLong Gold Antifade Mountant with DAPI (ThermoFisher Scientific). Fiber-associated satellite cells were then visualized using microscopy.
Cardiotoxin (CTX) injections
The left Tibialis Anterior (TA) muscle was injected with 50 μL of 10 μM cardiotoxin (Sigma).
The resulting muscle injury was given three weeks for regeneration, at which point mice were sacrificed for further muscle cross-sectioning and subsequent immunofluorescence analysis.
Tibialis Anterior cross-sections
The TA muscle was isolated from sacrificed mice using standard dissection techniques. The whole muscle was then fixed in 0.5% PFA for 2 hours at 40C. 20% sucrose was added to each sample, and they were left to equilibrate overnight. The 20% sucrose was decanted, and replaced with 1:1 20% sucrose to OCT in an aluminum foil cryomould. The muscle was then frozen in isopenttane that has been pre-cooled with liquid nitrogen. Once the muscle was completely frozen through, cross-sections were made using a cryomicrotome. Briefly, the cryostat was
$" cooled to -240C and 10 μM sections were taken from each sample and mounted onto microscope slides for immunofluorescence analysis.
Slides containing cross-sections were thawed to room temperature and washed twice in 1X PBS, followed by 5 minutes of permeabilization with 0.1% Triton-X 100 in 0.1M glycine at room temperature. After 3 washes with 1X PBS, samples were blocked with Mouse on Mouse
(M.O.M) blocking buffer solution (1 drop in 1.25 mL 1X PBS) for one hour at room temperature. Primary antibodies were incubated overnight at 40C (anti-Pax7 from DSHB at 2:3, and anti-dystrophin at 1:200 in blocking buffer). After 3 washes with 1X PBS, fluorescent secondary antibodies (AlexaFluor anti-mouse 568 and anti-rabbit 488) were diluted to 1:500 and incubated with samples for 45 minutes in the dark at room temperature. After 3 washes with 1X
PBS, samples were mounted with ProLong Gold Antifade Mountant with DAPI (ThermoFisher
Scientific), and visualized using the EVOS cell imaging system (ThermoFisher Scientific).
Statistical analysis
The student’s paired t-test was used to determine statistical significance, and p values less than
0.05 were accepted as significant.
$# 3.3 Results
Nedd4L protein is expressed during a specific window of time following satellite cell activation
Nedd4L has a dynamic gene expression signature throughout the stages of myogenesis
(Figure 5). During quiescence, the RNA levels are high, and then lower upon activation. Finally, during differentiation, the transcript level increases slightly before plateauing. These data suggest that Nedd4L is active during a certain period of time to regulate target proteins during the satellite cell’s lineage progression. In order to determine whether Nedd4L protein expression reflects the RNA-seq expression data, wildtype mice were sacrificed and individual muscle fibers were obtained from their EDL muscles. The fibers were either fixed immediately to observe quiescent satellite cells, or placed into growth culture to observe proliferating and differentiating myoblasts. For the purposes of comparison, both Nedd4L and Nedd4 were probed for using immunofluorescence, in parallel with Pax7 probing to identify satellite cells. Nedd4 does not have a dynamic expression pattern and is expressed at the RNA level consistently throughout quiescence, proliferation, and differentiation. It is therefore expected that Nedd4 protein will be present at equal levels during each time point, whereas Nedd4L will have a peak in protein expression.
Indeed, Nedd4 had a consistent level of protein expression in quiescence and at 24, 48, and 72 hours of activation (Figure 7B), and therefore reflected the gene expression data.
Interestingly, however, Nedd4L did not follow the trend observed with the gene expression data.
The microarray gene expression data had previously been validated with qPCR (Figure 7A), which clearly shows Nedd4L transcript is high in quiescence and is reduced in activation.
Surprisingly, there was no detectable Nedd4L protein in quiescence when Nedd4L RNA levels are at their highest. By 24 hours of activation, there was a slight increase in Nedd4L protein,
$$ followed by a peak at 48 hours. Finally, by 72 hours Nedd4L protein was expressed, but only at the periphery of the satellite cells (Figure 7B). Clearly, the protein expression pattern does not reflect the RNA expression pattern for Nedd4L.
Nedd4L conditional knockout mice produce more satellite cells following induced muscle regeneration
In order to assess the effect of Nedd4L on muscle regeneration, we deleted Nedd4L in satellite cells by crossing Nedd4Lf/f (obtained from Dr. Hiroshi Kawabe from the Max Planck institute in Germany118 ) with Pax7cre. In the knockout mice, the Pax7 promoter drives Cre expression, resulting in the excision of exon 15 of Nedd4L in Pax7-expressing satellite cells.
Exon 15 of Nedd4L forms a major part of the catalytic HECT domain. Therefore, Nedd4L conditional knockout (N4L-cKO) cells will possess a catalytically inactive form of Nedd4L
(Figure 8A). To assess the role of Nedd4L on muscle regeneration, we induced injury in the wildtype and N4L-cKO mice by CTX injection into the TA muscle. Following three weeks of regeneration, the mice were sacrificed and their TA muscles were dissected and cross-sectioned.
The resulting specimens were stained for Pax7 to quantify the number of satellite cells, and for dystrophin to delineate muscle fiber peripheries. We expect to see that the knockout will have more satellite cells than the wildtype control, due to the alleviation of Nedd4L’s repressive effects on myoblast proliferation as described previously in Aim 1.
The trend did indeed persist in the mouse model. The TA cross sections from the knockout had significantly more satellite cells than the wildtype (Figure 8B; p=0.007). The total number of satellite cells were tallied and then normalized to the total number of fibers in the field of view. There was no notable difference in the size and morphology of the fibers between the
$% knockout and wildtype samples, suggesting that muscle regeneration was not impaired in the knockout.
Nedd4L conditional knockout mice have more satellite cells per fiber and have more activated satellite cells than wildtype mice
To further characterize the effect of Nedd4L on satellite cells, the EDL muscle was dissected from the hind limbs of N4L-cKO and wildtype mice. Isolated individual fibers were immediately put in full growth media, and were cultured for 72 hours to allow for satellite cell proliferation. Using immunofluorescence techniques, the fibers were then probed for Pax7 to identify quiescent or early activate satellite cells, and Myogenin to identify more committed satellite cells.
Due to the large variation in total number of satellites per fiber, the results failed to reach significance. However, a few important trends were noted. The N4L-cKO on average had more satellite cells per fiber (22 per fiber compared to 12 in the wildtype; Figure 8E).
Additionally, the N4L-cKO had a higher proportion of activated cells (Pax7+/Myog+) than the wild type (Figure 8F). These data imply that in the N4L-cKO, more satellite cells have been activated and entered the cell cycle; consistent with the previous findings that Nedd4L inhibits this process.
Interestingly, the TA muscle cross-section images revealed that the N4L-cKO mice have more satellite cells in the satellite cell position than the wildtype following CTX injury
(Figure 8B). Additionally, the N4L-cKO had slightly more Myogenin+/Pax7- satellite cells
(Figure 8F), however there was no major trend noted.
$& 3.4 Discussion
Muscle regeneration requires a healthy satellite cell pool where the resident satellite cells maintain a critical balance between self-renewal and differentiation. In muscle wasting diseases, satellite cell function and muscle homeostasis is perturbed. Constant activation of the satellite cell pool is not conducive to sufficient self-renewal, and as a result the satellite cell pool quickly becomes depleted in various muscle wasting disorders. As the muscle continues to regenerate, the stem cells are unable to proliferate quickly enough, and eventually the satellite cell pool is exhausted. Therefore, it is important to elucidate what mechanisms direct a satellite cell’s decision to proliferate or differentiate. By understanding these mechanisms, molecular tweaks to the system can restore the balance in muscle wasting diseases to ensure the satellite cell pool is continually replenished and self-sustaining. It has already been clearly demonstrated that the removal of proteins through the UPS is paramount in regulating a wide variety of cellular processes including proliferation and differentiation. The E3 ubiquitin ligase Nedd4L is of interest because of its effects on cell proliferation and differentiation. In this aim, we first sought out to determine if Nedd4L is required for satellite cell proliferation and differentiation.
Standard immunofluorescence techniques were used to probe for Nedd4L protein in satellite cells associated with EDL muscle fibres. Interestingly, we found that the Nedd4L protein expression was in fact dynamic, however it did not align with the RNA expression data.
In quiescent satellite cells, Nedd4L RNA is at its peak (Figure 7A), yet there is no detectable protein (Figure 7B). This observation suggests that Nedd4L may be regulated by posttranscriptional mechanisms such as sequestration into an mRNP (messenger ribonucleoprotein) granule or repression of translation. Alternatively, in quiescent satellite cells the ubiquitin proteasome pathway may remove Nedd4L protein. Nedd4L protein appears by 24
$' hours of activation, and peaks by 48 hours. Then by 72 hours, Nedd4L protein is expressed only around the periphery of the cell (Figure 7B). Contrastingly, Nedd4 protein expression is uniform throughout quiescence and all time points of activation. This evidence supports the hypothesis that Nedd4L is acting during a certain window of time. Based on this time course study, Nedd4L protein expression peaks at 48 hours after activation, during which time the satellite cell is deciding whether to continue proliferating or to differentiate. By 72 hours, Nedd4L moves to the periphery of the cell where it most likely co-localizes with the inner cell membrane. This is a very important region as it contains all the cell surface receptors that receive extrinsic signals that guide cellular functions. Identifying the targets of Nedd4L at this location would be crucial for elucidating the molecular mechanisms through which Nedd4L elicits its effects on satellite cell proliferation and differentiation, and will be an exciting area of upcoming research.
Additionally in this aim, we assessed whether removing Nedd4L from the satellite cells of mice would re-establish the satellite cell pool following injury and regeneration. We also observed whether the Nedd4L knockout would increase the proliferation of activated satellite cells.
Following muscle injury and 3 weeks of subsequent muscle regeneration, cross sections of the TA muscle were probed for Pax7 and dystrophin using standard immunofluorescence techniques. We observed that the N4L-cKO mice had more satellite cells per fibre in the cross section compared to the wildtype (Figure 8B). This data is consistent with what was observed in Aim 1. When Nedd4L was overexpressed in C2C12 mouse myoblasts, their ability to proliferate was severely impaired (Figure 6A). It follows that genetic ablation of
Nedd4L function would enhance satellite cell proliferation following in vivo activation via CTX
$( injection. This trend is further supported in the following experiment where fibres were cultured ex vivo and satellite cells were counted.
EDL muscles were dissected from the N4L-cKO and wildtype mice, and individual muscle fibres were isolated and cultured in growth media for 72 hours to allow satellite cell proliferation. An overall trend was observed that the N4L-cKO mice had more satellite cells per fibre, but due to a large variance in the number of satellite cells per fibre, statistical significance was not reached. Again, this data suggests that removal of Nedd4L from satellite cells facilitates their proliferation in vivo.
Lastly, satellite cells on isolated EDL fibres were analyzed for their Pax7 and
Myogenin expression. Again, while not statistically significant, a trend was noticed. The N4L- cKO mice had a higher proportion of activated (Pax7+/Myogenin+) satellite cells than the wildtype. This observation suggests that deletion of Nedd4L creates an environment that is conducive to satellite cell activation and proliferation, thus more cells express Myogenin than in the wildtype control. This is a feature that would allow the knockout to respond to injury more efficiently and produce more satellite cells during the regeneration process (as observed in
Figure 8B).
% A
0hr 24hr 48hr 72hr B
Nedd4L Pax7 Pax7
DAPI DAPI
Nedd4
Pax7 DAPI DAPI
Figure 7. Aim 2: Gene expression and time course protein expression of Nedd4L
A) qPCR validation of Nedd4L transcript levels in quiescent (0hr) and activated (48hr) satellite cells from wildtype mice.
B) Nedd4L (top) and Nedd4 (bottom) protein expression in satellite cells associated with EDL fibres at 0, 24, 48, and 72 hours after culturing ex vivo. Pax7 (red) used to identify satellite cells.
DAPI (blue) used to stain nuclei.
%
A ! ' % # !
B C 0.6 p=0.007 0.5 *
Pax7
0.4
0.3
Dystrophin
# satellite cells/fiber # satellite 0.2
WT N4L-cKO 0.1
DAPI DAPI
WT N4L-cKO D
DAPI PAX7 Myogenin Overlay
DAPI PAX7 Myogenin Overlay
N4L-cKO Wildtype
E F 30
20
10 Wildtype
# satellite cells/fiber # satellite N4L-cKO 0 WT N4L-cKO
P7+ P7+/Myog+ Myog+
%! Figure 8. Aim 2: in vivo characterization of Nedd4L using a conditional knockout mouse model
A) Generation of the conditional knockout mouse and location of the loxP sites on the Nedd4L locus. qPCR validation of Nedd4L transcript reduction in the heterozygous and knockout mice, normalized to wildtype. B) TA cross-sections from N4L-cKO versus wildtype mice. Satellite cells are identified by Pax7 in red. C) Quantification of satellite cells in the TA cross sections between N4L-cKO and wildtype mice. n=2; 9 frames counted from each sample and averaged per fibre. D) Fibre-associated satellite cells from N4L-cKO and wildtype mice. E) Quantification of total number of satellite cells, normalized per fibre. F) Ratios of Pax7+/Myogenin-,
Pax7+/Myogenin+, and Pax7-/Myogenin+ satellite cells from N4L-cKO and wildtype mice.
%" 4.0 Summary and Future Directions
Muscle wasting presents as a symptom in a wide spectrum of diseases. In order to develop effective therapies aimed at sustaining muscle regeneration is degenerative muscle diseases, it is crucial to understand the molecular mechanisms that drive satellite cell behaviour.
With a better understanding of these intricacies, therapies that target specific interactions or pathways can be developed with minimal off target effects.
In order to understand the behaviour of any stem cell population, it is important to assess protein turnover in that system. Proteasomal degradation through the ubiquitin- proteasome system (UPS) is a key component in maintaining cellular homeostasis. E3 ubiquitin ligases are crucial for carrying out the function of the UPS, and as such we turn to their expression patterns to gain a window into how and when they regulate protein degradation and subsequent stem cell processes such as proliferation and differentiation. In muscle stem cells, there is only one E3 ubiquitin ligase that exhibits a dynamic, temporally defined expression pattern: Nedd4L. Through the research aims of this thesis, the role of Nedd4L was characterized in in vitro and in vivo model systems.
The in vitro experiments served as a good starting point to determine whether Nedd4L had any role in regulating myoblast behaviour. Through those experiments, it became clear that
Nedd4L is involved in the inhibition of proliferation, and its expression is activated during differentiation. Further analysis of the cell cycle revealed that overexpression of Nedd4L causes
C2C12s to accumulate in G2 phase, thus prohibiting them from entering S phase and subsequently, mitosis. Together, these data suggested that Nedd4L is somehow regulating muscle stem cell behaviour, and warranted further in vivo characterization.
%# To begin in vivo analysis, we first had to identify at what time points during the myogenic lineage Nedd4L protein was expressed. The resulting experiments revealed that
Nedd4L is expressed only during a defined window following quiescent satellite cell activation.
In contrast, Nedd4 protein expression is maintained uniformly through quiescence and various activation time points. This observation supported the hypothesis that specifically Nedd4L is the
E3 ubiquitin ligase that has a unique expression pattern during satellite cell lineage progression.
Further to protein expression analysis in wildtype mice, we conditionally deleted
Nedd4L in MuSCs. These mice had ablated expression of Nedd4L specifically in Pax7+ satellite cells. Interestingly, the conditional knockout mice showed enhanced ability to replenish the stem cell pool following CTX-induced injury and muscle regeneration. Additionally, individual muscle fibres were isolated from the knockout mice and were cultured ex vivo for 3 days to observe satellite cell proliferation. The knockout fibres showed the trend of having more satellite cells per fibre than the wildtype, and had more Pax7+/Myogenin+ satellite cells (i.e., Activated satellite cells) than the wildtype control. Together, these findings are consistent with the conclusions drawn from Aim 1. Specially, that Nedd4L expression inhibits proliferation of satellite cells. Thus by removing Nedd4L expression, satellite cells are able to enter the cell cycle and proliferate so as to replenish the satellite cell pool following regeneration or ex vivo fibre culturing.
The data gathered here provides adequate evidence to justify further investigation into the precise molecular mechanism of Nedd4L in satellite cells. The following will provide future directions that will thoroughly characterize Nedd4L in the muscle stem cell context.
While the data presented here provides valuable information on the effect of Nedd4L expression on satellite cell behaviour, it provides no mechanistic insight as how Nedd4L elicits
%$ those effects. Therefore, it would be extremely informative to identify all of the protein targets of
Nedd4L in satellite cells. This can be achieved using any one of several tools that are available for identifying the interactome of a given protein of interest such as BioID. The BioID method utilizes a promiscuous biotin protein ligase that is fused to the protein of interest. The fusion protein will biotinylate transiently and stably interacting proteins, and mass spectrometry is used to identify all of the biotinylated proteins following a biotin pull-down119 . Candidate interactors can then be further verified using proximity ligation analysis (PLA).
Additionally, it would be beneficial to further utilize the mouse model and isolate satellite cells using fluorescence activated cell sorting (FACS). The isolated myoblasts could then be cultured and assessed for proliferative and differentiation potential. Also, the proteome of the knockout can be compared to the wildtype to further identify Nedd4L downstream targets.
These data would provide information about how Nedd4L affects satellite cell processes in a relevant model.
From a clinical perspective, it would be interesting to elucidate whether attenuating
Nedd4L expression in dystrophic mice (mdx mice) would impart enhanced muscle regeneration.
This can be assessed by crossing Nedd4Lf/f:Pax7Cre with mdx mice and observing if there is an increase in satellite cell number and a lessening of the muscle wasting phenotype in the dystrophic mice lacking Nedd4L.
Altogether, the data presented in this thesis provided some characterization of the role of Nedd4L in satellite cell function both in vitro and in vivo. With further experiments that would investigate the precise molecular mechanistic through which Nedd4L acts, it may be possible to harness the satellite cell pool replenishing potential of Nedd4L and develop therapies targeted at attenuating the detrimental muscle wasting that occurs in degenerative muscle diseases.
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