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The Role of the p75 neurotrophin receptor in regulating oligodendrocyte progenitor cell differentiation Bedard, Simon 2014

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The Role of the p75 Neurotrophin Receptor in Regulating  Oligodendrocyte Progenitor Cell Differentiation by Simon Bedard  B.A., Acadia University, 2001 B.Sc., The University of British Columbia, 2011   A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (CELLULAR AND DEVELOPMENTAL BIOLOGY)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2014 © Simon Bedard, 2014 ii  Abstract  One major avenue towards repair of the damaged mammalian nervous system is an enhancement of the process known as remyelination.  This process restores to damaged neurons the protective  sheath that is critical not only for their survival but also for the conduction of their electrical signals.  However, remyelination is inhibited after many types of nervous system damage as well as in degenerative diseases of the nervous system.  This inhibition functions primarily through prevention of the capacity of progenitor cells to become remyelinating cells.  This work aims to address the role that the p75 neurotrophin receptor (p75) – an important signalling protein that stands at the intersection of several key signalling cascades – plays in mediating this inhibition of remyelination.  The intention of this work is to investigate p75 as a potential therapeutic target for clinical interventions aimed at enhancing remyelination. iii  Preface Dr. Matt Ramer identified the p75 neurotrophin receptor as a potential mediator of CNS  myelination and suggested I pursue it as a research question.  Based on this suggestion I identified and designed the research program with input from Drs. Matt Ramer and Wolfram Tetzlaff.  I was solely responsible for all aspects of the study execution with the exception of stereotaxic injections, for which Dr. Jie Liu was the lead surgeon and I offered surgical support.  This work was performed in accordance with animal ethics protocols approved by the UBC Animal Care Committee.  Specifically, animals were bred under protocol A09-0617 and surgeries were performed under protocol A09-0741.  iv  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iii Table of Contents ......................................................................................................................... iv List of Figures ............................................................................................................................. viii Acknowledgements ...................................................................................................................... ix Dedication .......................................................................................................................................x Chapter 1: Introduction ................................................................................................................1 1.1 Overview ......................................................................................................................... 1 1.2 Remyelination as a Therapeutic Target in the Mammalian Central Nervous System .... 1  Myelin ......................................................................................................................... 1 1.2.1 CNS Myelination, Demyelination, and Remyelination .............................................. 5 1.2.2 Target(s) .................................................................................................................... 12 The p75 Neurotrophin Receptor ................................................................................... 13  Discovery and Structure ............................................................................................ 13 1.3.1 Neurotrophins and Neurotrophin Receptors ............................................................. 16 1.3.2 Cell-Death Signalling................................................................................................ 17 1.3.3 Cell Migration, Axonal Targeting, and Oligodendrocyte Differentiation ................ 18 1.3.4 Myelination ............................................................................................................... 21 Murine Models of Demyelination and Remyelination ................................................. 24 1.5 Project Objectives ......................................................................................................... 27 Chapter 2: Methods .....................................................................................................................29 v  2.1 Experimental Overview ................................................................................................ 29 2.2 LPC-induced Demyelination of the Murine Corpus Callosum ..................................... 31  Breeding .................................................................................................................... 31 2.2.1 Pre-Surgical Care ...................................................................................................... 32 2.2.2 Surgical Procedures .................................................................................................. 32 2.2.3 Post-Surgical Care .................................................................................................... 33 2.2.4 Histological Analysis ................................................................................................ 34 Tissue Preparation and Sectioning .................................................................... 34 Immunohistochemistry ..................................................................................... 35 Microscopy, Stereology, and Statistical Analysis............................................. 35 Chapter 3: Results........................................................................................................................38 3.1 Characterization of Demyelination Acute to Injury...................................................... 38 3.2 Effect of p75 on Proliferative Capacity of OPCs Following Demyelination ............... 40 3.3 Effect of p75 on Differentiation Status of OPCs Following Demyelination ................ 43 Chapter 4: Discussion ..................................................................................................................51 Chapter 5: Conclusion .................................................................................................................59 5.1 Conclusions ................................................................................................................... 59 5.2 Strengths and Contributions .......................................................................................... 59 5.3 Recommendations for Future Work.............................................................................. 61 5.4 Concluding Statement ................................................................................................... 61 References .....................................................................................................................................63 Appendices ....................................................................................................................................73 Appendix A : Animal Monitoring Criteria and Recording Sheets ........................................... 73 vi  Appendix B : Genotyping Primers and PCR cycling protocol ................................................. 74 Appendix C Primary Antibodies and Immunohistochemistry Staining Worksheet ................. 75                      vii  List of Tables  Table 2.1 Total experimental animals competent for analysis ..................................................... 30  viii  List of Figures  Figure 1.1 Myelin structure in the PNS and CNS ........................................................................... 3 Figure 1.2 Antigenic markers of differentiation in the oligodendroglial lineage ........................... 6 Figure 1.3 Oligodendrocyte precursor cell differentiation and oligodendrocyte maturation ......... 7 Figure 1.4 Molecular structure of p75 .......................................................................................... 15 Figure 2.1 Experimental design overview .................................................................................... 30 Figure 3.1 LPC lesion characterization ......................................................................................... 39 Figure 3.2 Example image of OPC proliferation analysis ............................................................ 41 Figure 3.3: Effect of p75 on proliferative capacity of OPCs following demyelination ................ 42 Figure 3.4 Oligodendroglial recruitment to lesion and lesion volume ......................................... 46 Figure 3.5 Example image at 3dpi ................................................................................................ 47 Figure 3.6 Example image at 7dpi ................................................................................................ 48 Figure 3.7 Example image at 11dpi .............................................................................................. 49 Figure 3.8 Differentiation status of OPCs following LPC demyelination .................................... 50  ix  Acknowledgements  I thank Dr. Wolfram Tetzlaff for his enduring support, academic advice, and personal counsel as I have moved through my graduate training and on into my future endeavours.  I would also like to thank Dr. Matt Ramer for encouraging me to pursue graduate training, offering me the opportunity to enter into graduate studies, and gifting my novice hands with his extensive surgical expertise. Additionally, I thank Dr. Jie Liu for the many practical surgical tips he bestowed upon me, the surgical protocols he trained me in, and his diligent and generous work as a surgeon.   I owe an enduring debt of gratitude to Peggy Assinck and Greg Duncan, two colleagues whose innumerable consultations and intellectual generosity allowed me to complete this work and aided in my understanding of the field.  I am also thankful to Dr. Jason Plemel for the training in cell culture he provided that extensively expanded my understanding of oligodendroglial biology.  Special thanks are also owed to my wife, Kelly Gray, who has supported me throughout my years of science education, and extra-special thanks are owed to my three beautiful children Aveline, Tanis, and Fionn, all of whom have worked diligently throughout my graduate training to keep life both interesting and challenging.  x  Dedication  This work is dedicated to Nancy Hannah-Booth Anderson and her father   Dr. John Murray Anderson, both of who instilled in me from a young age a love of science, an enduring curiosity, and a fascination with the biological complexity of the world.1  Chapter 1: Introduction 1.1 Overview In the mammalian nervous system the peripheral nervous system (PNS) is able to effectively initiate repair following injury, a capacity that the central nervous system (CNS) largely lacks.  The lack of effective strategies to repair the damaged CNS remains a central impediment to the development of treatments for neurotrauma and a variety of neurodegenerative diseases.  Currently, several large fields of scientific endeavor seek to address this issue.  Although limitation of inflammatory-mediated secondary damage (i.e. cell death) and enhancement of the regenerative capacity of damaged neurons are both important aspects when addressing neurotrauma and neurodegeneration, this work focuses on addressing issues related to a less-discussed but no less important process when considering CNS repair, namely remyelination.  Indeed, in the absence of robust and appropriate remyelination, strategies to restore function may ultimately fail.  Thus a better understanding of the processes and molecular players contributing to remyelination are critical to the development of successful strategies to fully restore function to the damaged CNS.  1.2 Remyelination as a Therapeutic Target in the Mammalian Central Nervous System  Myelin  1.2.1In the development of a complex nervous system, one functional necessity is the ability to transmit signals over long distances.  The issue of conductance velocity and signal fidelity is one that inevitably rises to the forefront as the axon length of neurons increases, since the increased electrical resistance of a longer axon decreases the speed with which an action potential may propagate along its length.  There exist two major strategies to deal with the 2  problem of conductance velocity, axon gigantism and myelination  (1).  Although simply increasing axon diameter – the case in the giant axons of the squid – is an effective solution to the problem, vertebrates are precluded from this approach by the spatial restrictions of the internal skeletal structures for which their clade is named.  Rigid structures of cartilage and bone provide anchor points for muscle tissue to attach to and levers for it to act upon – allowing highly articulated movement – as well as providing protection and support for the more delicate tissues of the body, especially the nervous system.  However, the protection thus afforded comes at the cost of restricting the space available for neurons and their axons – most especially in the CNS – so an adaptation has necessarily emerged.  Within the vertebrate nervous system larger diameter axons of neurons are surrounded by myelin, a lipid-rich, multi-lamellar, spirally-wrapped sheath of plasma membrane (PM) provided by closely associating glial cells (2).  In the PNS an axon must possess a diameter > 0.2 µm in order to be myelinated while in the CNS certain regions of the brain have myelinated axons with diameters as small as 200-300 nm  (3).  This insulating sheath is regularly interrupted by Nodes of Ranvier, spaces between two myelinated segments of an axon created by the margins of the myelinating glia, which serve to focus ion channels and allow for saltatory (from the Latin saltare “to jump”) transmission of action potentials.  This type of transmission allows an action potential to leap from node to node rather than travelling the entire length of an axon and thus increases both the speed of signal transmission and the distance that an action potential may travel without an increase in axon diameter.  Myelin also serves to insulate axons from inappropriate cross talk with neighbors that could otherwise create short circuits and noise within the system.  Beyond the somewhat pedestrian task of insulating axons, however, the juxtaparacrine interactions of neurons and their myelinating glia have emerged as powerful mediators of neural cell survival, migration, and connectivity (2, 4).   3  In the peripheral nervous system (PNS) myelin is provided by Schwann cells (SCs) – derived from neural crest – with each Schwann Cell myelinating a single internode (5)(Fig. 1.1).   Figure 1.1 Myelin structure in the PNS and CNS Overview of the structure of myelin in both the central and peripheral compartments.  In the PNS myelin is provided by Schwann cells, with each cell myelinating a single internode.  In the PNS Schwann cells also secrete a basal lamina that confines the nucleus and the internode as a whole, a structure lacking in CNS myelin.  In the CNS myelin is provided by oligodendrocytes that myelinate multiple non-adjacent internodes on multiple axons (adjacent myelination is shown for convenience).  In the CNS astrocytic endfeet interact with nodes of Ranvier while in the PNS Schwann cells extend microvilli into the node.  The paranodal region is characterized by non-compact myelin in both the PNS and CNS, while the juxtaparanode (JXP) contains compact myelin similar to the internodal region.  [Modified from (6), used with permission]  In the CNS myelin is provided by oligodendrocytes (OLs), with each oligodendrocyte myelinating multiple internodes on adjacent axons (2).  Oligodendrocytes are descended from a large population of progenitor cells known as oligodendrocyte precursor/progenitor cells (OPCs) that are present both during development and into adulthood in mammals; unlike OPCs, OLs are non-migratory and post-mitotic (6).  Although SCs and OLs are similar in the function they   4  perform there are many differences between these cell types, ranging from their developmental sources, their response to trophic factors, or the molecular components of the myelin that each produces.  For instance, although SCs and OLs both produce myelin basic protein (MBP) – an identifying component of mature, compact myelin – they also produce proteins unique to their lineage that make it possible to distinguish OL-derived myelin from SC-derived myelin based on well-defined antigenic markers (7). Myelin is critical to the proper function of the vertebrate nervous system, a fact reflected in the severe and often terminal pathologies that result from dysregulation of myelination or outright demyelination, such as Multiple Sclerosis, Guillain-Barré Syndrome, and the Leukodystrophies.    Additionally, myelin has a powerful inhibitory effect on nerve regeneration following injury in the CNS and also inhibits the maturation of oligodendroglia in vitro (8, 9).  While these constraints are useful in the healthy CNS – a confined compartment where there is no room for inappropriate growth – they limit the body’s ability to repair it following injury or disease, through inhibition of not only axonal regeneration but also remyelination of regenerated axons and those spared axons that have been denuded of their myelin.  These concerns are less important in the periphery as myelin debris – a potent inhibitor of neural repair processes – is rapidly cleared by macrophages, allowing SCs to rapidly differentiate from resident precursor cells and myelinate regenerating or spared axons(5).  In the CNS, however, myelin debris is cleared much less efficiently and can persist for up to 3 years in humans with spinal cord injuries (10).  Indeed, when injured, the PNS has a robust regenerative capacity as compared to the CNS.  Whether damage occurs as a result of trauma or disease, this disparity is currently one of the greatest challenges facing efforts to repair the damaged CNS, and the deleterious effects of demyelination combined with the inhibitory effects of myelin debris on CNS repair is widely 5  regarded as a major challenge to be overcome (11).  Therefore this work focuses on CNS myelination, remyelination, the challenges CNS myelin debris poses to repair efforts, and how a better understanding of these processes will improve our ability to treat the injured CNS.    CNS Myelination, Demyelination, and Remyelination 1.2.2Key to the process of myelination is the generation of OLs at the correct time and place.  This is regulated via control of the differentiation of OPCs into OLs and then the subsequent maturation of those OLs from a pre-myelinating to a myelinogenic phenotype.  During murine development myelination is primarily a post-natal process, although the pools of OPCs that ultimately go on to myelinate the CNS are formed earlier on in embryonic development (12).  In the mouse, OPCs are first generated at around E9.5-12.5 in the ventral regions of the developing neural tube.  Later in development there are waves of OPC migration that emerge from more dorsal regions and from the post-natal period onward the sub-ventricular zone (SVZ) provides a source of OPCs (12–14).  The human case largely parallels the murine in terms of OPC generation, but insofar as the maturation of OPCs into MBP-producing OLs is concerned it is better approximated by considering mice to be born at the end of the second trimester of human gestation (14).  This is to say that although in humans (as in mice) a portion of developmental myelination does occur prenatally, developmental myelination is primarily a post-natal process.   The differentiation of OPCs into OLs is a process that may be assayed Immuno-histochemically using a suite of antigenic markers that characterize the major shifts in cellular character and have been defined over the last several decades of scientific exploration.  Common to all oligodendroglia and their precursors is the expression of the transcription factors Olig2 and Sox10, and they may thus be used as nuclear markers of the lineage (15)(Figure 1.2).  OPC 6  differentiation into OLs is characterized by a loss of PDGFRα immunoreactivity and an upregulation of the CC1 antigen while maturation of such immature OLs into myelinating OLs is defined by subsequent upregulation of myelin-specific genes such as MBP (Figure 1.2).    Figure 1.2 Antigenic markers of differentiation in the oligodendroglial lineage The differentiation status of the oligodendroglial lineage as defined by established antigenic markers. a) OPCs are defined by a bipolar morphology and expression of PDGFRα and the cell-surface proteoglycan NG2. b) OPC differentiation results the development of a branching, multi-polar morphology and down-regulation of PDGFRα and NG2 while CC1 – a marker of both immature and mature OLs – is upregulated. c) Myelination is characterized by the ensheathment of axons with OL membranes, the development of internodes, and the production of proteins specific to mature myelin such as MBP.  a-c) Throughout the oligodendroglial lineage the transcription factor Olig2 is expressed and may be used as a nuclear marker of the lineage. [Adapted from (16), used with permission]  7    OPCs are characterized by a bipolar morphology, high motility, a large proliferative capacity, and the expression of both Platelet-derived growth factor receptor alpha (PDGFRα) and the cell-surface proteoglycan NG2 (17).  OPCs are actually multi-potent progenitor cells themselves and may produce not only oligodendrocytes but also neurons, astrocytes, and Schwann cells, although the last are only produced following tissue damage (18–21).  Primarily, OPCs differentiate into pre-myelinating (or “immature”) OLs; these are post-mitotic cells that are characterized by reduced motility, a complex and multi-polar morphology,  the expression of the cell-surface marker O4, and a reduction in Wnt signaling (17, 22).  Upon contact with a competent axon and in the presence of necessary cues, these pre-OLs will mature into myelinating OLs, up-regulate myelin-specific genes, and begin the process of myelination.  This set of differentiation steps is summarized in Fig 1.3.  Figure 1.3 Oligodendrocyte precursor cell differentiation and oligodendrocyte maturation OPCs develop from Nestin+ neural precursor cells in response to Sonic Hedgehog and FGF signals, a step inhibited by BMP signaling.  OPCs are defined by expression of the transcription factors Olig2 and Sox10 as well as the cell-surface receptor PDGFRα and the proteoglycan NG2.  OPCs are multipotent progenitors themselves that most often produce OLs but may also develop into neurons, astrocytes, and Schwann cells.  Following exposure to pro-differentiation factors such as Insulin Growth Factor-1, Ciliary Neurotrophic Factor, and Triiodothyroxine, OPCs downregulate OPC markers and differentiate into Immature OLs, characterized by expression of the CC1 antigen and the proteoglycans O4 and Gal C.  Once an immature OL contacts an axon and begins myelinating it upregulates myelin specific genes such as MBP, MAG, and OMgp [Adapted from (17), used with permission].  8  The differentiation of OPCs into OLs and their subsequent myelination of axons is a process regulated by a host of different inter-, intra-, and extra-cellular signals.  These signals include trophic factors secreted by both axons and other glia, cell-surface molecules expressed by both axons and glia, elements of the extra-cellular matrix (ECM), and both the activity and size of an axon  (23–27).  Although much of it is beyond the scope of this work, parsing the multiplicity of signals that regulate OPC differentiation and OL myelination has evolved into an expansive field of scientific endeavor and  is well reviewed in the scientific literature (28, 29).   Once an OPC has migrated to the correct location, differentiated into a pre-myelinating OL, contacted an appropriate axon, and received the necessary pro-myelinating signals from both the axon and the surrounding glia, the process of myelination can begin.   This results in a massive increase in lipid metabolism in the OL as vast sheets of membrane are produced that then wrap around the axon multiple times (30).  In the CNS, the mechanics of this process have been somewhat obscure for many years, with several different potential models suggested.  Recently, an elegant study by Snaidero et al. used several methods – including 3D reconstruction of serial block face EM images – to show that in fact the developing myelin sheath is produced at the leading edge of the extending OL membrane.  This leading edge dives underneath itself after one wrap and continues to expand laterally as it wraps around the axon; compaction of the myelin proceeds in an “outside-in” manner, mediated by the translation of MBP mRNA in proximal (i.e. furthest from the axon) areas of the developing myelin sheath (31).    This process of myelination is time-critical as recently demonstrated by Czopka et al.  They showed, using two-photon live imaging in a zebrafish model, that an OL has only a very limited time window in which to myelinate an axon in vivo.  If a terminally differentiated OL does not successfully ensheath an axon within ~5 hours, thereby becoming exposed to the 9  reciprocal survival signals that the axon provides, it will not myelinate and will apoptose (32, 33).  This process appears linked to the process of synaptic pruning that occurs during development –  whereby an excess of synapses are produced with superfluous connections later removed – thereby ensuring that no more OLs survive in the mature CNS than are necessary to myelinate competent and functional axons (34).  A similar process of pruning was observed in Czopka et al’s work, where several (although always a small proportion of the total) of the axonal ensheathments established in the initial 5 hour window were retracted over the subsequent 1-2 days, presumptively as a result of differential, activity-related signals provided by the myelinated axons (32).  Demyelination is the process by which mature myelin is degenerated and breaks down, thereby denuding the axon.  This results in a dispersal of ion channels along the axon, rather than clustering at nodes of Ranvier, causing disruption of salutatory conduction and impaired signal conduction along the axon (35).  Demyelination can occur by a direct attack on the myelin itself, as is the case in multiple sclerosis (MS), or as a result of trauma.  In MS the immune system mounts an attack against components of the myelin sheath, resulting in demyelination, axon degeneration, and functional decline (36).  Demyelination can also occur as a sequela of spinal cord injury or other neurotrauma, with large numbers of axons in and adjacent to the injury site becoming demyelinated in the days following injury as a result of inflammation and axonal degeneration (10).  Irrespective of the mechanism causing demyelination, once an OL has lost contact with the axon that it myelinates it becomes deprived of axonal survival signals and will eventually apoptose.  The converse is also true, inasmuch as an axon deprived of its myelin sheath will ultimately begin to degenerate, a process which may eventually result in neuronal 10  loss (35).  However, the process of axonal degeneration in response to demyelination is not immediate and may be ameliorated if the myelin sheath is restored.  Remyelination is the process by which a myelin sheath is restored to a denuded axon.  By remyelinating an axon, it is possible to restore conductance to pre-injury levels, resulting in functional recovery after a spinal demyelination event (37–39).  Indeed, remyelination is a critical step in the repair of the injured CNS and contributes significantly to the restoration of function; this makes it is reasonable to seek strategies to enhance remyelination in order to facilitate efforts at neural repair (10).  Remyelination is a relatively robust process and occurs rapidly and readily in vivo, providing that there are not inhibitory elements impeding the process.  This is evidenced by the remyelination observed in many MS lesions during the early stages of the disease (known as “shadow plaques” for the altered appearance of the restored myelin), a process that can continue even into the chronic stages of the disease (40–42).  Eventually, however, the pathology of MS overwhelms the body’s ability to initiate tissue repair and the inevitable functional and cognitive decline that is a hallmark of the disease’s progression results (36).  Additionally, strategies to enhance remyelination have been shown promote functional recovery following spinal cord injury (SCI), making remyelination an important reparative process following neurotrauma as well (43, 44). As OLs are terminally differentiated cells and apoptose upon demyelination, the first step in remyelination is the production of new, myelinating OLs from OPCs.  This requires that resident OPCs adjacent to the lesion site migrate into the injured  area, proliferate, and then differentiate into myelinating OLs.  Through the release of inflammatory cytokines by astrocytes and microglia in response to tissue disruption or damage, OPCs are awakened from a quiescent 11  state to one in which they are responsive to a variety of chemoattractants and mitogens, PDGF and FGF2 key among them (45).  Once activated, OPCs are able to migrate into the damaged area and differentiate into OLs.  Once these OLs contact denuded axons, they will upregulate myelin-specific genes and begin the process of myelination.  The large numbers of apparently quiescent OPCs at the margins of MS lesions that fail to remyelinate stand as evidence that a major impediment to remyelination is not the result of a failure of OL myelination but rather the inhibition of OPC differentiation (46).  The cause of this inhibition is less clear, and may be multifactorial, but seems to be due in large part to persistent myelin debris at the site of injury.  This is evidenced by the fact that myelin debris persists for up to 8 years in humans with SCI, impairs OL maturation in vitro, and that OPCs at MS lesion borders are able to differentiate while those within the lesion are impaired (9, 10, 47, 48). Remyelination is generally perceived to be a recapitulation of developmental myelination, with the caveat that the myelin sheaths produced by remyelination are generally thinner and the internodes shorter than those produced during development (49).  It has been suggested that since the ratio between sheath thickness and internode length is maintained that the observed reduction in internodal size may be the result of the fact that the axons being remyelinated do not  expand due to body growth – with a concomitant growth of the myelin internode – the way developmentally myelinated axons do (50).  Recently, an elegant study by Young et al. lent support to this idea by showing that myelin is in fact dynamic throughout life, with new OLs born and myelinating even into late adulthood (51).  A Tamoxifen-inducible conditional knock-in model was used to fluorescently label the plasma membranes of OPCs in healthy young adult and aged mice, allowing visualization of any myelin produced by the progeny of those OPCs, thereby showing that new myelin sheaths continue to be generated in the 12  uninjured adult CNS (51). Young et al. further showed that the sheaths produced by these late-born OLs were thinner and the internodes shorter than their developmentally generated counterparts.  Thus it seems likely that the observed differences in the sheaths of remyelinated axons are a factor of adult myelinating processes and not necessarily intrinsic to remyelination as a process (51).  Indeed, this study highlights the fact that re-myelination is something of a misnomer – at least from the perspective of the individual OL – since each OL has only one chance to myelinate and any myelination that it does undertake will be the first myelination event it engages in. Remyelination – or perhaps more appropriately adult myelination – stands as a critical step to the repair of the damaged CNS and, without the trophic support provided to axons by their myelinating glia, degeneration and functional decline can ultimately result.  Whether the axon has become demyelinated as a result of a myelin-specific pathology or as a secondary result of neurotrauma, reinvesting the denuded axon with a myelin sheath is a key step in achieving sustained repair and functional recovery.     Target(s)  1.2.3Given the importance of remyelination in functional recovery and sustained repair of the nervous system, it is not surprising that strategies to enhance remyelination are currently at the forefront of scientific exploration.  Strategies to enhance remyelination may be divided into two categories: those that employ exogenous elements and those that attempt to enhance endogenous mechanisms.  The former encompasses strategies such as delivery of neurotrophins or other growth factors to the injury site either alone or in concert with either autologous or exogenous cellular transplants, approaches which show great promise in pre-clinical work (52–54).  13  Examples of endogenous strategies include the aforementioned LINGO-1 antibody trial and small molecule disinhibition of remyelination (55, 56).  Promising results have also been obtained with both exogenous and endogenous approaches to enhance remyelination, but it is the latter that may present the most opportunities for neurodegenerative disease.  While invasive interventions such as intrathecal delivery of growth factors and stem cell transplantation may be both justifiable and feasible following neurotrauma – a situation in which the site of neural damage is able to be discerned – in the case of systemic demyelinating diseases like MS such interventions are not currently translatable.  In the case of diffuse neurodegenerative disorders such as MS it is difficult to pinpoint the specific location of lesions with the precision necessary for a cellular transplant using currently available, non-invasive imaging technologies.  Thus in the case of demyelinating disease it is perhaps more clinically relevant to seek strategies to enhance the endogenous capacity of the CNS to initiate tissue repair following a demyelinating insult, whether by pharmacological or genetic means.  1.3 The p75 Neurotrophin Receptor  Discovery and Structure 1.3.1 The p75 neurotrophin receptor (p75) was first identified in 1973 as a mediator of Nerve Growth Factor (NGF) signaling and was therefore named the Nerve Growth Factor Receptor (NGFR)(57, 58).  The protein was later isolated and a complete cDNA clone of NGFR was produced, allowing characterization of NGFR as a 75kDa, type 1 transmembrane protein whose extracellular domain (ECD) contains a series of highly negatively-charged cysteine repeat domains (CRDs)(59, 60). When it was later realized that p75 binds not only to NGF, a potent trophic factor responsible for neuronal survival, but also to the other neurotrophins (NTs) the 14  name was changed to the p75 neurotrophin receptor, although the name of the gene that encodes p75 remains NGFR (61–63).  NTs are discussed in more detail in Section 1.3.2.  P75 was the first member of the Tumor Necrosis Factor Receptor (TNFR) family to be discovered and at the time of its identification was a novel receptor class.  As more members of the TNFR superfamily were discovered the highly conserved, ligand-binding CRDs in the ECD were identified as characteristic of TNFRs, although p75 is the only member of the family whose CRDs bind a secreted signaling molecule and not a membrane-bound one (64).  In the case of p75, these extracellular domains can bind to all 4 known NTs as well as their immature precursors, the pro-NTs.  A subset of TNFRs – p75 among them – contain an intracellular “death domain” (DD) that can induce Caspase-mediated apoptosis when cleaved and released into the cytoplasm (65).  In the case of p75 – as with many type I transmembrane proteins – the release of the intracellular domain (ICD) is achieved through regulated intramembranous proteolysis (RIP) at a presinilin-dependent γ-secretase cleavage site (66).  P75 possesses a type II DD, meaning that the DD need not bind to itself nor to other TNFR DDs in order to activate apoptotic signaling pathways (67, 68).  Additionally, there is an extracellular, juxtamembrane α-secretase cleavage site that may be cleaved by ADAM17/TACE, a metalloprotease, in order to release the ECD into the extracellular space (69).  Interestingly, Coulson et al. demonstrated that the Chopper domain – a  sequence of 29 amino acids that connects the death domain to the transmembrane domain – is both necessary and sufficient to induce p75-mediated apoptosis in vitro, indicating that the term “death domain” for the entire ICD of p75 is something of a misnomer and that the signaling capacities of the ICD may extend well beyond that of inducing apoptosis (70).  Indeed, Parkhurst et al. used a nuclear translocation assay combined with nuclear fractionation to demonstrate that, upon release from the PM, p75’s ICD translocates to the 15  nucleus and is capable of activating reporter gene expression (71).  Additionally, some p75 signaling occurs via endosomal pathways and is also mediated by the ICD (69).   Taken together, these findings suggest that p75’s apoptotic effects are achieved by different mechanisms than its TNFR siblings (i.e. the Chopper domain, not the ICD itself) and that apoptosis is only one outcome of the ICD’s release.  The structural components discussed herein are summarized in Figure 1.4.    Figure 1.4 Molecular structure of the p75 neurotrophin receptor The molecular structure of p75 as resolved by multiple X-ray crystallography experiments. In the extracellular domain p75 possess 4 cysteine-rich domains that are involved in binding pro-NTs and NTs.  Upper and lower blue lines demarcate cleavage sites for α-secretase and γ-secretase, respectively.  The chopper domain in the highly conserved Juxtamembrane domain is required for p75-dependent apoptosis, while the Death Domain is dispensable.  The Death Domain both trafficks to the nucleus to regulate gene transcription and associates with a number of other nuclear factors, such as FAK, TRAF2, and NFκβ.  Additionally, the site of palmitoylation allows tethering of them cleaved ICD to both the plasma membrane and the cytosolic aspect of endosomal signaling vesicles while the PDZ domain potentially allows the ICD to act as a scaffold for other intracellular signaling complexes. [Adapted from (72), used with permission] 16   Neurotrophins and Neurotrophin Receptors 1.3.2The neurotrophins (NTs) are a family of highly conserved, secreted signaling molecules that are potent mediators of neuronal survival and growth and are critical not only during development but also into adulthood in the vertebrate nervous system.  NTs are secreted by neurons, the cells they form synapses with, and glia (73).  There are currently four defined NTs in the family: Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), and Neurotrophin-4 (NT-4), all four of which form homodimers in order to activate their receptors(74).  NTs are derived from precursor molecules known as pro-NTs that are cleaved intracellularly in order to mature the protein and prepare it for extracellular release(75).  Mature NTs are generally released into the extracellular space via the constitutive secretory pathway, although in neurons and neuroendocrine cells BDNF is trafficked via the activity-dependent regulated secretory pathway available to these cell types (76). NT signaling is transduced by the three Tropomyosin-related kinase (Trk) receptors – A, B, and C – as well as p75 (68).  The Trk receptors are receptor tyrosine kinases that dimerize and autophosphorylate upon ligand binding (74).  Although there is a limited amount of cross-reactivity, each Trk receptor has a particular affinity for a cognate NT: TrkA binds NGF, TrkB binds BDNF or NT-4, and TrkC binds NT-3(69).  However, there are multiple isoforms of each Trk receptor that may arise from alternate splicing events and can alter the ligand-binding  and signaling capacity of each receptor (68).  In some cases this alternate splicing may result in a truncated Trk protein lacking the intracellular kinase domain while in others it may alter the ligand-binding affinity and/or specificity of receptor through modification of the extracellular domains.  Additional modulation of Trk signaling occurs when p75 either binds to the Trk receptors, thereby dramatically increasing their affinity and specificity for their cognate ligands, 17  or releases its ECD to negatively modulate Trk signaling through competitive binding (74).  It should be noted that p75 is able to bind all four NTs with roughly equal affinity but when it does so in the absence of the NT’s cognate receptor, conflicting evidence exists as to the outcome.  Several lines of in vitro evidence suggest that apoptotic signaling results in OLs, sympathetic neurons, and other neuronal subtypes when p75 binds NTs in the absence of the appropriate Trk receptor, yet NGF binding to p75 without TrkA present can be neuro-protective against gluta  44mate exitotoxicity for cortical neurons (77–80).  These apparently paradoxical results demonstrate the complexity of p75’s effects on NT signaling and highlighbn t that these effects are extremely context-dependent and may shift with cell type, sub-type, anatomical location, and specific transcriptome.   Cell-Death Signalling 1.3.3In addition to its interactions with the Trk receptors in the context of NT signaling, p75 is also essential to apoptotic signaling both during development and following injury to the CNS.  This apoptotic response is associated with a binding of p75 to Sortilin, a protein that regulates trafficking between the endoplasmic reticulum (ER) and the PM (81).  When complexed with Sortilin p75 will effectively bind pro-NGF and pro-BDNF, subsequently transducing a JNK-dependent apoptotic signal to the intracellular space (82, 83).  In this context p75 is now the primary actor, as Sortilin does not possess an intracellular signaling domain.  Release of the ICD by γ-secretase cleavage is generally accepted as necessary for the pro-apoptotic effects of p75 to be seen, but Provenzano et al. conducted an elegant study using antibodies specific to both the ECD and ICD of p75 and found that an apoptotic response was associated with the ICD 18  remaining at the PM.  Conversely, release of the ICD from the PM and translocation to the nucleus was associated with a pro-mitotic phenotype, further highlighting the multiple roles that p75 plays in determining cell fate (84).  Indeed, Urra et al. found that release of the ICD was necessary in order for TrkA-mediated signaling to occur (85).  This apparently contradictory finding may be reconciled with previous work by considering the palmitoylation site in the Chopper domain that can tether the ICD to the membrane even after γ-secretase cleavage, thus preventing nuclear translocation of the ICD yet allowing both endosomal survival signaling (via Trks) and apoptotic signaling mediated by membrane-bound effectors to occur (Figure 1.4) (69).  Overall, in the context of regulating cell death, p75 seems most important during development, as evidenced by the supernumerary of sympathetic neurons found in p75-null mice, presumptively as a result of impaired synaptic pruning, and the fact that p75 is generally down-regulated in the healthy adult CNS; this down-regulation is especially in populations of proliferative stem cells (78).  Following injury, however, p75 is upregulated by both neurons and glia and the p75-mediated apoptotic response to pro-NTs inappropriately released into the extracellular space by tissue damage may play an important role in secondary cell death following CNS trauma (82, 86).    Cell Migration, Axonal Targeting, and Oligodendrocyte Differentiation 1.3.4In recent years, p75 has been identified as a mediator of not only cell survival and death but also cellular migration and targeting.   These effects are the result of interactions between p75 and other signaling modules and are independent of p75-related NT signaling.  In 2002 Zhigang He’s research group identified p75 as a co-receptor of the Nogo-66 receptor (NgR), in which context p75 is a necessary component for signals that inhibit neurite outgrowth in 19  response to three myelin-based growth inhibitors (MBGIs): Nogo-A, oligodendrocyte myelin glycoprotein (OMgp), and myelin-associated glycoprotein (MAG) (87).  A third member of this signaling complex, LINGO-1 (LRR and Ig domain-containing, Nogo receptor interacting protein) was identified by Mi et al. two years later (88).  As NgR is a GPI-linked extracellular protein lacking a transmembrane domain and LINGO-1 has only a short cytoplasmic tail with no catalytic domain, p75 is the presumptive signaling member of this complex.  Indeed, p75 is found to be required for signaling from this complex to proceed, although the cytoplasmic domain of LINGO does contain a tyrosine phosphorylation site and there is some evidence that this domain may participate in lateral signaling through currently cryptic mechanisms (89).  Another TNFR family member, TROY, has also been found to be able to substitute for p75 as the signaling member of this complex, much as AMIGO3 has been shown to be able to stand in for LINGO-1 (90, 91).  Although both p75 and TROY are expressed in the CNS of adult mice and rats, as determined by in situ hybridization and immunohistochemistry, their expression domains overlap only slightly in the uninjured state (92).  It should be noted that the NgR/p75/LINGO-1 signaling module normally exists almost exclusively in neurons, as NgR is not expressed in glia in the intact adult CNS (93, 94).  NgR is upregulated in reactive astrocytes and microglia of active MS leasions but is never seen in OLs (94).  For this reason most of the work that has thus far been done on this aspect of p75-dependant signaling has focused on neurons and not glia. In relation to oligodendroglia, LINGO-1 signaling has shown to have an inhibitory effect on the differentiation of OPCs – and thereby myelination – both in vitro and in vivo, primarily  via inactivation of Fyn kinase and RhoA-GTP (19).  It is of note that expression of LINGO-1 on either axons or on OPCs is sufficient to inhibit the differentiation of OPCs into OLs, suggestive 20  of other signaling pathways being involved or unidentified ligands for LINGO-1 that are expressed axonally (95).  This inhibitory effect of LINGO-1 signaling on OPC differentiation is mediated by p75 but does not require the extracellular domain of LINGO-1, suggesting additionally that there is either another ligand-binding coreceptor present in OPCs or that p75 is able to bind previously unidentified ligands when bound to LINGO-1 (89).  In either case, application of LINGO-1 antagonists has been shown to promote both myelination and functional recovery when applied to animal models of demyelination and a Phase I clinical trial  of an anti-LINGO-1 antibody treatment in Multiple Sclerosis (MS) patients ( Identifier: NCT01244139) was completed in 2012, although the results of that study have not been reported (24, 96).   There are two other additional signaling modules that p75 has recently been identified as a co-receptor in that are of note as they both have effects on cellular migration and axonal targeting.  In the context of Ephrin/Eph signaling p75 appears to play an important role in “reverse” signaling, the process by which Ephrins (a large family of membrane bound ligands that are recognized by Eph receptors) send a signal back into the cell expressing them once they bind to a receptor on a nearby cell.  Lim et al. showed in 2008 that p75 was involved in transducing such a reverse signal during the Ephrin-A signaling responsible for repulsing the extending filopodia of retinal ganglion cells (97).  P75 is also required for the repulsive effect of Ephrin-B3 – a component of mature myelin expressed exclusively by OLs in the adult CNS – on cortical neurons expressing the EphA4 receptor (98).  Although unexplored as of yet, a similar role for p75 in transducing a reverse signal in OLs during Ephrin-B signaling events is conceivable.  Finally, sympathetic neurons experience growth cone collapse in vitro in response to Semaphorin 3A signaling through the neurpilin/Plexin receptor complex, an effect partially 21  relieved by ablation of p75 (99).  All of these effects appear tied at least partially to NT signaling as well, with p75’s reported effects on Sema3A signaling apparently requiring NGF binding to occur (99).  Semaphorin 3A and 3F signaling has also been shown to regulate oligodendroglial migration both during development and in response to demyelination events (100, 101).  As p75 is expressed widely during development (especially by neural crest cells and migratory neural stem cells), at low levels in the adult CNS, and is then up-regulated after injury, it seems that it  plays an important role in governing not only the patterning of the developing nervous system but also the CNS response to pathological perturbations (102).    Myelination 1.3.5Of particular relevance to this work is the role that p75 plays in mediating inhibitory signals in response to components of the myelin sheath as well as in regulating the process of myelination itself.  The role of p75 in regulating myelination differs between the PNS and the CNS, positioning p75 as one potential mediator of the observed disparity in repair capacity between the two compartments.  Technical challenges in the production of large numbers of myelinogenic OLs has meant that the effects of p75 on SC myelination have historically been more closely examined but, with the advent of more easily accessible in vitro models of CNS myelination, p75’s role in OL myelination has begun to be explored in recent years. In SCs, p75-mediated Trk signaling regulates proliferation, differentiation, axon ensheathment, and myelination (103).  Cosgaya et al. demonstrated in vitro that NT-3, via Trk C, inhibits myelination by promoting SCs proliferation and inhibiting differentiation (58).  Once NT-3 signaling is reduced, NGF and BDNF (depending on the subtype of neuron being myelinated) are able to up-regulate myelin-specific genes in SCs (58, 104).  Interestingly, in the 22  PNS NGF promotes myelination through TrkA in a p75-independent manner while BDNF-induced myelination is p75-dependent, drawing attention to the context specificity of p75’s influence (105).  It is also of note that NT signaling in both neurons and SCs is responsible for these effects, highlighting the large amount of communication between axons and glia that mediates the process of myelination in general.   In OLs the role of p75 in regulating myelination – primarily through its effects on NT signaling – is less well understood but is beginning to be more rigorously explored.  As might be expected, pro-myelinating NT signaling in OLs is modulated by both p75 and Trk receptors, although the relative role of each shifts contextually.  The contrast between the PNS and CNS in this regard is highlighted by the fact that NGF inhibits the myelination of NGF-dependent dorsal root ganglion (DRG) neurons by OLs rather that promoting it, as is the case with SCs (104).  NT-3 was one of the first factors identified as requisite for OPC survival and proliferation in vitro and mice lacking NT-3 exhibit severe deficiencies in CNS glia and a hypomyelinated phenotype, although it is unclear if this is as a result of a direct effect on myelinating OLs or as a result of a global decrease in OPCs (106, 107).  Much as is the case in the PNS, BDNF has a promyelinating effect on basal forebrain oligodendrocytes once they differentiate from OPCs (105).  However, Xiao et al. demonstrated that – unlike the PNS – this pro-myelinating effect is p75-independent, placing p75 critically at a point of differential myelinogenic potential between the CNS and PNS (108).  It must be said that these effects appear regional, as cortical OLs do not express TrkB and are sensitive instead to NGF, although the role of p75 in this latter process is unknown (109).  Nonetheless, it seems that p75 may be dispensable for the actual initiation of the myelination program in OLs, at least in some regions of the CNS. 23  In addition to p75’s role in modulating and transducing pro-myelinating NT signals, it also plays a role upstream of the process of myelination, inasmuch as LINGO-1 signals propagated by p75 inhibit the differentiation of OPCs (but do not cause apoptosis) and thus inhibit the production of myelin (24).  Given the fact that LINGO-1 is largely unexpressed in white matter of the adult CNS – but is widely up-regulated proximal to spinal cord contusions – and that increased p75 expression is found in both OLs and OPCs in and around active MS lesions, it is possible that this signaling plays a larger role in regulating remyelination following injury than during developmental myelination (92, 110).  Nonetheless, p75-dependent LINGO-1 signaling is relevant during development as well, since TrkA-mediated up-regulation of axonal LINGO-1 inhibits peripheral and central myelination (104).  Overall, the evidence seems to suggest that p75 is largely involved in setting the scene for CNS myelination to occur – through the migration, proliferation, and differentiation of OPCs – but may be unnecessary for myelination to proceed once myelinating OLs are produced.    Given the tight link of NT signaling to the process of myelination in both the PNS and the CNS, it is not surprising that p75 has a role to play.  Interestingly, p75’s role in the process of CNS myelination seems to be one that is primarily exerted upon OPCs as opposed to OLs.  This is to say that there are at least two populations of OLs thus far identified (basal forebrain and optic nerve) whose myelination programs are p75-independent, while there is little evidence to suggest that active CNS myelination is unable to proceed in the absence of p75.  These findings combine to position p75 as a potential therapeutic target when attempting to modulate the process of myelination after injury.  24  1.4 Murine Models of Demyelination and Remyelination In order to examine the processes that govern remyelination in the mammalian nervous system, a method of inducing demyelination experimentally is necessary.  There exist several different well-established models of experimental demyelination in mouse and rat models that may be divided into three categories: toxin-induced demyelination, autoimmune-mediated demyelination, and virus-induced demyelination.  Virus-induced demyelination is accomplished by the CNS inoculation of experimental animals with either oligodendroglia-specific corona viruses (such as the JHM strain of mouse hepatitis) or to Theiler’s murine encephalomyelitis virus (TMEV) (111, 112).  Autoimmune-mediated demyelination is accomplished by the induction of Experimental Autoimmune Encephamyelitis (EAE) through immunization of an animal with purified CNS myelin proteins – such as Myelin Oligodendrocyte protein (MOG) – in combination with Freund’s adjuvant (113).  This causes the body to mount an autoimmune attack on the myelin of the CNS with debilitating functional decline resulting.  However, virus-mediated demyelination and EAE both incorporate a significant immune component in order to achieve CNS demyelination.  While this immune component models the pathology of certain demyelinating diseases such as MS as well as the heightened inflammatory environment following neurotrauma, it also serves as a complicating and confounding factor when attempting to parse the role that non-immune cells play in regulating remyelination.  As activated astrocytes and microglia/macrophages can have both negative and positive modulating effects on the process of remyelination through both direct and indirect effects on OPCs and OLs, in vivo models that necessarily invoke these states through neuroinflammation may present challenges to interpretation (23, 114).  These concerns are somewhat ameliorated by using toxin-induced models of demyelination.  Systemic application of cuprizone – a potent chelator of copper – in 25  the diet of mice causes widespread CNS demyelination that is readily remyelinated once cuprizone is withdrawn (49, 115).  Cuprizone is quite specifically gliotoxic and the demyelination that occurs is not mediated by an immune or neuroinflammatory response.  Focal demyelination is also possible through stereotaxic injections of gliotoxic substances directly into the CNS, with ethidium bromide (a DNA interchelator) and lysophosphatidylcholine (LPC) being the most commonly used.  As ethidium bromide is highly cytotoxic and results in the focal depletion of both OLs and astrocytes it is in some ways a less desirable agent than LPC.  LPC is a naturally occurring lipid that is normally part of cellular membranes a very low concentrations but form micelles at high concentrations, thereby specifically disrupting myelin membranes while leaving astrocytes and microglia relatively intact (116).  However, it should be noted that a certain amount of microglial activation is unavoidable during CNS demyelination as a phagocytic response is mounted whenever myelin debris is produced.  Indeed, Ousman and David showed that even when LPC is used as a demyelinating agent the injection causes an acute disruption of the blood-brain barrier that allows some infiltration of T-cells and monocytes in the first 6hrs following injury, resulting in microglial activation (117).  This same study showed a remarkable capacity for such rapidly activated microglia to clear myelin debris following LPC injection into the white matter of the spinal cord at an even faster rate than occurs in the PNS following sciatic nerve injury, with most myelin debris being cleared by 4 days post injection (dpi) and large amounts of remyelination by 21dpi (117).  This demonstrates not only that the CNS is in fact capable of efficient myelin clearance and remyelination in the absence of widespread neuroinflammation but also that the LPC model allows examination of the effects of myelin debris acute to injection.  Thus, despite an inevitable involvement of the phagocytic cells of the CNS, LPC-mediated demyelination appears to avoid many of the major impediments to 26  myelin clearance (and thus remyelination) that are seen in other models, making it a good choice when attempting to assay the role of a given factor in non-immune-mediated remyelination processes.  It should be mentioned that a similar role for astro- and micro- glial activation and myelin debris clearance is clear in cuprizone-mediated demyelination as well.  However, the variation in demyelination between species and mouse strains, variation in demyelination between brain regions, extended time required for demyelination (5-7 weeks of cuprizone chow), and extremely rapid remyelination upon cuprizone withdrawal (<7 days) make this model in some ways better suited to the study of the roles astrocytes and microglia play in mediating remyelination rather than a direct examination of the myelinating glia themselves (118, 119). Given the several models of demyelination available, choosing the model most appropriate to the research question being asked is essential.  In contexts where an examination of the immune system’s role is desired, more systemic and immune-mediated approaches – such as EAE or TMEV – are perhaps more appropriate.  In contrast, if the focus is an understanding of oligodendroglial processes in remyelination is desired, a toxin-induced model may be preferable.  With these concerns in mind, the LPC model was chosen for this study.  Although myelin clearance is relatively rapid in the LPC model, at least as compared with immune-mediated models, it is still possible to examine the effects of myelin debris acute to injury (i.e. <4dpi).  As well, with LPC the off-target effects of the demyelination process are limited as much as is possible.  Thus this was determined to be the most appropriate model to address the experimental question posed in the present study.  27  1.5 Project Objectives Seeking strategies to enhance remyelination is a key goal in the quest for effective neural repair in the mammalian CNS.  A major impediment to remyelination is the inhibition of OPC differentiation into myelination OLs in the presence of myelin debris.  The p75/LINGO-1 signalling complex has been shown to transduce signals that inhibit OPC differentiation in response to myelin-based inhibitory factors and inhibition of LINGO-1 function enhances OPC differentiation and myelination in vitro (24, 88).  Further, while BDNF promotes PNS myelination in a p75-dependant manner, in the basal forebrain of the CNS the pro-myelinating effects of BDNF are p75-independent (105).  Thus p75 represents an amenable target to enhance CNS remyelination since inhibition of p75 function in OPCs may allow enhanced differentiation in the presence of inhibitory factors while not impairing the ability of OLs thus generated to initiate their myelination program.  I am therefore testing the hypothesis that ablation of p75 function will allow increased differentiation of OPCs into pre-myelinating OLs following chemical demyelination in mice.  In order to assess the validity of this hypothesis, focal demyelinating lesions were induced in the corpus callosum of wild-type (WT) and p75-null (p75 KO) mice.  The corpus callosum was chosen because it is one of the most densely myelinated tracts in the adult CNS, is relatively easy to access for stereotaxic injections, and lacks neuronal cell bodies.  In order to assess the role p75 plays in regulating OPC differentiation antigenic markers of OPC differentiation were assayed immunohistochemically at 3, 7, and 11 days post injection (dpi).  These time points were chosen because the correspond to the periods of greatest OPC migration, OPC differentiation, and  initiation of remyelination in the LPC injury model (116). 28  The aim of this work is to confirm p75 as a potential therapeutic target to enhance endogenous remyelination following demyelination.  P75 has demonstrated itself to be amenable to pharmacological inhibition of function, as shown by the myelin sparing and reduced secondary tissue damage observed when a small-molecule inhibitor of pro-NGF binding was administered orally following spinal cord injury in mice (55).  Although this effect was achieved by inhibiting p75’s pro-apoptotic function it is likely that a similar small-molecule inhibitor specific to p75’s ability to inhibit OPC differentiation could also be developed.  This thesis is therefore intended to address the question of whether or not p75 should be further pursued as a target in the search for strategies to enhance remyelination.  29  Chapter 2: Methods  2.1  Experimental Overview In order to evaluate what effects p75 has on OPC differentiation following a demyelinating insult, L-α-lysophosphatidylcholine (LPC) was used to induce a demyelinating lesion in the corpus callosum of both wild-type mice and mice with a germ-line knockout (KO) of the third exon of p75, a mutation that eliminates ¾ of the extracellular domains and abrogates p75 signaling (120).  At 3, 7, and 11 days post injection (dpi) 5 male and 5 female mice of each genotype were sacrificed for analysis.  Additionally, 2 females and 1 male received LPC injections and were analyzed at 1 dpi in order to characterize the lesion produced.  Due to variability in the stereotaxic injections, some animals were excluded post hoc as a result of inaccurate delivery of the LPC solution (i.e. too dorsally or too ventrally, resulting in the solution being released into the grey matter/lateral ventricle and little or no demyelination of the corpus callosum).  Figure 2.1 outlines the experimental workflow while Table 2.1 summarizes the total number of animals of each genotype and gender that were competent to be analyzed. In order to ensure that p75-mediated signaling does not regulate remyelination through an oblique effect on OPC responses to proliferative signals, 3 males of each genotype were analyzed at 3dpi using Olig2/PDGFRα/Ki67 immunoreactivity as a marker of proliferating OPCs (21, 121, 122). In order to determine if p75 plays a role in inhibiting OPC differentiation the differentiation status of OPCs within the lesion was immunohistochemically assayed at 3dpi, 7dpi, and 11dpi.  OPCs were defined as cells displaying Olig2+/PDGFRα+ double 30  immunoreactivity, while differentiated pre-myelinating OLs were defined as cells with Olig2+/CC1+ double immunoreactivity (123, 124).      Figure 2.1 Experimental design overview Animals were injected with LPC at 0 dpi and sacrificed at 1, 3, 7, and 11 dpi.  1 dpi animals were used to characterize the lesion acute to injury.  3dpi animals were used to assay OPC proliferation and differentiation status while 7dpi and 11 dpi animals were used only for differentiation analysis. Table 2.1 Total experimental animals competent for analysis 31  2.2 LPC-induced Demyelination of the Murine Corpus Callosum All procedures involving live animals were conducted in compliance with UBC’s institutional guidelines and within the constraints of Animal Ethics protocols approved by the UBC Animal Care Committee.   All animals were bred in-house and all surgical procedures and post-operative care procedures were performed in-house and by qualified UBC personnel.   Breeding 2.2.1In order to produce mice for this study three breeding pairs of mice heterozygous for a deletion of the third exon of NGFR were purchased from The Jackson Laboratory (JAX; B6.129S4-Ngfrtm1Jae/J ) and used to raise a breeding colony.  Mice from F5-F7 were used.  Genotypes were determined by duplicate PCR reactions using an Extract-N-Amp™ Tissue PCR Kit (Sigma-Aldrich XNAT2 SIGMA), primer designs provided by the manufacturer (Appendix B), and primers synthesized by UBC core services.  Although these mice were developed on a C57BlJ/6 background, the ablation of p75 function can be developmentally deleterious and JAX classifies this strain of mice as “a challenging breeder”.  Due to the low fecundity of the strain it was not possible to produce an entire experimental cohort of animals at a single time.  Rather, the experimental groups represented in Figure 2.1 were produced through serial matings that spanned 16 months of breeding.  In all cases where possible, littermate controls were used; where littermate controls were not possible controls were chosen from closely related animals (i.e. 1st cousins, never 2nd), age-matched to within 5 days.  In order to avoid confounds related to brain size and global metabolism, body size of p75-null mutants was considered.  Work completed during the generation of a conditional p75 allele indicates that by 10 weeks of age, 32  p75 mutants are equivalent in body size to their wildtype littermates (125), therefore only mice aged 70-92 days were used in this study.     Pre-Surgical Care 2.2.2All animals were weaned and raised in a group-housed breeding colony with environmental enrichment as well as food and water available ad libitum. In addition, high-calorie supplementation was provided via raw sunflower seeds and Froot Loops™.  Three days prior to surgery the diurnal cycle of the animals was shifted from breeding (light 0700-2100, dark 2100-0700) to experimental ( light 0700-1900, dark 1900-0700) in order to allow animals time to adjust.  In order to reduce stress levels and acclimatize animals to extensive handling, animals were provided with an enriched diet and additional daily handling prior to surgery.   Surgical Procedures 2.2.3Aerosolized Isoflurane was used to anaesthetize animals for surgery.  Anesthesia was induced with a flow-rate of 5% and then animals were maintained at a surgical level, as determined by loss of foot pinch and corneal reflexes, at a flow-rate of 1%-1.5%.  The dorsal aspect of the skull was shaved and sterilized using Hibitane and topical iodine solution.  Prophylactic pain control was administered in the form of a subcutaneous injection of 0.05 mg/kg Buprenorphine at the thoracic level concomitant with a subcutaneous injection of Lidocaine at the incision site.  In order to prevent dehydration during surgery, 1mL of Lactated Ringer’s solution was also provided subcutaneously.  A lubricating ophthalmic ointment was applied and maintained in order to prevent corneal damage during surgery.  Throughout surgery body temperature was monitored and animals were provided with supplemental heat in order to 33  maintain a constant body temperature.  Animals were mounted in a stereotaxic frame (KOPF) and a 1-1.5 cm incision was made on the dorsal aspect of the skull, exposing the bregma.  The head was determined to be level in the frontal plane by making measurements both rostral and caudal to the bregma – using a blunted Hamilton syringe mounted in the frame – and adjusted as necessary.  In the longitudinal plane the head is kept level by the ear bars of the frame.  The centre of bregma was determined as the point at which the sutures of the skull would meet if all were perfectly straight. At a point 1 mm rostral and 1.4 mm lateral to bregma a dentist’s drill with a fine bit was used to create a window of approximately 3 mm2 in the right frontal bone of the skull, with care being taken not to rupture the underlying meninges.  In order to demyelinate the corpus callosum a 10 μL Hamilton syringe with a sterilized, pulled-glass needle was loaded with 2 µL of 1% LPC (Sigma-Aldrich L1381) in sterile PBS, attached to an automated microinjection pump, and positioned at 1 mm rostral/1.4 mm lateral to bregma.  Zero depth was set by lowering the tip of the needle until the dura flexed and then raising it just until the dura returned to its original position. The needle was then lowered into the brain to a depth of 2.1 mm.  The LPC was injected at a flow-rate of 50 nL/min.  Animals were monitored for signs of distress or dehydration throughout the injection and, in order to prevent reflux of the injected fluid, a period of 8-12 minutes was allowed after the injection was complete before the needle was withdrawn.  Surgical incisions were closed using 6-0 nylon sutures.   Post-Surgical Care 2.2.4Following surgery, animals were placed in a 37Cº incubator and monitored until they had recovered from the anesthetic and were able to access food and water ad libitum.  Animals remained in the incubator for a minimum of 60 minutes to allow time for thermoregulation to be 34  re-established.  Animals were provided with a calorie-enriched diet following surgery in order to help with recovery.  Post-surgical pain control was provided in the form of 0.05mg/kg Buprenorphine injected subcutaneously at 12 hour intervals for 72 hours following surgery.  Animals were weighed and monitored daily for clinical signs in accordance with criteria previously approved by the UBC Animal Care Committee (Appendix A).     Histological Analysis Tissue Preparation and Sectioning Animals were humanely euthanized in accordance with approved protocols.  Animals were then perfused through the left ventricle of the heart, first with 15 mL of phosphate-buffered saline (PBS) to clear the circulatory system and then with 40 mL of an ice-cold solution of 4% paraformaldehyde to fix tissue.  The entire brain was removed and placed in 4% paraformaldehyde overnight at 4Cº.  Tissue was then cryoprotected by immersion in ascending concentrations of sucrose (12%, 18%, and 24%; 4Cº), spending 24 hours in each solution.  Following cryoprotection, tissue was flash-frozen over dry-ice and mounted on a cutting block using OCT compound (Tissuetek 4583) and stored at -80Cº.  Subsequently, tissue was thawed to -22Cº and 20 µm thin sections were produced on a cryostat (Thermo Scientific Microm HM-525).  Whole brains were sectioned in the transverse plane beginning at the olfactory bulb and moving caudally. Tissue sections began to be collected once the pyriform cortex – the structure rostral to the forceps anterior of the corpus callosum – was observed and continued to be collected until the third ventricle became evident.  Sections were collected serially onto 12 microscope slides such that each slide contained 10-12 serial sections at ~240 µm intervals and encompassing the entire lesion.   35 Immunohistochemistry Frozen slides were thawed for 60-90 min at room temperature before being rehydrated in 0.1M PBS (7.2% NaCl, pH 7.4) for 10 min.  Slides were then subjected to delipidation through ascending and descending ethanol washes (50%, 70%, 90%, 95%, 100%; 2 min ea.) followed by a triple wash in PBS.  Slides were blocked with 10% new donkey serum in PBS-Triton (BDH R06433) for 30 min, and then primary antibodies suspended in PBS-Triton were applied for 20-23 hrs.  Slides were then triple-washed in PBS before the application of secondary antibodies (Jackson Immunoresearch, Alexa Fluor Affinipure F(ab’) fragments, donkey host), also suspended in PBS-Triton, which were applied for 2 hrs.  Slides were then triple washed in PBS before being coverslipped with Shandon Immu-Mount (Thermo-Scientific 9990402).  A list of primary antibodies used and a detailed worksheet outlining Immunohistochemical workflow is available in Appendix C. Microscopy, Stereology, and Statistical Analysis All imaging was performed on a Zeiss Axioplan Observer confocal microscope fitted with a Yokogawa spinning disc.  Zeiss Zen software was used for image processing, including tile stitching and shading correction, as well as for image analysis and cell counting.  Large, tiled images encompassing the entire lesion area were captured utilizing a z-stack with optical sections spaced 1.25 μm apart.  Lesion borders were defined using MBP immunoreactivity to identify damaged myelin.  The uninjured contralateral ventricle was used as a reference to ensure that only areas that had been myelinated were included in the defined lesion border while aggregations of cells and debris in the lateral ventricle were excluded.  Upper and lower z- limits 36  were set 1 optical section beyond the point at which no tissue was in the focal plane to ensure all tissue was imaged. Cell counts were obtained using the 3D counting method described by Williams and colleagues, with slight modification to update the method for use with a confocal microscope and a large tiled area (126).  Briefly, a confocal stack was chosen in which the entire lesion area was in focus.  Due to tissue warping and differential tissue shrinkage during delipidation this usually comprised the central 6-8 optical sections of the z-stack.  The upper and lower z-limits were then reduced by one optical section (i.e. one section from the upper and one from the lower were removed) to define the upper and lower “guard zones” of the counting box.  The top, bottom, and sides of the counting box were defined as the lesion borders, delineated on the central optical section.  Cells whose nuclei crossed the top and right sides of the lesion border were counted while those that crossed the bottom and left borders were excluded.  Contrary to Williams et al. cells that cross both the upper and lower boundaries were counted.  Although this results in a slight overestimation of the total cellular density within the lesion volume, the densities so obtained are not statistically different from densities previously observed in our lab or reported in the literature (Greg Duncan, unpublished data)(127).  For each animal, 4 sections on two separate slides were analyzed, resulting in good coverage of the lesion rostro-caudally.  All cell counts were performed by a single rater, blinded to the identity of the slides by triply obscuring each slide’s ID information with opaque tape from a time before imaging commenced until after all cell counts were complete.   IBM SPSS Statistics software was used for all statistical analyses.  To compare WT to p75 KO groups within a time point, data were confirmed to be normally distributed with Levene’s Test for Equality of Variances and then group means were compared using Student’s 37  independent t-test.  Where data were non-parametric, a Mann-Whitney U-test was used to compare group means instead.  To compare means between time points a one-way ANOVA was conducted with post-hoc Bonferroni correction to t-tests.  For all tests α-level for P-values was set at 0.05.  Statistics are reported in the text as mean (SD) while data are reported in figures as mean (SEM). 38  Chapter 3: Results 3.1 Characterization of Demyelination Acute to Injury To establish a qualitative baseline for the LPC injury and confirm that the injury was comparable to those created previously, two female and one male mouse were killed and analyzed at 1 day post injection (dpi).   Staining for Myelin Basic Protein (MBP) shows heavily disrupted myelin membranes and myelin debris (Figure 3.1, top panel).  Approximately 80% of axons were spared, consistent with previously obtained results (Figure 3.1 middle panel; Greg Duncan, unpublished observations).  The lesion epicenter lacked Olig2 immunoreactivity and only minor staining at the lesion border was seen (Fig.3.1 bottom panel).                          39  Figure 3.1 Example image of LPC lesion at 1dpi IHC characteriza-tion of the lesion caused by LPC injection.  (Top) MBP, marking healthy myelin, shows complete demyelination in epicenter and disrupted and degenerating myelin at lesion borders, (Middle) The panoligoden-droglial marker Olig2 is complete-ly absent from the centre of the lesion, while (Bottom) the SMI312 axonal marker shows preservation of axons within the lesion.  Scale bar = 50µm         40  3.2 Effect of p75 on Proliferative Capacity of OPCs Following Demyelination In order to address effects of p75 on the proliferative capacity of OPCs acute to LPC injection 3 males of each genotype were analyzed at 3dpi.  Lesion borders were defined using MBP immunoreactivity to identify damaged myelin (Figure 3.2).  Mean lesion volume analyzed (µm3) was not different between groups [WT 2.77x106 (8.43x105),  p75KO 2.35x106 (7.65x105), p>0.05 Student’s independent t-test](Figure 3.3).  Total oligodendroglial recruitment was measured by counting Olig2+ nuclei/mm3 and no difference was seen between groups [WT 1.99x106 (6.5x103, p75KO 1.90x106 (3.95x104), p>0.05 Student’s independent t-test].  Proliferation status of OPCs was measured by Olig2+/PDGFRα/Ki67+ triple labelling (Figure 3.2).  No difference was seen between group means either in total proportion of Olig2+ cells that were OPCs [WT 41.9% (3.3%), p75KO 38.2% (3.0%), p>0.05 Student’s independent t-test] or in the proportion of total Olig2+ cells that were proliferating OPCs [WT 22.8% (3.1%), p75KO 19.9% (7.7%), p>0.05 independent samples Mann Whitney U-test](Figure3.3).             41   Figure 3.2 Example image of OPC proliferation analysis At 3dpi LPC injection into the corpus callosum causes widespread demyelination and a large amount of myelin debris is still present.  The lesion has been repopulated with Olig2+/ PDGFRα OPCs (arrowheads) by 3dpi, many of which also co-label with Ki67 (arrows), identifying them as proliferating OPCs.  cc: corpus sallosum, lv: lateral ventricle, svz: sub-ventricular zone, scale bar = 100µm.  42  Figure 3.3: Effect of p75 on proliferative capacity of OPCs following demyelination Lesion volume and total oligodendroglial recruitment were comparable between WT and p75KO. For both WT and KO groups the total number of OPCs was not different, nor was the proportion of OPCs that were in active cell growth (~50%). Data shown are group means, error bars are SEM.    43  3.3 Effect of p75 on Differentiation Status of OPCs Following Demyelination  First, the effects of p75 KO on OPC recruitment were analyzed by counting total density of Olig2+ nuclei (cells/mm3) within the lesion borders.  No differences were seen between groups at 3dpi [WT 1.88x105 (4.74x104), p75KO 1.78x105 (3.28x104), p>0.05 Student’s independent t-test], 7dpi [WT 1.88x105 (4.74x104), p75KO 1.78x105 (3.28x104), p>0.05 Student’s independent t-test], or 11dpi  [WT 2.67x105 (3.69x104), p75KO 2.41x105 (4.55x104), p>0.05 Student’s independent t-test](Figure 3.4a).  When time points were pooled and oligodendroglial recruitment was compared, 3dpi was significantly less than both 7dpi (p<0.001) and 11dpi (p<0.05), while there was no difference between 7dpi and 11dpi (p>0.05) [ 3dpi 1.83x105 (3.95x104), 7dpi 2.76x105 (8.08x104), 11dpi 2.45x105 (4.56x104), One-way ANOVA with post-hoc Bonferroni correction]. It was confirmed that there was no difference in total lesion volume analyzed (µm3) and no differences were seen between groups at 3dpi [WT 2.5x106(7.37x105), p75KO 2.82x106 (9.9x105), p>0.05 Student’s independent t-test], 7dpi [WT 1.97x106(7.787x105), p75KO 1.8x106 (2.53x105), p>0.05 Student’s independent t-test], or 11dpi [WT 1.78x106(7.12x105), p75KO 2.09x106 (1.28x105), p>0.05 Student’s independent t-test] were observed (Figure 3.4b).   When volume lesion analyzed was compared across time points, a difference was seen between 3dpi and 7dpi (7dpi<3dpi, p=0.019), a trend  towards difference was seen between 3dpi and 11dpi (p=0.1), and no difference was seen between 7dpi and 11dpi (p=1) [ 3dpi 2.67x106 (8.7x105), 7dpi1.89x106 (6.28x105), 11dpi 1.99x106 (1.12x106), One-way ANOVA with post-hoc Bonferroni correction]. In order to examine the effects of p75 on OPC differentiation, both male and female animals were analyzed at 3dpi, 7dpi, and 11dpi (Figures 3.5-3.7).  Total animals competent to be 44  analyzed at each time point are summarized in Table 2.1.  Differentiation was assessed as a shift in character from Olig2+/PDGFRα+ to Olig2+/CC1+ and quantified as a percentage of total Olig2+ cells within the lesion borders.  No difference between groups was seen at 3dpi [WT PDGFRα 37.3% (4.6%) WT CC1 58.9% (5.0%), p75KO PDGFRα 41.8% (7.9%) p75KO CC1 54.3% (7.6%), p>0.05 Student’s independent t-test], 7dpi [WT PDGFRα 33.8% (8.4%) WT CC1 63.3% (8.9%), p75KO PDGFRα 32.0% (8.5%) p75KO CC1 65.1% (9.0%), p>0.05 Student’s independent t-test], or 11dpi [WT PDGFRα 33.0% (10.8%) WT CC1 63.3% (10.4%), p75KO PDGFRα 32.2% (14.1%) p75KO CC1 64.4% (13.4%), p>0.05 Student’s independent t-test](Figure 3.8).  When groups within each time point were pooled and compared across time points the proportion of CC1+ positive cells at 3dpi was significantly different from 7dpi (p<0.05), but not different from 11dpi (p>0.05), while no difference was observed between 7pi and 11pi (p=1) [3dpi 56.5% (6.7%), 7dpi 64.4% (8.0%), 11dpi 63.6% (13.4%), One-way ANOVA with post-hoc Bonferroni correction].  When the groups in each time point were pooled and the proportion of PDGFRα+ cells was compared across time points no difference was seen between any time point [3dpi 39.7% (6.8%), 7dpi 32.5% (7.7%), 11dpi 33.1% (13.9%), One-way ANOVA with post-hoc Bonferroni correction].  When the above comparisons were conducted on male and female groups separately, similar results were obtained (Figure 3.8).   For males, no difference between groups was seen at 3dpi [WT PDGFRα 35.5% (5.4%), WT CC1 61.33% (6.0%), p75KO PDGFRα 44.6% (9.2%) p75KO CC1 52% (9.2%), p>0.05 Student’s independent t-test], 7dpi [WT PDGFRα 31.2% (9.7%) WT CC1 65.6% (10.7%), p75KO PDGFRα 27.9% (8.9%) p75KO CC1 69.1% (10.2%), p>0.05 Student’s independent t-test], or 11dpi [WT PDGFRα 22.3% (5.8%) WT CC1 65.5% (1.9%), p75KO PDGFRα 30.2% (3.9%) p75KO CC1 72.2% (6.5%), p>0.05 Student’s independent t-test]. 45  For females, no difference between groups was seen at 3dpi [WT PDGFRα 38.8 (3.8%), WT CC1 57% (3.5%), p75KO PDGFRα 38.9% (6.1%) p75KO CC1 56.6% (5.7%), p>0.05 Student’s independent t-test], 7dpi [WT PDGFRα 37.1% (6.2%) WT CC1 60.5% (6.5%), p75KO PDGFRα 36.1% (6.6%) p75KO CC1 61.2% (6.5%), p>0.05 Student’s independent t-test], or 11dpi [WT PDGFRα 35.7% (15.9%) WT CC1 61.2% (15.9%), p75KO PDGFRα 40.1% (14%) p75KO CC1 57.7% (14.2%), p>0.05 Student’s independent t-test]. The group means of males and females were also compared at each time point in order to identify any gender differences, however no significant differences in relative proportions of OPCs to OLs between genders emerged at any time point.      46   Figure 3.4 Oligodendroglial recruitment to lesion and lesion volume Top) No difference between wild type (WT) and p75 knockout (KO) groups at 3dpi (red), 7dpi (green), or 11dpi (blue) was seen in total oligodendroglial recruitment, although there was an increase in total Olig2+ cells between  3dpi and later time points.  Bottom) Total lesion volume analyzed was not different between groups at any time point.  Total lesion volume decreased from 3dpi to 7dpi and trended towards a decrease from 3dpi to 11dpi while no difference was seen between 7dpi and 11dpi.  Data shown are group means, error bars show SEM. 47   Figure 3.5 Example image at 3dpi  At 3dpi significant amounts of myelin debris remains in the lesion but the lesion has been repopulated with oligodendroglia comprising OLs (arrowheads) and – primarily – OPCs (arrows).  cc: corpus callosum, lv: lateral ventricle, svz: sub-ventricular zone, scale bar= 100µm.  48   Figure 3.6 Example image at 7dpi  By 7dpi myelin debris has been largely cleared and the lesion is populated with pre-lesion levels of Olig2+ cells and the proportion of OLs (arrowheads) is now greater than OPCs (arrows).  cc: corpus callosum, lv: lateral ventricle, svz: sub-ventricular zone, scale bar= 100µm.   49   Figure 3.7 Example image at 11dpi  By 11dpi remyelination at the lesion borders is visible and the lesion is densely populated with OPCs (arrows) and OLs (arrowheads).  cc: corpus callosum, lv: lateral ventricle, svz: sub-ventricular zone, scale bar= 100µm.  50    Figure 3.8 Differentiation status of OPCs following LPC demyelination a) No differences were seen between WT and p75KO groups within time points but there was a shift in the identity of Olig2+ cells as OPCs differentiated into OLs over time.  b,c) There were no differences found between WT and p75KO when males and females were considered separately.  Data shown are group means, error bars show SEM       51  Chapter 4: Discussion This study was designed to investigate the role of p75 in regulating CNS remyelination in vivo using a murine model of toxin-induced demyelination.  Specifically, the role of p75 in OPC differentiation was examined following injection of the demyelinating agent lysophosphatidylcholine (LPC) into one of the most richly myelinated tracts of the adult brain, the corpus callosum.  This model was chosen for its low inflammatory component relative to other murine models of demyelination, thereby allowing a more precise examination of the role that p75 plays in oligodendroglia while minimizing the confounding effects of large-scale neuroinflammation.  In order to examine p75’s role, LPC was injected into both wild-type mice and mice possessing a germ-line knockout of the third exon of NGFR, a hypomorphic mutation that results in a non-functional p75 protein (p75 KO) (120).  To the authour’s knowledge this study is the first to specifically address the role of p75 in the regulation of OPC differentiation.  Since OPC differentiation is a major rate-limiting step to neural repair following neurotrauma and demyelinating disease, identification of amenable molecular targets that effect enhanced OPC differentiation is a key step to developing therapeutic approaches for clinical translation (10, 35).  Given that small molecule inhibitors of some p75 function have been developed and shown to have efficacy in preventing OL death and enhance myelin sparing following spinal cord injury, the potential to disinhibit OPC differentiation through a similar mechanism is promising, provided that p75 plays a role in inhibiting OPC differentiation (55). In order to assess the demyelination caused by the LPC injection a small cohort of mice were analyzed at 1dpi.  Within the lesion boundary almost complete demyelination was observed (Figure 3.1, top panel), with remaining MBP immunoreactivity being attributable to still degenerating myelin and persisting myelin debris.  However, consistent with previously reported 52  results, excellent preservation of axons within the lesion was observed (Figure 3.1, middle panel) indicating that the while LPC is effective at disrupting the myelin sheaths it has only minor effects on axonal survival acute to injury (116).  Most importantly, a complete lack of Olig2 immunoreactivity within the lesion was observed (Figure 3.1, bottom panel).  The complete absence of oligodendroglia within the lesion at 1dpi demonstrates that any Olig2+ cells that are subsequently observed within the lesion must be freshly recruited and not the result of oligodendroglial sparing.  Taken together, these results show that the LPC injury model results in robust demyelination through a direct action on OLs while sparing the majority of axons. A potential confound in assessing the effect of an experimental intervention on the differentiation capacity of OPCs is the potential for an effect on the proliferative response of OPCs to skew results.  Inhibition of OPC proliferation, whether through direct or indirect mechanisms, can have the same deleterious effect on remyelination as an inhibition of differentiation (116).  In either case the ultimate result is a decrease in the absolute number of remyelinating OLs available to initiate tissue repair.  P75 plays a large role in modulating neurotrophin signaling, the effects of which are known to affect the proliferation of OPCs (75, 106).  In order to address this, the effect of p75 on OPC proliferative capacity was examined at 3dpi (Figure 3.2).  After confirming that there were no statistical differences in total lesion volume or total OPC recruitment between groups, no differences between WT mice and p75 KO mutants were observed (Figure 3.3) Both groups showed around 50% of OPCs co-labelling with the proliferative marker Ki67, as reported previously (127).  Since the proliferative response of OPCs is most important acute to injury – during the phase of OPC recruitment and proliferation prior to OPCs differentiating into OLs – it is reasonable to conclude from these data that p75 is not affecting OPC proliferation (128, 129).  This is rectified with p75’s known effects on NT 53  signalling in OPCs by considering that NT signalling can and does continue in the absence of p75, although it may not be as powerful, and that in vitro the proliferative effects of NT-3 on OPCs (when combined with PDGF) are mediated directly by full-length TrkC (129–131).   The main purpose of this study was to assess the effects of p75 on OPC differentiation in response to chemical demyelination.  Because p75 is known to play a role in signalling complexes that regulate OPC migration, total oligodendroglial recruitment to the lesion was first examined in order to exclude these effects (100, 101).  No differences between WT and p75 KO groups were observed at any time point (Figure 3.4).  Total Olig2+ density increased 50% between 3dpi and 7dpi, returning Olig2+ cell densities to levels commensurate with the uninjured state and consistent with kinetics of oligodendroglial recruitment and remyelination reported previously in the LPC injury model (116, 127).  As total lesion volume could potentially skew results, it was confirmed at each time point that there were no differences between WT and p75KO groups (Figure 3.4).  No statistical difference in lesion volume was seen between WT and p75 KO at any time point, although when WT and p75KO groups were pooled at each time point and compared across time points there was a small but statistically significant reduction in total lesion volume between 3dpi and 7dpi (7dpi < 3dpi, p=.019) but not between 3dpi and 11dpi (p = 0.1) or between 7dpi and 11dpi (p=1).  This slight reduction in lesion size by 7dpi is consistent with remyelination at the borders of the lesion beginning, thereby reducing the lesion size (116).  The fact that this reduction is not statistically significant between 3dpi and 11dpi may be attributed to increased variability and the relatively small sample size in the latter group.   In order to examine p75’s role in regulating OPC differentiation the ratio of OPCs to OLs (i.e. Olig2+/PDGFRα+ cells to Olig2+/CC1+ cells) was used as a measure of OPC differentiation (21, 124).  Triple-stained cells (i.e. Olig2+/ PDGFRα+/CC1+) were observed only 54  extremely rarely ( <0.1%) and only 2-4% of cells were Olig2+/PDGFRα-/CC1-, indicating that these antigenic markers are almost entirely mutually exclusive and are a good measure of OPC differentiation.  OPC differentiation was analyzed at 3 dpi, 7 dpi, and 11dpi (Figure 3.5-7) and was considered with genders pooled and with males and females independently.  Statistical differences in the ratio of OPCs to OLs were not observed between WT and p75 KO at any time point, nor was there a difference between the OPC differentiation status between males and females at any time point (Figure 3.8).  Interestingly, at 11dpi p75 KO males trended (0.05> p <0.1) towards having a greater proportion of differentiated OLs than p75 KO females but this may be the result of increased variability in the latter group.  For all groups there was a shift in the ratio of OPCs to OLs over time, with the proportion of OPCs decreasing and the proportion of OLs increasing (Figure 3.8).  This shift in character is consistent with a large influx of OPCs migrating into the lesion site acute to injury and then rapidly differentiating into CC1+ pre-myelinating OLs.    At 3dpi OPCs comprise almost half of the Olig2+ population but by 7dpi the OPC proportion has decreased to approximately 25-30% of Olig2+ cells, proportions that are supported by previous studies using this injury model (132).  Taken together these data suggest that p75 signalling does not affect the differentiation of OPCs following demyelination. The data generated in this study indicate that p75 is not required for the normal recruitment, proliferation, and differentiation of OPCs into pre-myelinating OLs following LPC-mediated demyelination of the murine corpus callosum.  Thus it appears that p75 is entirely dispensable for the initiation of remyelination in this injury model.  These findings are surprising since p75 forms signalling complexes with LINGO-1 – a known inhibitor of OPC differentiation whose disinhibition allows for enhanced differentiation in vitro  and in vivo – and is the putative signalling member of the complex (24, 88, 105).  Further, Bourikas et al. showed in vitro that 55  p75 antagonists were able to relieve LINGO-1 mediated inhibition of differentiation in human OPCs (89).  Of course, the situation in vivo is much more complex than the isolated and controlled conditions of cell culture but the results of the current study nonetheless seem to contradict these previous findings.    It is possible that the failure of this study to show an effect of p75 KO on OPC differentiation following LPC-mediated demyelination could be a result of the genetic model that was used.  The p75 KO mice that were used in this study (Jackson Laboratories B6.129S4-Ngfrtm1Jae/J) are the standard genetic model that has been used in the field of p75 research since their generation by the lab of Rudolf Jaenisch more than 2 decades ago; there is a large body of literature comprising both in vitro and in vivo studies that have used this genetic model.  However, these mice carry a germline deletion of exon III of NGFR and while mice homozygous for the mutation are viable the manufacturer classifies them as “challenging breeders” and they display a host of deleterious phenotypes.  These include, but are not limited to: delayed post-natal growth, reduced fecundity, defects in the descending aorta, development of edematous ulcers on the extremities by 4 months of age, deficits in peripheral innervation (especially of the skin, footpads, and tongue), abnormal reflexes, impaired mobility caused by hindlimb ataxia, peripheral hypomyelination, central hypermyelination (in certain tracts), and a peri-natal death rate of approximately 40% (120, 125).  Taken together, this host of anatomical abnormalities demonstrates the importance of p75 function during normal embryonic and post-natal development, primarily through its role in modulating NT signalling but also through its pro-apoptotic role as a mediator of appropriate cell-death.  However, these mice are viable and so it must be considered that some redundancy is partially compensating for the loss of p75 function, thereby allowing these animals to survive and potentially confounding the results of this study. 56  The most likely candidate for p75 redundancy is another TNFR family member known as TROY that has high homology to p75 and – in contrast to p75 – is widely expressed in the uninjured adult CNS (68, 133).  Consistent with this, TROY has been shown to be able to replace p75 in the NgR/p75/LINGO-signalling complex that mediates repulsion of neurites and growth cone collapse both in vitro and in vivo (90, 134).  In addition, Sun et al. have just recently published work showing that not only do OPCs express TROY but also that pharmacological or RNAi-mediated inhibition of TROY function promotes OPC differentiation in vitro and enhances the remyelination produced by grafted OPCs following spinal cord injury; this information was unavailable at the time that the current study was being designed and executed (135).  Thus it now appears that compensatory mechanisms allowing signals inhibiting OPC differentiation in response to myelin to be transduced in the absence of p75 do exist.  Since the p75 KO mice used in this study lacked p75 function throughout their life and p75 is an extremely promiscuous and important signalling molecule during development, it is entirely conceivable that TROY may have been abnormally upregulated to fill the gap left by p75 deletion and substituted some of p75’s functions, at least in the LINGO-1 signalling complex, thereby allowing a measure of appropriate axonal targeting and OPC differentiation inhibition to occur,  a possibility that should be explored in future work.  While it is not possible to parse the relative contributions of p75 and TROY in regulating remyelination using the exon III p75 KO mouse, the use of a more specific genetic model would allow this.   In the time since the exon III p75 KO mouse was developed the suite of genetic tools available to basic researchers has expanded greatly.  With the advent of inducible, conditional genetic manipulations afforded by Cre-LoxP recombination technologies it is now possible to ablate gene function immediately prior to an experimental intervention, thus allowing the mice to 57  develop normally.  A more precise genetic model could be generated by crossing mice containing a p75 allele in which the transmembrane and cytoplasmic domains (exons 4-6) have been floxed with mice containing a knock-in allele of tamoxifen-inducible Cre recombinase expressed under a high-efficiency PDGFRα promoter (>95% recombination in corpus callosum); both of these are strains currently available to interested academic researchers (125, 136).  This model would allow for p75 to be ablated specifically in OPCs directly before a demyelinating insult and would allow for a more precise examination of the role p75 actually plays in OPC differentiation after injury to the CNS.  Additionally, the recent development of CRISPR/Cas-mediated genome editing and the Jaenisch lab’s development of a streamlined protocol for implementing this technology to rapidly generate mice with up to 5 genome edits at once mean that it is now both technically feasible and practical to investigate the specific, regional, and relative roles of p75 and TROY in mediating OPC differentiation and remyelination in the murine CNS (137).  Future studies using such genetic technologies will allow a clarification of the results obtained in this study and a rectification of these results with previous and current findings. In addition to a more precise genetic model, it will be informative to examine p75’s role in mediating remyelination in other murine models of demyelination.  The transgenic mice suggested above should be exposed to both the cuprizone model and to EAE as well.  Cuprizone-induced demyelination is widespread, has delayed remyelination, lacks a large inflammatory component, and does not activate the phagocytic response of microglia in the way that LPC does, all of which would allow a better examination of the role p75 plays in mediating OPCs’ responses to myelin debris.  The EAE model is entirely immune mediated and would allow for 58  an examination of p75’s role in regulating OPC differentiation in the presence of a severe inflammatory response and widespread auto-immune attack. Overall, this study may have raised more questions than it answered.  While the data produced are unequivocal it may be that the genetic model used contains within it confounds that are capable of masking a subtle effect of p75 on OPC differentiation.  While it is tempting to accept the findings of this study prima facie, it is likely that if p75 is playing a role in OPC differentiation that its role is – like other p75 signalling – nuanced, subtle, and contextual.  Thus a more precise examination of p75 function in this context, utilizing the most modern genetic tools available, is warranted before the matter is deemed closed.  59  Chapter 5: Conclusion 5.1 Conclusions In order to query the role of p75 in the regulation of OPC differentiation the LPC injury model was applied to both wild-type (WT) mice and mice transgenic for a deletion of the third exon of p75 (p75KO).  These two cohorts of mice were then analyzed immunohistochemically at 1, 3, 7, and 11 days post injection (dpi). The validity of the model was shown and LPC injections were found to cause almost complete demyelination within the injection site at 1dpi, via elimination of oligodendroglia, while leaving left axons relatively unharmed.  Consistent with previous characterizations of the LPC injury model, there was a large amount of myelin debris visible at 3dpi, this debris had largely been cleared by 7dpi, and remyelination was visible at lesion borders by 11dpi (116).  Thus the injury model worked well in our hands. Based on the findings of this study, p75 is not required for the appropriate migration, proliferation, or differentiation of OPCs necessary for remyelination following LPC-induced demyelination of the murine corpus callosum.  The data generated herein therefore fail to provide support for the hypothesis that abrogation of p75 signalling allows disinhibition of OPC differentiation in the presence of myelin debris, thereby enhancing remyelination, and the null hypothesis fails to be rejected.    5.2 Strengths and Contributions This study is the first to specifically address the role of p75 in regulating the inhibitory effect of myelin on OPC differentiation in the LPC injury model.  Both male and female mice were used in the study, allowing examination of gender-specific effects that might exist.  Time 60  points of 3dpi, 7dpi, and 11dpi were chosen to correspond with the height of  OPC recruitment/proliferation, differentiation, and initiation of remyelination, respectively (116).  Additionally, this work was conducted entirely in vivo.  While this does introduce the complexity of the complete organism it also makes any findings more immediately relevant to clinical translation. The work contained in this thesis may have ultimately resulted in more questions being asked than answered when it comes to the role of p75 in the regulation of OPC differentiation.  This, however, is not necessarily a shortcoming of the study.  In light of the fact that the findings of this study contradict results produced previously one of the contributions that this study has made to the larger field of p75 research is to call into question the validity of the standard genetic model (i.e. the p75 exon III KO mouse) when assessing p75’s role in oligodendroglia.  While this model has been extremely useful and generated reams of data in the examination of p75’s role in neuronal function, the contradictory findings of the current study suggest that a more precise genetic model may be necessary in order to assay the more subtle role that p75 may play in regulating oligodendroglial function. It is hoped that the uncertainties raised by the current work as to p75’s role in regulating OPC differentiation will spur further inquiry into the matter.  Although there now seem to be other molecular elements that are critical, p75 may still represent a valid target to enhance CNS repair via remyelination.  This possibility is highlighted by the fact that p75 is relatively well characterized and a number of small molecule inhibitors of p75 function exist.  Thus even if p75 alone is insufficient to alleviate myelin-mediated inhibition of OPC differentiation, it may nonetheless be a key component in a multi-factorial intervention aimed at enhancing endogenous remyelination. 61  5.3 Recommendations for Future Work The results of this thesis suggest that in future more precise genetic models should be used when examining the role of p75 in oligodendroglial biology in vivo.  Specifically, a tamoxifen-inducible conditional knockout model should be used wherein the cytoplasmic domain (i.e. the signalling domain) of p75 is specifically ablated in OPCs prior to experimental interventions.  This would eliminate the various and often severe phenotypes associated with a global knockout of p75 function.  Since p75’s functions in both neurons and glia are relevant to the processes that mediate remyelination, a global knockout is less than desirable.  With the advent of CRISPR/Cas genome editing technology and the ability to introduce multiple genome edits simultaneously, it is also now possible to create genetic models that are specific at both the temporal and cellular level and can thus examine the relative roles of several genes on a given process.  Given the recent work by Sun et al. that identifies TROY as a mediator of OPC differentiation, future work should explore the relative roles of p75 and TROY in regulating CNS remyelination using such precise genetic models (135).  As well, in future studies of p75’s function in the context of remyelination, both cuprizone-mediated demyelination and EAE-mediated demyelination should be examined.  This will allow a more nuanced understanding of the role that p75 signalling plays in mediating the neuronal and oligodendroglial response to neuroinflammation and large-scale monocytic infiltration of the CNS.   5.4 Concluding Statement  Remyelination is a potent endogenous repair process that is maintained throughout adult life in the mammalian CNS and strategies to enhance remyelination after CNS damage represent 62  promising avenues for clinical interventions.  This thesis has examined p75 as a potential therapeutic target for such interventions.  The results obtained in this study suggest that p75 may be excluded as such a target.  However, the findings of this thesis contradict some previous findings regarding p75’s role in OPC differentiation and so further and more targeted exploration using more precise genetic models would be prudent.  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Cell 153:910–8.   73  Appendices Appendix A  : Animal Monitoring Criteria and Recording Sheets 74  Appendix B  : Genotyping Primers and PCR cycling protocol  Protocol Primers      Primer 5' Label Sequence 5' --> 3' 3' Label Primer Type oIMR7617 - GCT CAG GAC TCG TGT TCT CC - Common oIMR7618 - CCA AAG AAG GAA TTG GTG GA - Wild type Reverse oIMR8162 - TGG ATG TGG AAT GTG TGC GAG - Mutant Retrieved from : Cycling  Step # Temp °C Time Note 1 94 3 min - 2 94 30 sec - 3 50 1 min - 4 72 1 min repeat steps 2-4 for 35 cycles 5 72 2 min - 6 10 - hold  Retrieved from :          75  Appendix C  Primary Antibodies and Immunohistochemistry Staining Worksheet STAINING DETAILS              Date:____________________ Slide #s: 1. Dry Time 60-120 mins  2. PAP Pen   3. Rehydration (In cylinder) (PBS)10mins 4. Delipidation:   None / Alcohol / Chloroform 5. Wash (if delip. - PBS w/ agitation) 10 mins X3  6. Serum Block:               serum;  conc.___1:10__   time: _______     # of slides________ X 500 (full) µL = _______ µL Total     ______ µL Total – _____ µL Serum = ______ µL PBSTriton  Total # of slides per combo of 1˚ Ab’s µL of 1˚ solution / slide Total volume of µL of 1˚ solution Colour 1˚ Ab Dilution µL 1˚ Ab in Total µL of 1˚ solution  µL PBSTriton (Total volume – Total µL of all 1˚ Ab’s)  X 300 = GREEN     RED    BLUE    FAR RED     Primary Ab’s   OVERNIGHT Next Morning 1. Wash (PBS w/ agitation) 10 mins X3 2. Apply 2˚ Ab    2 HOURS  Total # of slides per combo of 2˚ Ab’s µL of 2˚ solution / slide Total volume of µL of 2˚ solution Colour 2˚ Ab Dilution µL 2˚ Ab in Total µL of 2˚ solution  µL PBSTriton (Total volume – Total µL of all 2˚ Ab’s)  X 300 = GREEN     RED    BLUE    FAR RED    3. Wash (PBS w/ agitation) 10 mins X3 5.Coverslip and Label with colour specific names of primary antibodies 76  List of Primary Antibodies   Antibody Host Supplier Catalogue #MBP Ch Aves MBPSMI312 Ms Covance SMI-312R-100Olig2 Rb Millipore AB9610MBP Ch Aves MBPOlig2 Ms Millipore MABN50PDGFRα Gt R&D Systems AF1062Ki67 Rb Abcam ab15580MBP Ch Aves MBPOlig2 Rb Millipore AB9610CC1 (APC) Ms Abcam ab16794PDGFRα Gt R&D Systems AF1062Lesion characterizationProliferation AssayDifferentiation Assay


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