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The role of p75NTR in axonal regeneration and intraspinal plasticity following spinal deafferentation Scott, Angela Lee 2009

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THE INHIBITORY ROLE OF P75NTR IN AXONAL REGENERATION AND INTRASPINAL PLASTICITY FOLLOWING SPINAL DEAFFERENTATION  by ANGELA LEE SCOTT B.Sc., The University of Saskatchewan, 2001 B.A., The University of Saskatchewan, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2009  © Angela Lee Scott, 2009  Abstract Following spinal cord injury (SCI), functional recovery is extremely limited in adult mammals. This lack of recovery is reflective of the failure of axons to regenerate and the incapacity of uninjured neurons to compensate for the lost connections. In animal models of SCI, the administration of neurotrophic factors such as neurotrophins promotes both the regeneration and sprouting of injured and uninjured axons. Neurotrophins elicit these growth-promoting effects through tropomyosin-related kinase (Trk) receptors. However, neurotrophins also interact with a pan neurotrophin receptor, p75NTR. Several functions of the p75NTR receptor have been reported, but its role in neurotrophin-mediated signalling following SCI remains unclear. To determine the role of p75NTR in neurotrophin-mediated axonal regeneration and sprouting within the CNS, I assessed anatomical changes and functional outcomes following spinal deafferentation in mice lacking the neurotrophin-binding domain of p75NTR (p75-/-) and in wild-type littermates (p75+/+). Regeneration of sensory axons was significantly greater in p75-/- mice, and resulted in functional re-connection with dorsal horn neurons. Axonal regeneration in the p75-/- mice was neurotrophin-dependent, and dis-inhibited by the absence of p75NTR expression on glial cells following injury. These findings indicate that glial expression of p75NTR restricts neurotrophin availability to the extent that it prevents spontaneous sensory axon regeneration into the spinal cord. Intraspinal sprouting of uninjured neuronal processes following dorsal root injury was also evaluated in p75+/+ and p75-/- mice. The density of sprouting axons was significantly enhanced by the application of exogenous neurotrophins and dis-inhibited by the absence of p75NTR. Dendritic density was also promoted by exogenous  ii  neurotrophin treatment, but was not affected by the absence of p75NTR. Together, these results demonstrate that p75NTR restricts the intraspinal sprouting of neurotrophinresponsive axons, but not dendrites, within the injured spinal cord. Dis-inhibition of dendritic plasticity was, however, correlated to the antagonism of truncated TrkB receptor (TrkBT1) expression. Thus, the neurotrophin receptors p75NTR and TrkBT1 may differentially inhibit intraspinal sprouting of pre- and post-synaptic processes within the spinal cord following injury.  iii  Table of Contents Abstract.......................................................................................................................... ii Table of Contents .......................................................................................................... iv List of Figures............................................................................................................... ix List of Abbreviations .................................................................................................... xi Acknowledgements .................................................................................................... xiii Dedication ................................................................................................................... xv Co-authorship Statement ............................................................................................ xvi 1. Introduction ............................................................................................................. 1 1.1 Overview ........................................................................................................... 2 1.2 Dorsal rhizotomy: a model for the investigation of axonal regeneration and intraspinal sprouting ........................................................................................... 4 1.2.1 Anatomical changes to sensory processes following dorsal rhizotomy ....... 5 1.2.2 Wallerian degeneration in the spinal cord .................................................. 7 1.2.3 Wallerian degeneration in the dorsal root .................................................. 9 1.3 Inhibition of axonal regeneration and sprouting following injury ..................... 11 1.3.1 Astrocytic-derived inhibition ................................................................... 11 1.3.2 Myelin-derived inhibition ........................................................................ 12 1.3.3 Inhibition of axonal regeneration and plasticity following dorsal rhizotomy ................................................................................................ 15 1.4 Promotion of axonal regeneration and sprouting following injury .................... 17 1.4.1 The Neurotrophin family ......................................................................... 17 1.4.2 Tropomyosin-related kinase (Trk) receptors ............................................ 18  iv  1.4.3 Promoting regeneration and sprouting following dorsal rhizotomy .......... 20 1.5 Growth-promoting and growth-suppressing factors converge on p75NTR ........... 21 1.5.1 p75NTR is a pan neurotrophin receptor ..................................................... 21 1.5.2 p75NTR interaction with Trk receptors ...................................................... 23 1.5.3 p75NTR-mediated inhibition of axonal growth .......................................... 25 1.5.4 p75NTR is a co-receptor for myelin-associated inhibitory proteins ............ 25 1.5.5 Consequences of p75NTR expression following dorsal rhizotomy ............. 26 1.6 Experimental objectives and hypotheses .......................................................... 27 1.7 Bibliography .................................................................................................... 34 2. Schwann cell p75NTR prevents spontaneous sensory re-innervation of the adult spinal cord ................................................................................................... 47 2.1 Introduction ..................................................................................................... 48 2.2 Materials and methods ..................................................................................... 49 2.3 Results ............................................................................................................. 58 2.3.1 Injured sensory axons regenerate into the spinal cord in p75-/- mice ......... 58 2.3.2 Regeneration is accompanied by functional recovery in p75-/- mice ......... 59 2.3.3 Successful regeneration in p75-/- mice depends on endogenous neurotrophins .......................................................................................... 61 2.3.4 Regeneration of primary afferent axons in p75-/- mice is only marginally augmented with the addition of neurotrophic factors ............................... 62 2.3.5 Absence of axonal p75NTR does not promote regeneration ....................... 63 2.3.6 P75-/- Schwann cells enhance neurotrophin-dependent regeneration in vitro .................................................................................................... 65  v  2.4 Discussion ....................................................................................................... 66 2.5 Bibliography .................................................................................................... 81 3. Deafferentation and neurotrophin-mediated intraspinal sprouting: a central role for the p75 neurotrophin receptor ................................................................ 84 3.1 Introduction ..................................................................................................... 85 3.2 Materials and methods ..................................................................................... 86 3.3 Results ............................................................................................................. 89 3.3.1 Primary afferent axons ............................................................................ 90 3.3.2 Serotonergic axons .................................................................................. 91 3.3.3 Tyrosine-hydoxylase-expressing axons ................................................... 93 3.4 Discussion ....................................................................................................... 94 3.4.1 Spinally-derived neurotrophins may initiate intraspinal sprouting ............ 96 3.4.2 Altered neurotrophin-tropomyosin-related kinase affinity/specificity and intraspinal sprouting ......................................................................... 97 3.4.4 Intracellular tropomyosin-related kinase and p75 signaling cascades and intraspinal sprouting................................................................................. 98 3.4.4 Myelin-derived inhibitory protein signaling and intraspinal sprouting ..... 99 3.5 Bibliography .................................................................................................. 116 4. Differential effects of endogenous neurotrophins on dorsal rhizotomy induced dendritic plasticity reveal a novel interaction between NGF and BDNF signaling ................................................................................................................... 121 4.1 Introduction ................................................................................................... 122 4.2 Materials and methods ................................................................................... 124  vi  4.3 Results ........................................................................................................... 129 4.3.1 Exogenous BDNF induces sprouting of intraspinal dendritic processes................................................................................................ 129 4.3.2 p75NTR does not account for the inhibition of dendritic plasticity following injury .................................................................................... 130 4.3.3 The expression of TrkBT1 is increased in the deafferented spinal cord .. 131 4.3.4 Endogenous NGF regulates the expression of the truncated TrkB receptor ................................................................................................. 131 4.3.5 Endogenous NGF limits dendritic sprouting in the lateral spinal nucleus .................................................................................................. 132 4.3.6 Deafferentation-induced serotonergic sprouting is enhanced by TrkA-Fc ................................................................................................ 133 4.4 Discussion ..................................................................................................... 134 4.4.1 Injury-induced dendritic plasticity in the CNS ....................................... 134 4.4.2 Regulation of dendritic plasticity by neurotrophins ................................ 135 4.4.3 TrkB isoforms and dendritic sprouting in the deafferented spinal cord .. 136 4.4.4 p75NTR does not suppress dendritic plasticity following spinal deafferentation ...................................................................................... 138 4.4.5 Conclusions .......................................................................................... 139 4.5 Bibliography .................................................................................................. 149 5. Discussion ............................................................................................................ 153 5.1 Summary of thesis ............................................................................................... 154 5.2 The multifaceted role of p75NTR in axonal regeneration ....................................... 156  vii  5.2.1 Glial p75NTR inhibits neurotrophin-mediated regeneration across DREZ ..... 156 5.2.2 Axonal p75NTR promotes Trk-mediated axon regeneration .......................... 157 5.2.3 P75NTR regulates cellular death following axonal injury .............................. 159 5.2.4 P75NTR controls neurotrophin-mediated myelination ................................... 161 5.3 Differential regulation of intraspinal sprouting via p75NTR ................................... 162 5.3.1 MAIPs inhibit intraspinal axonal plasticity via p75NTR ................................ 162 5.3.2 P75NTR influences neurotrophin-responsiveness of spared axons ................. 162 5.3.3 Glial p75NTR sequestration of neurotrophins in the CNS .............................. 163 5.3.4 P75NTR induces oligodendrocyte cell death .................................................. 164 5.3.5 Intraspinal plasticity of dendrites is not governed by p75NTR ....................... 165 5.4 Final comments ................................................................................................... 165 5.5 Bibliography ........................................................................................................ 168  viii  List of Figures Figure 1.1 Sensory afferent innervation of the dorsal horn ........................................... 31 Figure 1.2 Sites of potential manipulation of myelin-signaling pathways for promoting axonal plasticity ......................................................................................... 33 Figure 2.1 P75NTR prevents spontaneous regeneration into the CNS ............................. 71 Figure 2.2 Functional recovery in rhizotomized p75-/- mice ......................................... 73 Figure 2.3 Regeneration in p75-/- mice is neurotrophin-dependent ............................... 75 Figure 2.4 P75NTR expression by the axonal environment, not the axon, limits regeneration ................................................................................................................ 77 Figure 2.5 Enhanced regeneration in a p75-/- environment is neurotrophin-dependent in vitro ............................................................................................................................ 79 Figure 2.6 Regeneration is limited in wild-type mice due to neurotrophinsequestering Schwann cell p75NTR ............................................................................... 80 Figure 3.1 Time-course of primary afferent axon sprouting in the dorsal horn after rhizotomy in p75+/+ and p75-/- mice ........................................................................... 102 Figure 3.2 Effects of neurotrophins on primary afferent axon sprouting in the dorsal horn 7 days after rhizotomy in p75+/+ and p75-/- mice ................................................ 104 Figure 3.3 Time-course of serotonergic axon sprouting in the dorsal horn after rhizotomy in p75+/+ and p75-/- mice ........................................................................... 106 Figure 3.4 Effects of neurotrophins on serotonergic axon sprouting in the dorsal horn 7 days after rhizotomy in p75+/+ and p75-/- mice ................................................ 108 Figure 3.5 Time-course of tyrosine hydoxylase-expressing axon sprouting in the dorsal horn after rhizotomy in p75+/+ and p75-/- mice ................................................. 110 Figure 3.6 Effects of neurotrophins on tyrosine hydoxylase-expressing axon sprouting in the dorsal horn 7 days after rhizotomy in p75+/+ and p75-/- mice ............. 112 Figure 3.7 Summaries of deafferentation and neurotrophin-mediated changes in axon density after septuple dorsal rhizotomy ............................................................. 113 Figure 3.8 Positive and negative influence on intraspinal plasticity mediated by p75NTR ....................................................................................................................... 115  ix  Figure 4.1 Dendritic density in the lateral spinal nucleus ........................................... 141 Figure 4.2 Injury-induced TrkBT1 expression was significantly upregulated 1.5-fold following injury ........................................................................................... 143 Figure 4.3 TrkA-Fc treatment substantially promoted the increase of MAP2positive dendrite density in both p75+/+ and p75-/- mice following injury ................... 145 Figure 4.4 Deafferentation-induced serotonergic sprouting is enhanced by TrkA-Fc treatment ..................................................................................................... 147 Figure 4.5 Nerve growth factor expression is upregulated following spinal deafferentation ................................................................................................ 148  x  List of Abbreviations 5-HT BDNF BSA cAMP CGRP CNS CsA CSPG CST CTB DMEM DREZ DRG ECL FGF2 GAG GAP43 GAPDH GFAP GFP GDI GDP GPI HBSS IB4 LSN MAIPs MAG MAPK MAP2 NF200 NFκB NGF NgR NTs NT3 NT4/5 Omgp p75NTR p75-/p75+/+ PBS PNS RAGs  - 5-hydroxytryptamine (serotonin) - brain derived neurotrophic factor - bovine serum albumin - cyclic adenosine monophosphate - calcitonin gene-related peptide - central nervous system - cyclosporin A - chondroitin sulfate proteoglycan - corticospinal tract - cholera toxin subunit B - Dubelco’s Modified Eagle Medium - dorsal root entry zone - dorsal root ganglia - enhanced chemiluminescense - fibroblast growth factor 2 - glycosaminoglycan - growth-associated protein 43 - glyceraldehyde-3-phosphate dehydrogenase - glial fibrillary acidic protein - green fluorescent protein - GDP dissociation inhibitor - guanosine 5'-diphosphate - glycosylphosphatidylinositol - Hank’s balanced salt solution - isolectin B4 - lateral spinal nucleus - myelin-associated inhibitory proteins - myelin-associated glycoprotein - mitogen-activated protein kinase - microtubule-associated protein 2 - neurofilament 200 - nuclear factor kappa B - nerve growth factor - Nogo receptor - neurotrophins - neurotrophin-3 - neurotrophin-4/5 - oligodendrocyte myelin glycoprotein - p75 neurotrophin receptor - p75 knockout - p75 wild-type - phosphate buffered saline - peripheral nervous system - regeneration-associated genes  xi  SCs SCI SCT SERT TH TMB Trk TNFR WGA  - Schwann cells - spinal cord injury - spinocervical tract - serotonin transporter - tyrosine hydroxylase - tetramethylbenzidine - tropomyosin-related kinase - tumor necrosis factor receptor - wheatgerm agglutinin  xii  Acknowledgements I would like to begin by thanking my family. Throughout my academic pursuits, my parents have been a constant source of love and support, and for this, I owe my deepest graditude. I also wish to thank my brothers for their understanding, respect and unconditional friendship. I will never be able to express how much I have appreciated their encouragement and unwavering confidence in me throughout it all. I would also like to thank my supervisor Dr. Matt Ramer for allowing me the opportunity to pursue this scientific endeavor. Over the years, he has guided me through the highs and lows of academic research and graduate school. I’d like to believe that I stayed the course and landed on my feet as a result. I would also like to thank him for the trust and scientific freedom that he bestowed on me. By allowing me the liberty to expand my boundaries and make mistakes, I was able to learn more about the science and develop as a researcher. I will always appreciate that. Finally, he has led by example, and in doing so, persuaded me to always do the best that I can. I would like to thank my supervisory committee (Dr. Wolfram Tetzlaff, Dr. Vanessa Auld, Dr. Patricia Schulte, and Dr. Tim O’Conner) for thier patience, encouragement and advice. I was always welcomed with an open door when I needed it. A special thanks goes to Dr. Wolfram Tetzlaff and Dr. Patricia Schulte for extending the open door policy to their labs. Without the use of these facilities, many of the experiments required for my thesis work would have been impossible. To the past and present Ramerites and adoptees, I thank you for the many laughs, the heartfelt cries, the countless stories, the shared pitchers, and wonderful memories. Your friendship made the lab a wonderful place to be and will always be highly valued. I would like to add a special thank you to Leanne Ramer, for continuing to be an inspiration in her pursuit of excellence in science and motherhood; to Jessica Inskip, for always reminding me of the positive side in every situation; to Victoria Claydon, for all the talks and cheeky pints; to Lesley Soril, for her faith in me and endless encouragement; and to Andrew Gaudet, for his willingness to explore the many places and conferences with me over the years – it was always a blast. I would like to thank my co-authors (Leanne Ramer, Lesley Soril, Jacek Kwiecien, Jaimie Borisoff, and Matt Ramer) for the privilage of working with them and their assistance with the manuscripts. I am also indebted to Emily Lipinski for excellent animal care support and her much needed assistance with genotyping; to Jeremy Toma for his gracious assistance in a set of the behavioral studies; to Alan Gillet, for his work with me during his directed studies; to Dr. D. Hampton, Dr. P. Wood and Andrew Gaudet, for their helpful direction during the tissue culture experiments; and to Clarrie Lam, for being an incredible resource for molecular biology techniques and her willingness to always help. I must also thank my experimental subjects. I hope that I will never take for granted their crucial contribution to our quest of understanding biological systems. To my fellow ICORDians, so many of you have helped me along this journey to get to this final destination. To the fellow graduate students, post-docs and technicians at ICORD, who have offered their support in so many ways, have been my extended scientific family and a brilliant group of friends since I began graduate school. I would like to thank the ICORD administration staff, especially Cheryl Niamath and Jeremy  xiii  Green, for being an excellent liaison between students and the university. I would also like to thank the Zoology administration staff, Allison Barnes and Alice Liou in particular, for all their assistance over the years. Finally, I would like to thank my husband Graham Scott. His everlasting patience, unwavering support and repeated encouragement, has been a cornerstone for each step that I have been able to take during the past several years. I owe much of my success to him and thank him from the bottom of my heart.  xiv  Dedication  To my parents and my loving husband  xv  Co-authorship Statement The work presented in Section 1.3 of Chapter 1, and Chapters 2 - 4 of this thesis has been previously published or submitted for publication. Scott, A.L.M., Ramer, L.M., Soril, L.J.J., Kwiecien, J.M. and Ramer, M.S. (2006) Targeting myelin to optimize plasticity of spared spinal axons. Molecular Neurobiology 33: 91-111. Scott, A.L.M. and Ramer, M.R. (2009) Schwann cell p75NTR prevents spontaneous sensory re-innervation of the adult spinal cord. Submitted. Scott, A.L.M, Borisoff, J.F. and Ramer, M.S. (2005) Deafferentation and neurotrophinmediated intraspinal sprouting: a central role for the p75 neurotrophin receptor. European Journal of Neuroscience 21: 81-92. Scott, A.L.M and Ramer, M.S. (2009) Differential effects of endogenous neurotrophins on dorsal rhizotomy-induced dendritic plasticity reveal a novel interaction between NGF and BDNF signaling. Submitted. The thesis author Angela Scott was the primary author of the review article subsection presented in Section 1.3 of Chapter 1. Leanne M. Ramer, Lesley J.J. Soril and Dr. M. Ramer assisted in the preparation of this manuscript. Lesley J.J. Soril and Dr. J. Kwiecien conducted the experiments for this manuscript that were not included in this thesis.  The thesis author Angela Scott was the primary researcher for all results presented in the research articles presented in Chapters 2-4 under the guidance of Dr. M. Ramer. Dr. J. Borisoff assisted in the statistical analysis of the data presented in Chapter 3.  xvi  CHAPTER ONE  Introduction1  1  A portion of this chapter has been published. Scott, A.L.M., Ramer, L.M., Soril, L.J.J., Kwiecien, J.M. and Ramer, M.S. (2006) Targeting myelin to optimize plasticity of spared spinal axons. Molecular Neurobiology 33: 91-111. 1  1.1 Overview Spinal cord injury (SCI) is a debilitating neurological condition that can often result in permanent autonomic, motor and sensory disorders. In Canada, more than 40,000 people live with chronic disabilities caused by SCI, and over 1,000 new cases arise each year (Canadian Paraplegic Association, 2009). Current treatments for patients with SCI, such as surgical manipulation of the vertebral column and anti-inflammatory strategies (Bagnall et al., 2008; Hurlbert and Hamilton, 2008), help to minimize the damage caused by SCI but do not reverse it. Thus, there is a prominent and growing need for the development of treatment strategies that will restore neurological function. Restoration of neurological function following SCI is dependent on the successful regeneration of injured axons and/or the compensatory sprouting of uninjured processes to re-establish lost connections. In the adult injured CNS, axonal regeneration and neuroplasticity is severly limited by the low intrinsic growth capacity of CNS axons, and the presence of inhibitory extrinsic factors (reviewed in Jacobs and Fehlings, 2003). Recent treatment strategies have attempted to increase the regenerative vigor of growing axons by exposing them to growth factors, or neutralize the restrictive environmental constituents in the CNS with the application of blocking peptides (reviewed in Fawcett and Asher, 1999). Unfortunately, these treatments used individually or in combination have achieved only limited success in adult mammalian models. The ineffectiveness of these treatment strategies suggest that our understanding of the factors that govern functional recovery in the injured CNS is far from complete. Thus, the aim of the research presented in this thesis was to further elucidate the underlying mechanisms that prevent axonal regeneration and sprouting and investigate alternative  2  treatment strategies. In particular, I examined the role of p75 neurotrophin receptor (p75NTR) in the injured spinal cord. p75NTR is a compelling target for SCI research since it is a point of convergence for both growth-suppressing and growth-promoting molecules within the CNS. For instance, p75NTR is a receptor for myelin-associated inhibitory protiens (MAIPs), which inhibit axonal growth (Wang et al., 2002a; Yamashita et al., 2002). On the other hand, neurotrophins are a family of growth factors that enhance axonal growth (reviewed in Kaplan and Miller, 2000), but also interact with p75NTR. The inhibitory role of p75NTR in MAIP-mediated signaling was established; however, the role of p75NTR in neurotrophin-mediated axonal growth was relatively unknown. My goal, therefore, was to examine the effects of p75NTR expression to axonal regeneration and intraspinal sprouting following injury and how its interaction with neurotrophins, in particular, affected functional recovery. In the first study (Chapter 2), I investigated the effect of p75NTR on the failure of axonal regeneration in the spinal cord, and the influence p75NTR expression had on neurotrophin signaling following injury. In the second and third study (Chapter 4 and 5), I explored the possible inhibitory role of p75NTR on intraspinal sprouting of uninjured pre- and post-synaptic processes following injury, and the potential effect of p75NTR on neurotrophin-mediated sprouting. In each study, I used dorsal rhizotomy as the injury model to assess axonal regeneration or intraspinal sprouting. A comprehensive description of this model is given in Section 1.2. of this chapter. The remainder of Chapter 1 provides a detailed discussion of the positive and negative influences on functional recovery following dorsal rhizotomy, the molecular relationships of p75NTR in the context of axonal growth, and the experimental objectives of the data chapters.  3  1.2 Dorsal rhizotomy: a model for the investigation of axonal regeneration and intraspinal sprouting Dorsal rhizotomy, or dorsal root injury, results in an interuption in the transmission of sensory information from the periphery (e.g. skin) to the spinal cord. The neurons responsible for the transmission of sensory information are located outside the spinal cord in dorsal root ganglia (DRG). The DRG contains a heterogeneous population of sensory neurons including large-diameter Aβ mechanoreceptors and proprioceptors, as well as small-diameter Aδ and C nociceptors (Fraher et al., 2002). These primary sensory neurons are pseudounipolar: they have one axon that bifurcates, sending one branch to the periphery and the other to the spinal cord. Axons emerging from the distal pole of the DRG form bundles, which merge with autonomic and motor axons to form the spinal nerves. Those that emerge from the proximal pole are purely sensory, form the dorsal roots and innervate the dorsal horn of the spinal cord. The typical termination of these axons within the dorsal horn is patterned such that small-diameter nociceptors innervate the superficial laminae (I and II), and the large-diameter axons innervate the deeper dorsal horn laminae (III, IV) (Fig. 1.1a, b) (reviewed in Bradbury et al., 2000). Injury to the axons in the dorsal root leads to the degeneration of these central-projecting axons and subsequent spinal deafferentation. In each study, dorsal rhizotomy was performed at the level of the brachial plexus. The brachial plexus is a network of sensory, motor and autonomic nerves that connect the lower cervical and higher thoracic spinal cord (C4-T2) with the forelimbs. Although I have used dorsal rhizotomy as a means to investigate the mechanisms underlying limits to regeneration and the extent of intraspinal sprouting, it may also be considered a  4  clinically relevant model of brachial plexus avulsions. These injuries, often caused by motorcycle accidents or complications during childbirth, can leave patients with loss of sensory and motor function to the forelimb (Samii et al., 2001; Flores, 2006). The rhizotomy model used in Chapters 2, 3 and 4, in which all seven dorsal roots of the brachial plexus were injured unilaterally, resulted in the complete disruption of sensory communication between the forelimb and spinal cord. Dorsal rhizotomy provides an ideal setting in which to study axonal regeneration and the sprouting of intact neural processes within the spinal cord. Following dorsal rhizotomy, the axons or dendrites that are normally innervated by DRG axons within the dorsal horn are spared from the initial insult and changes in their density can be easily assessed over time. Axons regenerating from the periphery towards the spinal cord can also be readily identified and distinguished from other populations using neural tracers applied to peripheral nerves (Ramer et al., 2000). In addition, desirable characteristics of this injury model include a high level of reproducibility, few surgical complications, and a straightforward assessment of sensory recovery given the persistence of motor and autonomic function.  1.2.1 Anatomical changes to sensory processes following dorsal rhizotomy Following dorsal rhizotomy, axonal degeneration of the central projections of DRG neurons occurs in both the dorsal root (PNS) and the spinal cord (CNS). The boundary between PNS and CNS tissue occurs within the dorsal root entry zone (DREZ), the cord-adjacent portion of the dorsal root, which contains both PNS and CNS tissue. The CNS component occurs as a conical projection of CNS tissue into the proximal  5  portion of the root, and the surface of this cone contains ‘transitional’ nodes, where axons go from being ensheathed by Schwann cells in the periphery to astrocytes and oligodendrocytes in the CNS at a single node of Ranvier (Fraher et al., 2002; see Fig. 1.1c). Under normal conditions, many of these axons successfully regenerate within the dorsal root but fail to re-enter the CNS component of the DREZ (Fig. 1.1d) (Bradbury et al., 2000; Fraher et al., 2002; Tessler, 2004). Spinal deafferentation and the chronic loss of somatic sensory function following this injury are attributed to the inability of these regenerating axons to penetrate the CNS. Axonal degeneration caused by dorsal rhizotomy is also accompanied by the spontaneous sprouting of specific uninjured neural populations within the deafferented spinal cord. Small, peptidergic sensory axons sprout in the superficial lamina of the dorsal horn following dorsal rhizotomy (Polistina et al., 1990; Belyantseva and Lewin, 1999; Wong et al., 2000; Darian-Smith, 2004). In addition supraspinal monoaminergic populations such as serotonergic and dopaminergic neurons, known to modulate sensory processing within the spinal cord, undergo sprouting of their axons in response to the loss of primary afferent innervation (Polistina et al., 1990; Wang et al., 1991a; Zhang et al., 1993; Belyantseva and Lewin, 1999; Darian-Smith, 2004; MacDermid et al., 2004). Responses of higher-order sensory neurons following deafferentation injuries, although far less characterized, have also been described. In particular spinocervical tract neurons in the dorsal horn, which are primarily involved in mechanosensation (Brown et al., 1987), demonstrate changes to their dendritic architecture such as increased dendritic diameter and branch number (Sedivec et al., 1986).  6  The anatomical changes of sensory processes following dorsal rhizotomy occur in response to a collection of tightly regulated events that take place within the degenerating axons and the environment where degeneration takes place. This process is referred to as Wallerian degeneration (Waller, 1850). The changes associated with Wallerian degeneration involve several different cell types that contribute to the degeneration of neural projections, and are highly dependent on the location and extent of the injury. Since the centrally-projecting axons that are injured following dorsal rhizotomy extend into the dorsal root and the spinal cord, these axons undergo Wallerian degeneration in both the PNS and CNS.  1.2.2 Wallerian degeneration in the spinal cord Neural trauma induces a time-dependent series of intra-axonal events that initiate the process of axonal degeneration. Immediately following injury, there is a significant drop in blood flow and a resultant reduction in oxygen, glucose and ATP levels that lasts for several hours (Walker et al., 1979; Hayashi et al., 1983). This decrease in ATP levels leads to ionic imbalances within the injured axon, including the accumulation of both sodium and calcium as well as a loss of potassium. Through a reversal of the Na+- Ca2+ exchanger, Ca2+ levels can accumulate as early as 45 minutes after injury, and reach a maximum within 8 hours (LoPachin and Lehning, 1997; Craner et al., 2003). The intraaxonal Ca2+ initiates the activation of a variety of proteases and phospholipases. For example, phospholipase C and A2 are involved with the breakdown of the axoplasm and axolemma (Schlaepfer and Bunge, 1973; DeCoster, 2003).  7  The activation of phospholipases acts as a chemoattractant for the infiltration of leukocytes to the lesion site (Lopez-Vales et al., 2008). Following injury to the spinal cord, and disruption of the blood brain barrier, infiltrating neutrophils arrive within the first few hours of injury and disappear by 3 days (Kigerl et al., 2006). The second wave of infiltrates consists of haematogenously-derived and resident macrophages, which arrive by the 3rd day post-injury and reach maximal accumulation by 10 days (Dusart and Schwab, 1994). The primary purpose of these cells is to clean up the cellular debris resulting from the disintegration of axonal processes and the degeneration of myelin sheaths. Following dorsal rhizotomy, axonal degeneration occurs in the dorsal grey matter as well as in the dorsal columns and dorsolateral fasciculus (Ling, 1979). Although the blood-brain barrier is not directly disturbed following dorsal rhizotomy, peripherally derived leukocytes are present in areas of degeneration within the first week (Ling, 1979; Rutkowski et al., 2002). However, the accumulation of blood-borne macrophages is significantly delayed (until the 3rd week post-injury) following dorsal rhizotomy (George and Griffin, 1994), and as such thier contribution to Wallerian degeneration is likely minimal. CNS glial cells are primarily responsible for Wallerian degeneration following dorsal rhizotomy. In response to injury in the CNS, microglia proliferate within the first few days post-injury and begin to express markers for phagocytosis (Lawson et al., 1994; Liu et al., 1998). Dorsal root lesion elicits these microglial responses predominantly in the dorsal horn and dorsal column nuclei and, to a lesser extent, in the central component of the DREZ and dorsal funiculus (Persson et al., 1995; Liu et al., 1995; Liu et al., 2000).  8  Astrocytes, which also have phagocytic properties, significantly proliferate within the spinal cord in response to dorsal rhizotomy (Liu et al., 2000; Magnus et al., 2002), suggesting that these cells may also contribute to the removal of axonal and myelin debris. Despite the contribution of these glial cells in the CNS, they display a subdued phagocytic phenotype in comparison to the macrophage response in the PNS (Avellino et al., 1995; Zee-Brann et al., 1998).  1.2.3 Wallerian degeneration in the dorsal root Following dorsal rhizotomy, Wallerian degeneration occurs at a much faster rate in the lesioned dorsal root than in the spinal cord. In several studies using the dorsal rhizotomy model, macrophage infiltration into the dorsal root was observed by 3 days post-injury and continued to accumulate for 2 weeks (George and Griffin, 1994; Avellino et al., 1995). In contrast, the presence of macrophages in the spinal cord was negligible by comparison at all time points, and by 14 days post-injury macrophage accumulation in the dorsal root was nearly 100-fold greater than in the spinal cord (Avellino et al., 1995). This difference was correlated to the complete removal of axonal and myelin debris clearance observed in the dorsal root by 30 days post-rhizotomy, and the continued presence of degenerated tissue in the spinal cord by 90 days following rhizotomy (George and Griffin, 1994). The presence of Schwann cells, another phagocytic cell-type, also contributes to Wallerian degeneration in the PNS. Schwann cells are the myelin-forming glial cells of the PNS, and undergo degeneration of their processes following peripheral axonal injury. However, before the appearance of macrophages, these cells de-differentiate and undergo  9  autophagy of degenerating myelin processes (Salzer and Bunge, 1980; Stoll et al., 1989). In addition, Schwann cells transiently proliferate within the remnant basal lamina tubes at the proximal tip of the lesioned axon and create cellular columns called bands of Büngner (Büngner, 1891). These Schwann cell columns provide permissive scaffolding and may help direct regenerating axons to their appropriate targets. However, before axonal regeneration can occur during these periods of Wallerian degeneration, the injured neurons must convert from a signal-transmitting mode into one for growth. Within the neuron, this involves a change in the collective expression of genes that correlates with growth periods. These genes are referred to as regenerationassociated genes (RAGs) (reviewed in Plunet et al., 2002). The upregulation of RAGs differs between the PNS and CNS. For example, growth-associated protein (GAP-43), a cytoplasmic protein correlated with neurite outgrowth and axonal regeneration, is significantly increased in DRG neurons following peripheral axotomy, but only transiently expressed in injured CNS neurons, and even then, at a lower level than in the PNS (Chong et al., 1994; Fernandes et al., 1999). These intrinsic differences may account, at least in part, for the differences in regenerative success in the CNS versus the PNS; however, the properties of the environment play a significant role. A classic set of experiments by Aguayo and colleagues (1981), showed that injured CNS axons in the spinal cord are indeed capable of regenerating when presented with a peripheral nerve graft. However, when the conditions were switched, axotomized PNS neurons, with a normally higher regenerative capacity, did not grow into a CNS graft. These findings exemplify the inhibitory environment of the CNS and demonstrate that the changes to the cellular milieu during  10  Wallerian degeneration are a major determining factor to the success of axonal growth following injury.  1.3 Inhibition of axonal regeneration and sprouting following injury Following SCI there is an accumulation of migrating and proliferating cells at the lesion site including microglia, mengingeal cells, projenitor cells and astrocytes. In addition, the myelinating processes of oligodendrocytes degenerate within the lesion site immediately following CNS injury. The combination of these cell types leads to the formation of a cellular meshwork known as the glial scar (reviewed in Fawcett and Asher, 1999). The presence of the glial scar, which is absent in the PNS, largely underlies the inhibitory nature of the CNS environment and the lack of axonal regeneration and sprouting observed following SCI. Many of the cell types that compose the glial scar restrict axonal growth at the lesion site. In the following sections, I have focused my discussion on two relatively prominent cell types in the glial scar - astrocytes and oligodendrocytes. Under normal conditions, these glial cells do not restrict axonal growth; however, following injury they express a multitude of inhibitory molecules that significantly prevent axonal regeneration and limit neuroplasticity in the CNS (Davies et al., 1999; Bradbury et al., 2002; McDermid et al., 2004; McPhail et al., 2005).  1.3.1 Astrocytic-derived inhibition Astrocytes are one of the chief constituents of the glial scar. Following injury astrocytes become ‘reactive’, which is characterized by hypertrophy, process extension,  11  upregulation of intermediate filaments, and proliferation (reviewed in Renault-Mihara et al., 2008). At the boundary of the glial scar, reactive astrocytes delineate the edge of the lesion site and confine it by one week post-injury (Faulkner et al., 2004). Although this boundary of reactive astrocytes protects the surrounding tissue from secondary damage, it also creates a biochemical barrier for regenerating axons. Reactive astrocytes express a number of extracellular matrix molecules, including chondroitin sulfate proteoglycans (CSPGs), which contribute to this inhibitory barrier (reviewed in Fawcett and Asher, 1999). CSPGs come in several varieties that all possess a chondroitin sulfate core and glycosaminoglycan side chains. Although the mechanism of CSPG-signaling is largely unknown, these side chains have been shown to directly inhibit neurite outgrowth in vitro (Borisoff et al., 2003; Monnier et al., 2003). Furthermore, CSPGs are upregulated by astrocytes following SCI (Lemons et al., 1999) and significantly impede both axonal regeneration and sprouting at the glial scar (Davies et al., 1999; Bradbury et al., 2002; Barritt et al., 2006).  1.3.2 Oligodendrocyte-derived inhibition In the CNS, degenerated myelin is present almost immediately and remains in the lesion site up to several months post-injury (George and Griffin, 1994). These degenerating oligodendrocyte processes express surface proteins that are involved with the inhibition of axonal growth (Domeniconi and Filbin, 2005). One of these proteins is a member of the Reticulon family – Nogo (Chen et al., 2000; GrandPre et al., 2000). Nogo exists in three isoforms NogoA, NogoB and NogoC, which are expressed in all eukaryotes (Oertle et al., 2003). Distinct from other reticulon proteins and other Nogo  12  isoforms, NogoA contains a long amino terminus that appears only in higher vertebrates such as birds or mammals, and correlates to the regenerative failure in these species (Oertle et al., 2003). In the adult mammal, expression of NogoA is localized to oligodendrocytes, and has not been detected in the PNS (Chen et al., 2000; GrandPre et al., 2000; Prinjha et al., 2000). Antagonism of NogoA significantly increases neurite outgrowth of several CNS neural populations such as retinal ganglion neurons (Oertle et al., 2003) and cerebellar neurons (Simonen et al., 2003). In addition, neurite extension of DRG neurons was greater on NogoA-/- or NogoA/B-/- myelin extracts in comparison to growth on wild-type myelin (Kim et al., 2003). Myelin-associated glycoprotein (MAG) is another inhibitory molecule that has been identified in the CNS (Everly et al., 1973). MAG is sialic acid-binding protein and a member of the immunoglobulin superfamily that is expressed on both CNS and PNS myelin sheaths (Willison et al., 1987). It exists in two alternatively spliced isoforms, large and small, dictated by the length of their cytoplasmic domains (Salzer et al., 1987). In vitro studies show that neurite extension of adult DRGs, postnatal cerebellar neurons and motor neuron-like cells (NG108-15 cells) is significantly diminished when plated on monolayers of MAG-expressing cells (McKerracher et al., 1994; Mukhopadhyay et al., 1994). Similarly, adult DRG and neonatal cerebellar axonal growth is enhanced when cultured on MAG-/- CNS and PNS myelin sheaths (Shen et al., 1998) or on fractionated myelin extract derived from MAG-/- mice (Li et al., 1996). An additional inhibitory myelin-associated protein identified is the glycosylphosphatidylinositol (GPI)-linked oligodendrocyte-myelin glyocoprotein (OMgp). Initially described as a myelin-specific protein, OMgp expression was later  13  discovered in diverse populations of neurons, in particular large projection neurons (motoneurons, dorsal spinal neurons, Purkinje cells, and pyramidal cells) (Habib et al., 1998). This 110- to 120-kDa GPI-linked protein contains a cysteine-rich amino terminal domain, a series of leucine-rich repeats, a serine/theonine-rich region, and a potential carboxyl terminal cleavage site (Mikol et al., 1990). Similarly to the other myelin inhibitory proteins, OMgp was found to inhibit neurite outgrowth of cerebellar neurons, hippocampal neurons, DRG neurons, retinal ganglion neurons, and cell lines NG108 and PC12 (Kottis et al., 2002; Wang et al., 2002b). All three myelin-derived inhibitory proteins bind to the NogoA receptor (NgR) (Gonzenbach and Schwab, 2008). NgR is a glycosylphosphatidylinositol (GPI)-linked receptor that is expressed on several populations of neurons within the CNS and PNS (Barrette et al., 2007). NEP1-40, a competitive antagonist of NgR, enhances neurite outgrowth of embryonic chick DRG neurons plated on CNS myelin (GrandPre et al., 2002). Similarly, NgREcto, a soluble, truncated form of NgR, increases outgrowth of embryonic DRG neurons over myelin (Fournier et al., 2002) and rescues neurite outgrowth of adult DRG neurons over MAG (Liu et al., 2002). However, the genetic deletion of NgR has had equivocal results. In vitro studies show that deletion of functional NgR from DRG neurons prevented growth cone collapse in vitro in response to NogoA (Kim et al., 2004); yet in a second study, deletion of NgR did not enable DRG neurite outgrowth on myelin inhibitory substrates (Zheng et al., 2005). Likewise, axonal regeneration in vivo is not improved within the injured corticospinal tract of NgR-/- mice (Zheng et al., 2005) but is improved within raphespinal and rubrospinal tracts of NgR-/- mice following SCI (Kim et al., 2004).  14  Two other isoforms of NgR (or NgR1) were recently discovered (Barton et al., 2003; Lauren et al., 2003; Pignot et al., 2003). Now called NgR2 and NgR3, these share 55% homology with NgR1 and encode the leucine-rich domain, the cysteine-rich carboxyl terminal, and the GPI-linkage site. Whereas NgR3 does not interact with any of the known myelin-associated inhibitory proteins, NgR2 binds to MAG with high affinity and is sufficient to confer MAG inhibition in neonatal rat DRG neurons (Venkatesh et al., 2005). MAG is also known to interact with complex gangliosides on the surface of neurons (Yang et al., 1996; Collins et al., 1997; Vinson et al., 2001); of these gangliosides, GT1b and GD1a have been implicated in MAG inhibition (Vyas et al., 2002).  1.3.3 Inhibition of axonal regeneration and sprouting following dorsal rhizotomy Following dorsal rhizotomy, the first barrier regenerating sensory axons must face are the reactive astrocytes. Dorsal rhizotomy not only causes astrocytic proliferation (Liu et al., 2000), but also hypertrophy such that extension of astrocytic processes reach up to 700µm in the dorsal root (Nomura et al., 2002; Zhang et al., 2001). The astrocytic boundary at the DREZ is normally permissive to axonal growth; however, following rhizotomy axon growth is completely halted (McPhail et al., 2005). As previously discussed, reactive astrocytes upregulate the expression of CSPGs (Beggah et al., 2005) and create a strong chemical boundary to sensory axon regeneration. Removal of this chemical barrier through the digestion of the inhibitory CSPG side chains has been shown to induce functional regeneration of sensory axons beyond the DREZ (Cafferty et al., 2007).  15  The myelin-derived inhibitory proteins create a second barrier to sensory axonal regeneration. DRG neurons possess all of the known receptors that interact with the myelin-associated inhibitory proteins (Ahmed et al., 2006), expressed by oligodendrocytes and thier degenerating myelin processes within the dorsal horn. Previous studies have shown that DRG neurite outgrowth is inhibited by MAIPs in vitro (Domeniconi et al., 2002; Wang et al., 2002a; Kim et al., 2003). A recent report also demonstrated that sensory axonal regeneration across the DREZ is successful following the antagonism of NgR in vivo (Harvey et al., 2009), although studies from our lab oppose these findings (M.S. Ramer, J.F. Borisoff, L.M. Ramer, J.W. Kwiecien, V.E. MacDermid, unpublished observations). The contribution of myelin-associated inhibitory proteins to the inhibition of intraspinal sprouting following rhizotomy, however, has not been disputed. NgR is expressed on primary afferents as well as on descending monoaminergic populations involved with sensory modulation (Barrette et al., 2007). Sequestration of the myelinassociated proteins, from endongenous receptors, with a soluble form of NgR (sNgR) resulted in significant increases to the sprouting of smaller-diameter sensory afferents, serotonergic, dopaminergic, and noradrenergic fibres following dorsal rhizotomy (MacDermid et al., 2004). Because NgR1 is not expressed by spinally projecting monoaminergic axons (Hasegawa et al., 2005), it appears likely that the effects of sNgR are attributable to its ability to sequester MAG from NgR2.  16  1.4 Promotion of axonal regeneration and sprouting following axonal injury In the PNS, Schwann cells also express the inhibitory myelin-associated proteins MAG and Omgp, but significantly downregulate these proteins during de-differentiation (Apostolski et al., 1994; Paivalainen and Heape, 2007). This downregulation coincides with an upregulation of several RAGs that encode growth-promoting adhesion molecules and neurotrophic factors (Ide, 1996). One particular family of neurotrophic factors - the neurotrophins - has been extensively studied since the discovery of nerve growth factor (NGF), which has dramatic effects on the growth of both sensory and sympathetic neurons (Levi-Montalcini et al., 1954).  1.4.1 The Neurotrophin family Neurotrophins are pleiotropic growth factors that regulate neuronal survival, proliferation, differentiation, axonal growth, guidance and branching, synaptic plasticity, and neuronal repair (Kaplan and Miller, 2000; Zhou and Shine, 2003; Teng and Hempstead, 2004). The family members include NGF, as well as brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin 4/5 (NT-4/5). They are synthesized in precursor forms called pro-neurotrophins, which are either cleaved by convertases within the cytoplasm (Mowla et al., 2001), or by plasmin and matrix metalloproteinases extracellularly (Lee et al., 2001). The mature forms are noncovalently bound dimers that share 90% homology, including a cysteine-rich motif within the structural core of the dimer and 3 beta hairpin-loops that form the distinctive regions involved in determining receptor specificity (Wiesmann and de Vos, 2001).  17  Neurotrophin expression within the PNS and CNS is maintained at low levels in the adult nervous system. Following injury in the PNS, however, the expression of both NGF and BDNF have been shown to significantly increase within peripheral neurons (Sebert and Shooter, 1993), as well as in proliferating Schwann cells (Johnson et al., 1988; Funakoshi et al., 1993). Changes to neurotrophin levels are much less dramatic in the CNS, and occur mostly within the first week before returning to control levels (Li et al., 2007; Hajebrahimi et al., 2008). The relative lack of neurotrophin expression within the CNS following injury is another contributing factor to the failure of regeneration and limitation of plasticity. Increasing trophic support has been one of the strategies used to induce axonal regeneration and enhance axonal sprouting in the injured CNS (Lu and Tuszynski, 2008).  1.4.2 Tropomyosin-related kinase (Trk) receptors Each neurotrophin dimer interacts with a specific tropomyosin-related kinase (Trk) receptor. The Trk receptor subfamily has three members, TrkA, TrkB and TrkC (Martin-Zanca et al., 1986; Martin-Zanca et al., 1989; Klein et al., 1989; Klein et al., 1990; Middlemas et al., 1991; Lamballe et al., 1991; and reviewed in Huang and Reichardt, 2003). Each of these members is a cell-surface transmembrane receptor that possesses ligand-dependant tyrosine kinase activity. However, alternative splicing of TrkB (TrkBT1, TrkBT2, and TrkBT4) and TrkC (TrkCTK-) produces additional truncated isoforms of these receptors that lack the tyrosine kinase domain (Squinto et al., 1991; Valenzuela et al., 1993; Forooghian et al., 2001). Each full-length receptor and truncated isoform share a conserved extracellular domain that includes 2  18  immunoglobulin-like C2 domains that not only prevent the auto-activation of the fulllength receptors (Arevalo et al., 2000), but also determine neurotrophin binding specificity (Urfer et al., 1995; Urfer et al., 1998). This specificity has translated into high affinity interactions of NGF with TrkA, BDNF and NT-4/5 with TrkB isoforms, and NT3 with TrkC isoforms (Chao, 2003). Thus, the cellular sensitivity and responsiveness to neurotrophins is dictated by the expression of their respective Trk receptors. Trk receptors are differentially expressed on neurons and glial cells throughout the CNS and PNS. In the periphery, expression of Trk receptors is diverse among the different neural populations. For example, motoneurons express TrkC and TrkB (Copray and Kernell, 2000); sympathetic neurons only express TrkA (Majdan et al., 2001); and sensory neurons express all three in varying degrees (Mu et al., 1993). In the adult, expression of Trk receptors on neurons is relatively low, and following injury, it has been shown to remain low or even decrease (Sebert and Shooter, 1993; Krekoski et al., 1996). However, in contrast to the neurons, Schwann cells significantly upregulate both TrkC and TrkB following axotomy (Funakoshi et al., 1993). In the CNS, intraspinal neurons as well as those projecting to the spinal cord primarily express TrkB and TrkC (Frisen et al., 1992; Muragaki et al., 1995; Giehl and Tetzlaff, 1996; King et al., 1999). Following SCI, Trk receptor expression within the lesion site in both neurons and glial cells has been shown to decrease and remain at low levels (Liebl et al., 2001; Widenfalk et al., 2001; Hajebrahimi et al., 2008). The one exception to this is the truncated form of TrkB, TrkB-T1. Unlike the other isoforms, TrkBT1 is significantly upregulated at the injury site by astrocytes and ependymal cells (Funakoshi et al., 1993; King et al., 2000; Liebl et al., 2001; Widenfalk et al., 2001).  19  1.4.3 Promotion axonal regeneration and sprouting following dorsal rhizotomy Successful regeneration of sensory neurons beyond the astrocytic boundary at the DREZ has been achieved with the application of exogenous neurotrophins. Trk receptors are differentially expressed on sensory neurons, such that smaller-diameter nociceptors primarily express TrkA, and larger-diameter mechanoreceptors and proprioceptors primarily express TrkC and to a lesser extent, TrkB (Mu et al., 1993; Wright, 1995). Given these expression patterns it is not surprising that both NGF and NT-3 have been shown to induce the functional regeneration of small and large primary afferent axons, respectively (Zhang et al., 1995; Ramer et al., 2000; Romero et al., 2001). These findings demonstrate that increased availability of neurotrophins is sufficient to promote sensory axonal regeneration and overcome the inhibitory environment of the CNS. It is important to note, however, that this neurotrophic effect is time-dependent. If neurotrophin treatment following rhizotomy is delayed by one week, axons regenerate beyond the astrocytic barrier, but stall within the degenerating white matter (Ramer et al., 2002). Neurotrophin treatment has also been shown to enhance axonal sprouting in the spinal cord following SCI or dorsal rhizotomy (Tessler, 2004). For example, NGF promotes the collateral sprouting of undamaged nociceptive fibres within the dorsal horn (Krenz et al., 1999; Krenz and Weaver, 2000; Romero et al., 2001; Tang et al., 2004). Likewise, NT-3 enhances trauma-induced sprouting of Aβ afferents (Bradbury et al., 1999), corticospinal projections (Schnell and Schwab, 1993; Schnell et al., 1994; Jeffery and Fitzgerald, 2001; Zhou and Shine, 2003), and raphespinal axons (Tobias et al., 2003). Additionally, exogenous BDNF increases the sprouting of corticospinal axons (Vavarek et al., 2006), and dopaminergic or noradrenergic processes (M.S.R., unpublished  20  observations). In these studies, the influence of neurotrophins coincides with their distribution of corresponding Trk receptors.  1.5 Growth-promoting and growth-suppressing factors converge on p75NTR P75NTR is a member of the tumour necrosis factor receptor (TNFR) family and, like other members, possesses an intracellular motif of six α helices (death domain), and cysteine-rich modules in its extracellular portion (Liepinsh et al., 1997). However, p75NTR has some distinguishing features that are not consistent with other TNFRs, including the propensity to dimerize rather than trimerize, and bind to structurally unrelated TNFR ligands, such as neurotrophins (Roux and Barker, 2002).  1.5.1 P75NTR is a pan neurotrophin receptor P75NTR binds to all neurotrophins with similar affinity, and is described as the low-affinity neurotrophin receptor, due to the high dissociation rate of this interaction (Rodriguez-Tebar et al., 1990; Squinto et al., 1991; Rodriguez-Tebar et al., 1992). The interaction between p75NTR and neurotrophins has been recently reported to have a symmetrical 2:2 stoichiometry in both rodents and humans (Aurikko et al., 2005; Gong et al., 2008). Neurotrophins bind p75NTR at sites located in the four cysteine loop repeats and the C-terminus of this receptor (Gong et al., 2008). P75NTR is not only involved with the signaling of neurotrophins, but also their precursors. The pro-neurotrophin form of both NGF and BDNF are secreted along with the mature forms (Lee et al., 2001). In conjunction with another transmembrane protein, Sortilin, pro-neurotrophins bind with high-affinity to p75NTR, and induce neuronal,  21  Schwann cell, and oligodendrocyte apoptosis following injury (Ferri and Bisby, 1999; Beattie et al., 2002; Harrington et al., 2004; Nykjaer et al., 2004; Teng and Hempstead, 2004). This is at odds with the pro-survival activation of the NF-κB pathway via p75NTR that occurs during development (Hamanoue et al., 1999; Gentry et al., 2000). Significant neural loss in mice with mutations of full-length p75NTR confirms the importance of p75NTR to the viability of neurons (Lee et al., 1992; Lee et al., 1994; Kawaja, 1998). These effects are seemingly contradictory, but may depend on Trk receptor coexpression: apoptotis occurs in glial cells that express p75NTR but no detectable TrkA and in neurons when co-localization of p75NTR with Trk receptors is minimal or absent. Similar to neurotrophins and Trk receptors, the expression of p75NTR is distributed in both neural and glial cell types throughout the CNS and PNS, and maintained at low levels in the adult. Schwann cells, but not neurons, substantially increase their expression of p75NTR following injury in the PNS (Johnson et al., 1988; Syroid et al., 2000). Although Schwann cell expression of p75NTR plays a role in cellular death, its influence on neurotrophin-mediated axonal growth is unknown. One prevailing model proposed that p75NTR expressed by Schwann cells acts to ‘present’ neurotrophins to Trk receptors on growing axons and, in this way, improve regenerative success (Johnson et al., 1988). Currently, there is no evidence to support this model, and recent structural studies of ligand - receptor interactions suggest, that it is more likely that non-neuronal p75NTR competes with Trk receptors for available neurotrophins (Gong et al., 2008). Although the full-length p75NTR is the dominant isoform in the nervous system, a previous report identifies an alternative splice product resulting in a weakly-expressed short isoform that lacks the cystiene loops involved in neurotrophin binding (von Schack  22  et al, 2001). A transgenic mouse with a targeted deletion to the Exon III domain (Lee et al., 1992), a domain that is responsible for encoding the extracellular cysteine loops, was initially described as expressing the short isoform. This prompted the creation of another p75NTR transgenic mouse with a deletion of Exon IV domain (transmembrane domain), which notionally resulted in a complete deletion of all p75NTR isoforms (von Shack et al., 2001). However, later reports failed to detect any isoforms in p75NTR wild-type animals or the Exon III transgenic mice, but did uncover an alternative p75NTR gene product that encodes the entire intracellular region of the receptor in the Exon IV transgenic (Paul et al., 2004; Dhanoa et al., 2006). Overexpression of this receptor fragment led to the activation of pro-apoptotic signalling cascades, including Jun Kinase activation and procaspase 3 cleavage (Paul et al., 2004). Given the multiple roles of p75NTR in neurotrophin signalling, including apoptosis (as described above), the interpretation of findings using the Exon IV transgenic may be complicated by the gain-of-function mutation. For this reason, the use of mice containing mutations of the Exon III domain of the p75NTR receptor are routinely used in current studies examining the role of p75NTR.  1.5.2 P75NTR association with second messengers The expression of p75NTR can influence axonal growth through the small guanine triphosphate (GTP)-ase Rho family. One particularly important member of the GTPase family that is involved in axonal growth is RhoA. RhoA activates Rho-associated kinase, an effector kinase that, in turn, activates LIM-kinase (Maekawa et al., 1999; Redowicz, 1999). LIM-kinase phosphorylates cofilin to reduce actin turnover, thereby inhibiting neurite growth (Ghosh et al., 2004; Ng and Luo, 2004). Yamashita and colleagues (2003)  23  found that p75NTR is involved with the activation of Rho kinase through its interaction with a Rho-GDP dissociation inhibitor (Rho-GDI) at the fifth α helix on its intracellular domain. Unbound Rho-GDI associates with Rho-GDP and prevents it from being converted to its active form, Rho-GTP. However, the interaction of Rho-GDI with p75NTR prevents the interaction of Rho-GDI with Rho-GDP, and results in activation of Rho kinase. Cell lines transfected with p75NTR have enhanced Rho-GTP expression and reduced neurite outgrowth (Yamashita et al., 1999; Madura et al., 2007). Additionally, neurite outgrowth of cultured sensory neurons from mice possessing a targeted deletion of p75NTR (Exon III knockout; p75-/-) is enhanced, and levels of activated Rho in these mice is lower than in wild-type mice both before (Gehler et al., 2004) and after SCI (Dubreuil et al., 2003). Recently, Pincheira and colleagues (2008; 2009) discovered another factor involved with neuronal growth that associates with the intracellular death domain of p75NTR. Sall2, a neuronal transcription factor, is constitutively bound to p75NTR and consequently tethered to the membrane. Once Sall2 dissociates from p75NTR, it translocates to the nucleus to potentiate neuronal growth. NGF catalyzes the dissociation of Sall2 from p75NTR and Trk-mediated activation of Erk1/2 is required for its nuclear transport. Inhibition of Sall2 signaling with siRNA caused a 50% decrease in neurite outgrowth of primary neuron cultures in the presence of NGF. From these studies, the emerging role of p75NTR in neurite outgrowth is that of a ‘gate keeper’. With Sall2 and Rho-GDI bound to the intracellular domain, p75NTR prevents neurite outgrowth; but if p75NTR is bound to NGF, Sall2 dissociates and stimulates neurite outgrowth (Pincheira et al., 2009), and the activation of Rho-GDP is inhibited (Yamashita et al., 1999; Nakamura  24  et al., 2002).  1.5.3 P75NTR interaction with Trk receptors Co-expression of p75NTR and Trk receptors occurs in various neuron populations, and influences neurotrophin interactions with these neurons. Co-localization of p75NTR with Trk receptors alters their affinity and ligand specificity through interactions of the receptors’ intracellular domains (Huber and Chao, 1995; Bibel et al., 1999; Gong et al., 2008). In the presence of p75NTR, TrkA binds primarily to NGF (Mahadeo et al., 1994; Esposito et al., 2001), and TrkC specificity is reduced, binding to NT-3, as well as BDNF and NT-4/5 (Vesa et al., 2000). However, in the absence of p75NTR, the binding affinity of NGF for TrkA is reduced (Mahadeo et al., 1994; Esposito et al., 2001), and NT-3 binds all Trk receptors (Vesa et al., 2000; Mischel et al., 2001). In addition, p75NTR attenuates neurotrophin-dependent ubiquitination of Trk receptors, which results in delayed internalization and degradation of the receptors (Makkerh et al., 2005). These results suggest that the expression of p75NTR may also influence neuronal growth by manipulating neurotrophin signaling via Trk receptors.  1.5.4 P75NTR is a co-receptor for myelin-associated inhibitory proteins The transduction of inhibitory signaling via MAIPs occurs through a ternary receptor complex that consists of NgR1 as well as Lingo-1 (Mi et al., 2004) and p75NTR (Wang et al., 2002a; Wang et al., 2002b). Stimulation of this receptor complex by myelin-associated inhibitory proteins leads to the enhanced association of p75NTR with Rho-GDI, promoting the activation of Rho-GTP and inhibiting DRG neuron outgrowth in  25  vitro (Wang et al., 2002a; Yamashita et al., 2002). In line with this, in vitro studies have shown significantly enhanced neurite outgrowth with siRNA targeting p75NTR and NgR1 NTR  (Ahmed et al., 2005). It is unknown whether NgR2 interacts with p75  in the same way  as NgR1, but the structural homology between NgR1 and NgR2 and the fact that both are GPI-linked to the cell membrane (i.e. they lack intracellular signaling components) suggests that it does. NgR-independent inhibition via myelin-associated inhibitory proteins is also dependent on p75NTR. In addition to the NgR complex, MAG binds to the glycoprotein ganglioside GT1b, and inhibits both neurite outgrowth and axonal regeneration (Vinson et al., 2001; Vyas et al., 2002). The co-receptor for ganglioside GT1b in neurons is p75NTR (Yamashita et al., 2002). The inhibitory signaling of MAG through either the NgR-Lingo1-p75NTR or GT1b-p75NTR receptor complexes is dependant on the neural population. For example, sensory neurons undergo MAG-induced neurite outgrowth inhibition primarily via the NgR-Lingo-1-p75NTR receptor complex, whereas cerebellar granule neurons are exclusively inhibited via GT1b-p75NTR (Mehta et al., 2007).  1.5.5 Potential significance of p75NTR expression following dorsal rhizotomy P75NTR is expressed in 100% of TrkA-positive DRG neurons and 50% of TrkCpositive DRG neurons (Wright and Snider, 1995). Likewise, p75NTR is expressed in large percentage of bulbospinal populations (Barrette et al., 2007), and therefore, poised to act directly on axonal regeneration and sprouting following dorsal rhizotomy. In addition, P75NTR is significantly upregulated in Schwann cells (Syroid et al., 2002) and oligodendrocytes (Chu et al., 2007) in response to injury. Given the extensive expression  26  of p75NTR in both neurons and glial cells following dorsal rhizotomy, p75NTR-mediated affects to axonal regeneration and sprouting may include: i) prevention of axonal growth by association of Rho-GDI and/or Sall2; ii) sequestration of neurotrophins by glial cells; iii) alteration of Trk receptor affinities and specificities for neurotrophins; iv) induction of secondary damage due to the apoptosis of glial cells; v) or interaction with myelinassociated protein receptors, NgR or GT1b (illustrated in Fig. 1.2). Since p75NTR appears to be a point of convergence for these possible effectors, the aim of my dissertation was to examine the influence of p75NTR in axonal regeneration and intraspinal sprouting within the CNS following injury.  1.6 Experimental objectives and hypotheses As discussed above, p75NTR is involved in many signaling complexes, which produce a variety of cellular consequences. However, how these interactions relate to axonal regeneration and intraspinal sprouting following SCI has yet to be elucidated. To directly test the contribution of p75NTR on axonal regeneration, I compared the regeneration of DRG axons following rhizotomy in mice with a targeted deletion to the p75NTR receptor (p75-/-), to those in a wild-type background strain (p75+/+). Since neurotrophin signaling is required for axonal regeneration across the DREZ, and is influenced by p75NTR expression, I examined regeneration in these two genotypes in situations of high and low neurotrophin availability, by treating with either exogenous neurotrophins or sequestering Trk receptor fractions. I hypothesized that the absence of p75NTR expression would dis-inhibit neurotrophin-mediated axonal regeneration across the DREZ.  27  In addition, I also aimed to determine the relative contribution of neuronal versus glial expression of p75NTR in neurite outgrowth or axonal regeneration of DRG neurons. Neurite outgrowth of adult DRG neurons in vitro was examined in the presence of p75+/+ or p75-/- Schwann cells, and in the presence or absence of endogenous neurotrophins. In vivo, these neuronal and non-neuronal contributions were examined using p75+/+ or p75-/transplants. Specifically, adult DRGs from either mouse genotype were transplanted into a host rat DRG, and axonal regeneration of the transplanted neurons was assessed following dorsal rhizotomy in the host. In a second transplant experiment, axonal regeneration within a p75+/+ or p75-/- peripheral nerve graft of p75+/+ sensory neurons was assessed following SCI. Given the large changes to p75NTR expression in Schwann cells following injury, and the potential competition for endogenous neurotrophins by p75NTR, I hypothesized that the glial expression of p75NTR would inhibit neurotrophinmediated neurite outgrowth and axonal regeneration. In chapter 4, I examined the influence of p75NTR on the spontaneous intraspinal sprouting of neuronal populations known to express p75NTR, including small-diameter peptidergic afferents, as well as raphespinal and dopaminergic projections. I compared the sprouting of these populations in p75+/+ and p75-/- mice, again with and without the addition of exogenous neurotrophins. In line with my previous hypotheses, I predicted that intraspinal sprouting would be enhanced in the p75-/- mice and the addition of exogenous neurotrophins would potentiate the plasticity of these axons in the absence of p75NTR. Given the possible effects of p75NTR on axonal sprouting, I examined the potential involvement of p75NTR in post-synaptic plasticity following injury. Very little work has  28  focused on dendritic changes following SCI or dorsal rhizotomy, but like axons, they are neurotrophin responsive, and thus, may be influenced through similar mechanisms. In Chapter 5, I measured the intraspinal plasticity of dendritic processes following deafferentation in p75+/+ and p75-/- mice, again in the presence and absence exogenous neurotrophins and Trk-Fc proteins. Here, I hypothesized that dendritic sprouting could be promoted by the application of exogenous neurotrophins and antagonism of p75NTR.  29  Fig. 1.1. Sensory afferent innervation of the dorsal horn. (a) Large-diameter DRG (CTB positive, inset) axons (red) project into laminae III-IV of the dorsal horn (DH) from the dorsal root (DR), demarcated by laminin (green). (b) Small to medium-diameter (WGApositive, inset) axons (red) innervate the superficial laminae of the dorsal horn, through the astrocytic CNS boundary (green). (c) Organization of the dorsal root entry zone (DREZ). (d) Axon regeneration (arrows) stops at the DREZ (*) one week following dorsal rhizotomy. Scale bars: 100 µm.  30  31  Fig. 1.2. Sites of potential manipulation of myelin-signaling pathways for promoting axonal regeneration and plasticity. Myelin-derived inhibitors (MAG, OMgp, NogoA) bind to a growing number of receptors: MAG interacts with the gangliosides GT1b and GD1a as well as the NgR1 and NgR2 receptors. OMgp and NogoA interact with the NgR1 receptor, which requires Lingo-1 and p75 or TROY for signal transduction. The amino (intracellular) terminal of NogoA may also bind to an as yet unidentified receptor (indicated by “?”). Other coreceptors for NgR1 and/or NgR2 may also exist (indicated by “?”). In addition to its interaction with NgR, the p75 receptor binds neurotrophins (NTFs) directly and alters affinity-specificity properties of NTF–Trk interactions. On its own (i.e., in the absence of other receptors or ligands), p75 is involved in the conversion of RhoA-GDP (inactive) to RhoA-GTP (active). The NgR/Lingo-1/p75/TROY complex enhances RhoA activation, whereas NTF binding to p75 inhibits it. MAG/NgR2 complexes probably also enhance RhoA activation. Trk–NTF binding decreases RhoA activation through intracellular messengers such as protein kinase A and Grit. Activated RhoA results in Rho-associated kinase activation. LIM-kinase is directly phosphorylated by Rho-associated kinase to act on cofilin, thus restricting neurite outgrowth. Experimental and potential therapeutic targets include dysmyelinated mutant animals (1); molecules which interfere with myelin-derived inhibitory molecules and their receptors, such as sNgR1 (2); altering p75 function, as is the case for p75 knockout (exon III deletion) mice (3); promoting Trk-mediated effects with exogenous NTFs (4); and interfering with intracellular small GTPase-dependent signaling with molecules such as C3 or Y-27632 (5).  32  33  1.7 Bibliography Aguayo AJ, David S, Bray GM (1981) Influences of the glial environment on the elongation of axons after injury: transplantation studies in adult rodents. J Exp Biol 95:231-240. 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Zheng B, Atwal J, Ho C, Case L, He XL, Garcia KC, Steward O, Tessier-Lavigne M (2005) Genetic deletion of the Nogo receptor does not reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivo. Proc Natl Acad Sci U S A 102:1205-1210. Zhou L, Shine HD (2003) Neurotrophic factors expressed in both cortex and spinal cord induce axonal plasticity after spinal cord injury. J Neurosci Res 74:221-226.  46  CHAPTER TWO  Schwann cell p75NTR prevents spontaneous sensory re-innervation of the adult spinal cord2  2  A version of this chapter has been submitted for publication. Scott, A.L.M. and Ramer, M.S. Schwann cell p75NTR prevents spontaneous sensory re-innervation of the adult spinal cord. 47  2.1 Introduction Neurotrophins such as nerve growth factor (NGF) and neurotrophin-3 (NT-3) are potent inducers of neurite outgrowth when applied to adult sensory neurons in vitro (Gavazzi et al., 1999). However, neither NGF nor NT-3 are required for regeneration of sensory axons in vivo: after peripheral nerve injury, regeneration occurs normally in the presence NGF and NT-3 function-blocking antibodies (Diamond et al., 1987; Diamond et al., 1992; Wright and Snider 1995). Similarly, peripherally-injured motor axons do not require endogenous neurotrophins for successful elongation (Streppel et al., 2002). Thus, while neurotrophins are clearly sufficient for axonal regeneration, they are not necessary in the peripheral nervous system (PNS). More definitive roles for endogenous neurotrophins during peripheral nerve regeneration in the adult include apoptosis of supernumary Schwann cells (SoiluHanninen et al., 1999; Syroid et al., 2000), Schwann cell migration (Yamauchi et al., 2004) and myelination (Chan et al., 2001; Cosgaya et al., 2002). These are mediated by Schwann cell-derived neurotrophins (Funakoshi et al., 1993; Raivich and Kreutzberg 1987), and the p75 pan-neurotrophin receptor (p75NTR), which is robustly-upregulated by reactive Schwann cells after peripheral nerve injury (Taniuchi et al., 1986). Unlike the expression of p75NTR by Schwann cells, the expression of neurotrophin receptors by sensory neurons (including p75NTR, NGF-binding TrkA and NT-3-binding TrkC) remains relatively unaffected (Ernfors et al., 1993), or is down-regulated (Zhou et al., 1996; Krekoski et al., 1996) following nerve injury. Therefore, increased Schwann cell p75NTR might be expected to reduce the availability of endogenous neurotrophins for axonal Trk signaling. This notion is supported by enhanced regeneration of injured  48  peripheral motor axons in mice lacking the neurotrophin-binding domain of p75NTR (Ferri et al., 1998; Boyd and Gordon 2001). Therefore, we hypothesized that regeneration of sensory axons would be enhanced in the absence of p75NTR. Here we test this hypothesis using dorsal rhizotomy, after which axons regenerate successfully within the Schwann cell-containing peripheral compartment of the dorsal root, but fail to cross the PNS:CNS interface (Ramon y Cajal 1991). Treatment of rhizotomized animals with exogenous NGF or NT-3, but not brainderived neurotrophic factor, prompts functional sensory regeneration into the spinal cord (Ramer et al., 2000). We show that mouse sensory axons, under the influence of endogenous NGF and NT-3, regenerate into the spinal cord and restore function in the absence of the neurotrophin-binding domain of p75NTR. In sensory neuron/Schwann cell co-culture experiments, as well as in complementary tissue-grafting experiments in vivo, we further show that it is the absence of p75NTR from Schwann cells, not from neurons, which effects regeneration.  2.2 Materials and methods Mice. Mice that express truncated p75NTR (p75-/-, exon III deletion, 8-10 weeks old) were compared to C57Bl/6 wild-type littermates (p75+/+). The mice were offspring of commercially available p75+/+ and p75-/- breeders (Ngfrtm1Jae, version 1, Jackson Laboratory). Triplicate ear punches were used for genotyping. Reagents for DNA extraction and polymerase chain reaction (PCR) were obtained from the REDExtract-N-Amp Tissue PCR kit (Sigma-Aldrich, St. Louis, MO). Two neo generic primer sequences and two Ngfr wild-type primer sequences (oIMR0013, oIMR0014, oIMR0710, oIMR0711; JAX®  49  Mice) were produced by Integrated DNA technologies. PCR products were separated by electrophoresis on a 2% agarose gel and visualized with ethidium bromide under UV light. For DRG transplant and DRG/SC co-culture experiments, we used p75+/+ or p75-/littermates, which had been crossed with mice expressing eGFP in all cells driven by the actin promotor (C57BL/6-TgN, Jackson Laboratory). Dorsal rhizotomy. A left dorsal laminectomy exposed the dorsolateral cervical spinal cord, and the dura mater was opened with iris scissors. The C4-T2 dorsal roots were either crushed repeatedly with fine forceps (#5) (three times, 10 s each) or completely transected with iris scissors. Treatments included: fibrin glue (FG) alone (n=6/genotype), FG + TrkA-Fc (n=10 p75-/- mice), FG + TrkC-Fc (n=10 p75-/- mice), FG + NT-3 (n=4 mice of each genotype), or FG + NGF (n=4 mice of each genotype). FG was prepared as previously described (Scott et al., 2005). TrkC-Fc or TrkA-Fc (R&D Systems), was combined with FG, and applied to the spinal cord at final concentrations of 100µg/ml (TrkC-Fc: n=4; TrkA-Fc: n=5) or 1mg/ml (TrkC-Fc: n=6; TrkA-Fc: n=5). NT-3 and NGF (gifts of Rinat Neuroscience Inc.) were combined with FG for a final delivery concentration of 100 µg/ml. Two days prior to sacrifice, the median nerve was injected with 1% cholera toxin B fragment (CTB; List Biological Laboratories), 5% wheat germ agglutinin (WGA; Vector Laboratories), and 2% IB4 lectin (Vector) via a Hamilton syringe. Mice were killed 7 (vehicle and Trk-Fc treatments) or 28 days (untreated) postsurgery. All surgical procedures were performed according to the guidelines of the University of British Columbia Animal Care Committee and the Canadian Council on Animal Care. For the following surgical procedures, animals were anesthetized with an intraperitoneal injection of ketamine hydrochloride (80-100 mg/kg; Bimeda-MTC,  50  Cambridge, ON) and xylazine hydrochloride (5-10 mg/kg; Bayer Inc., Etobicoke, ON). Post-operatively, they were given an intramuscular injection of buprenorphine (0.08 mg/kg for mice, 0.02 mg/kg for rats; Animal Resource Center, Montreal, QC), and a subcutaneous injection of Lactated Ringer’s solution (1.5 ml for mice and 5 ml for rats). Animals were monitored daily. Behaviour. All behavioural experiments were carried out by an individual who was unaware of mouse genotype. In 10 p75+/+ and 11 p75-/- mice, we assessed response threshold to progressive punctate force (Dynamic Plantar Aesthesiometer, Ugo Basile, cut-off: 3g), response latency to a cooling stimulus (5ml acetone squirt, cut-off: 60s) of the ipsilateral forepaw before and four weeks following rhizotomy according to previously published procedures (Ramer et al., 2007). Because of the differences between uninjured p75+/+ and p75-/- mice (Bergmann et al., 1997), responses at 4 weeks were expressed as a difference score (ipsilateral minus contralateral). For the formalin test, uninjured (n=4/genotype) or rhizotomized (four weeks earlier) p75+/+ (n=9) or p75-/(n=7) mice were acclimatized for 1h in a clear ventilated box above a 45° mirror, then lightly anesthetized (~30s) with 3% isoflourane. Five µl of 2% formalin was injected into the ipsilateral palmar forepaw. The time the mice spent licking or biting the injected paw was recorded for 1 hour. Mice were killed two hours post-injection. DRG transplants. DRGs were incubated in 0.1% collagenase (Sigma-Aldrich) for 1 hour. Dissociated cells were centrifuged (1000 rpm) for 3 minutes, and then re-suspended in 2ml of DMEM (Invitrogen, Burlington, ON). The cell suspension (1ml/tube) was then  51  gently added to 2ml of 15% BSA, creating a gradient. The cell gradients were centrifuged (650rpm) for five minutes. The resulting pellet was re-suspended in DMEM. The dorsal roots (C6-C8) of 8 male Sprague Dawley rats (200-250g; n=4 per group) were exposed and crushed repeatedly with fine forceps (#5). The attached dorsal root ganglion (DRG) was injected with 2µl of Dubelco’s Modified Eagle Medium (DMEM, Invitrogen) containing dissociated DRG neurons (40,000 cells/ml) isolated from eGFP-expressing p75+/+ or p75-/- littermates. Injected rats received cyclosporin A (CsA, Novartis Pharmaceuticals, 10mg/kg/day, i.p.) for 2 days before and immediately after surgery. Rats then recieved CsA in their drinking water (150mg/L) until they were killed two weeks later. Dorsal column injury and peripheral nerve grafting. Nerve grafts were prepared from either p75+/+ or p75-/- mouse donors, which received a unilateral sciatic nerve ligation one week earlier. The pre-degenerated sciatic nerve was removed, bathed in ice-cold Hank’s balanced salt solution (Invitrogen) and cut into 1-2 mm pieces. A 1-2 mm segment of the thoracic dorsal columns were excised from p75+/+ mice. The peripheral nerve graft was implanted into the cavity and covered with fibrin glue. Host mice (n=9 with p75+/+ grafts; n=5 with p75-/- grafts) were treated with CsA (10mg/kg/day, i.p.) until they were killed 28 days later. Hosts’ sciatic nerves were injected with 1ml of 1% CTB via a Hamilton syringe three days prior to sacrifice. Immunohistochemistry. Animals were euthanized with chloral hydrate (100 mg/kg, i.p.) and perfused with 4% paraformaldehyde. Spinal cords were removed, post-fixed overnight, cryoprotected in 20% sucrose in 0.1 M phosphate buffer for 24 h at 4°C,  52  frozen and stored at -80 °C. Transverse sections (16µm for mouse tissue; 20µm for rat tissue) and longitudinal sections (20µm) were processed immunohistochemically. Immunohistochemical characterization of tissue sections included visualization of p75NTR (1:500, goat, Neuromics Inc., Edina, MN), CTB (1:2000, goat), IB4 (1:2000, goat), βIII tubulin (1:500, mouse), and Fos (1:5000, rabbit) (all from Cedarlane); WGA (1:100, goat), laminin-1 (1:1000, rabbit), and NF200 (1:500, mouse) (all from Sigma-Aldrich); neuron-specific nuclear protein (NeuN, 1:100, mouse monoclonal, Chemicon, Temecula, CA) and glial fibrillary acidic protein (GFAP, 1:1000, rabbit, Dako, Glostrup, Denmark). Sections were incubated in 10% normal donkey serum for 1 hour, then in primary antibodies overnight. Secondary antibodies (raised in donkey), conjugated to Alexa 488 (1:400; Invitrogen), aminomethylcoumarin amide (1:400) or Cy3 (1:200; both from Jackson Immunoresearch) were applied the following day for 2 hours. Cell culture. SCs were derived from p75+/+ or p75-/- sciatic nerves from chloral hydrate (Sigma-Aldrich) -killed mice. p75+/+ or p75-/- nerves were bathed in cold Hank’s Balanced Salt Solution (HBSS, Invitrogen, Burlington, ON), de-fasiculated and cut into 2-3mm segments, and dry-plated in 24-well culture plates (Sigma-Aldrich). Nerves were maintained in growth media: DMEM (Invitrogen), 10% fetal calf serum, 0.25 mg/ml fungoside, 1% penicillin-streptomycin (all from Invitrogen), 0.001% forskolin and bovine pituitary extract (both from Sigma-Aldrich), and 10ng/ml basic fibroblast growth factor (FGF-2) (recombinant human; Invitrogen). Media were replaced every 2-3 days for two weeks. Nerve segments were then removed from the wells and dissociated in 0.1% collagenase, 0.25% trypsin and 0.1% DNase (all from Sigma-Aldrich) for 3-4 hours in 37°C. Cells were dissociated with a flame-polished Pasteur pipette, and centrifuged  53  (1000rpm) briefly. The pellet was re-suspended in 1ml of triturating solution: 1% bovine serum albuminin (BSA), 0.0002% DNase, and 0.05% trypsin inhibitor (all from SigmaAldrich), and 2ml DMEM. After several minutes, the cell suspension was transferred to another tube, centrifuged for 3 minutes (1000 rpm), and re-suspended in media containing 1% BSA. Cells (~20,000/well) were then plated on laminin/poly-L-lysinecoated 8-well Labtek slides (NuncTM, Rochester, NY) for 24 hours and maintained in growth media (replaced every 2-3 days) for 5-7 days. Monolayers were lifted from slides in 0.25% trypsin and centrifuged (1000rpm) briefly. Cells were re-suspended in triturating solution and DMEM, transferred to Petri dishes (60x15mm, Nunclon™, Mississauga, ON) coated with 50µg/ml of goat anti-mouse IgG Fab’ fragment (Jackson Immunoresearch, West Grove, PA), and incubated for 40-50 minutes at 37°C. This was followed by incubation with monoclonal anti-mouse CD90 (Thy 1.2, 1:40; Cedarlane, Hornby, ON) for 2 hours at 37°C, and phosphate buffer washes. Unbound cells were collected and centrifuged (1000 rpm) for 3 minutes, resuspended in 1% BSA growth media and plated on laminin/poly-L-lysine coated slides as described above. SC cultures were 94-96% pure and were maintained in growth media until confluent. Suspensions of dissociated eGFP-expressing p75+/+ or p75-/- DRG neurons (prepared as described previously) were plated (10,000 cells/ml, 200ml/well) on SC monolayers, and maintained at 37ºC for 18 hours. In some cultures, a mixture of TrkA-Fc and TrkC-Fc (500ng/ml) was added. Cells were then rinsed with cold PBS, fixed with 4% paraformaldehyde (VWR) for 20 minutes, washed and stored in PBS at 4ºC prior to immunocytochemistry.  54  Immunocytochemistry. Cultures were washed repeatedly with PBS before being incubated in 10% normal donkey serum for 1 hour. Primary antibodies against GFP (1:500; host mouse; Abcam, Cambridge, MA), TrkA (1:500; host goat; Neuromics, Edna, MN) and S100b (1:2000; host rabbit; Sigma-Aldrich) were applied for 2 hours. Secondary antibodies (listed above) were applied for 1 hour. Image analysis (in vivo experiments). Digital images were captured with an Axioplan 2 microscope (Zeiss, Jena, Germany) using a 20x or 40x objective, a digital camera (Q Imaging, Burnaby, BC) and Northern Eclipse software (Empix Imaging Inc., Mississauga, ON). SigmaScan Pro 4 software (SPSS, Chicago, IL) was used for all image analysis. We traced the peripheral PNS:CNS interface, defined by laminin-1 immunoreactivity. The trace was overlaid onto pseudocoloured images of CTB and NF200 or WGA and βIII tubulin immunoreactivity. Only structures that were positive for both tracer and axonal markers were measured. The penetration distance of each traced axon was measured from its point of entry at the dorsal root entry zone. All analysis was done at the microscope where the observer was able to follow axons through and between sections. In situations where the entry point of an axon could not be determined, the nearest laminin-1 positive region was assigned as the point of entry. Note that this method resulted in the absolute minimum possible CNS penetration distances. Regeneration of CTB-labeled dorsal column axons into peripheral grafts was also assessed. The graft:spinal cord boundary was defined by laminin-1 immunoreactivity, manually outlined and overlaid on matched images of CTB. To account for variations in tracer labeling between animals, a ‘Regeneration Index’ was calculated from the relative density of CTB-labeled axons that entered the first 200mm of the graft to the density of 55  all CTB-labeled fibres 200mm outside the graft. At least 3 sections per animal were analyzed. Functional activation of dorsal horn neurons was determined using Fos immunohistochemistry from formalin-injected mice. Dorsal horn cells that co-expressed Fos and NeuN were counted in six sections 64 µm apart for each of C6, C7, C8 and T1. Image analysis (in vitro experiments). To begin, all grayscale images were passed through an edge-detection filter and converted to a monochrome image by setting a constant threshold determined empirically. The percent of neurons bearing neurites was determined, and neurite outgrowth was analyzed according to a previously described method (Gardiner et al., 2007). Briefly, individual neurons were outlined and the coordinates of the cell body in the image were determined. A concentric circle overlay was applied to each neuron (centred on the soma), and the number of neurite cross-points was then recorded for a number of pre-set distances from the cell body ranging from 33 mm to 366 mm. From these data we calculated the total number of neurites and their branches for each neuron. Four separate experiments were analyzed for each group. Western Blots. Sciatic nerves of p75+/+ and p75-/- adult mice were removed three days following sciatic nerve ligation, and the tissue was homogenized in 0.01M Tris(hydroxymethyl)aminomethane with protease inhibitors (Invitrogen). Protein concentration was determined with BCA protein tritration, and 20µg of protein per lane was separated by 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a PVDF transfer membrane (Amersham Corp., Arlington Heights, IL). Each lane represented one animal for each genotype (p75+/+, n=3; p75-/-, n=3). The  56  membranes were then blocked in 5% Bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO), and incubated overnight with anti-NGF (1:5000, host goat), anti-NT-3 (1:2000, host goat; both from Neuromics), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:500, host mouse) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for an internal loading control. The membranes were then washed in 0.05M Tris, 0.15M NaCl, and 0.1% Tween 20, and incubated with affinity purified goat or mouse anti-horseradish peroxidase antibody (Jackson Immunoresearch Laboratories Inc., West Grove, PA) for 2 hours at room temperature. The membranes were washed again, treated for 5 minutes with enhanced chemiluminescence reagents (ECL; Amersham Corp.), exposed to X-ray film for 10 minutes, and developed with an automatic processor (Kodak M35A XOMAT). Protein density was calculated from digital images with Sigma Scan Pro 4 Software (Chicago, IL), and normalized to background density and to the loading control. Statistics. In rhizotomy experiments, dorsal root axon penetration distances were pooled from spinal sections C6-T1 from each mouse. Pair-wise statistical comparisons of regeneration between genotypes were carried out using a one-way ANOVA followed by the Holm-Sidak post hoc test. For functional assessment of regeneration, the average number of Fos-labeled nuclei per section was determined and comparisons were made between genotypes using Student’s t-test. Significant differences between groups in responses to progressive punctate force and cooling stimuli were assessed with Student’s t-test. Significant differences between groups in formalin responses were determined independently for early and late phases using Student’s t-test. In peripheral nerve grafting experiments, regeneration indices were compared using Student’s t-test. In dissociated DRG/confluent SC cultures, neurite number and percentages of neurite-bearing neurons  57  were compared using a one-way ANOVA with the Holm-Sidak pairwise comparison procedure. In all cases error bars represent the standard error of the mean.  2.3 Results 2.3.1 Injured sensory axons regenerate into the spinal cord in p75-/- mice p75NTR immunohistochemistry in wild-type mice illustrate the substantial upregulation of the receptor in Schwann cells (SCs) following dorsal root injury, and elimination of most p75NTR-positive axon terminals from the dorsal horn (Fig 2.1a). Seven and 28 days following multiple rhizotomy (C4-T2 dorsal root transection, which eliminates all sensory input to the spinal cord from the forelimb), p75+/+ NT-3-responsive mechanosensory and proprioceptive axons labeled by intraneural cholera toxin B (CTB) injection (McMahon et al., 1994; Wright and Snider1995; Tong et al., 1999) had rarely penetrated the central nervous system (CNS). In contrast, CTB-labeled p75-/- axons had grown significantly farther into the CNS by 7 days post-injury (representative image shown in Fig 2.3a), and by 28 days had not only extended through degenerating white matter (Fig 2.1b, e), but had also successfully entered the superficial dorsal horn (7d, n=6/group, p<0.001; 28d, n=4/group, p=0.013). There was no evidence of SC migration into the cord from the dorsal roots in either genotype. Note that ‘minimum distances’ – the straight-line distances between the nearest PNS tissue and labeled axon endings – are reported, and are thus highly conservative estimates of the extent of regeneration. Intraneural injection of wheat germ agglutinin (WGA), which is transported primarily along NGF-responsive small-caliber peptidergic nociceptors (Swett and Woolf 1985; McMahon et al., 1994; Wright and Snider, 1995), was also used to assess  58  regeneration. Rhizotomized, WGA-labeled p75+/+ axons rarely entered the spinal cord (Fig 2.1c, d). On the other hand, p75-/- WGA-labeled axons had successfully penetrated the cord by both 7 days (representative image shown in Fig 2.3a) and 28 days post-injury (7d, n=6/group, p=0.002; 28d, n=4/group, p<0.001), at which point axons had also entered grey matter. Interestingly, axons that had regenerated did so at a constant rate for four weeks following injury, supporting a role for axonal p75NTR in transducing inhibitory myelin signaling (Wang et al., 2002a). Transganglionic tracing of nonpeptidergic nociceptors with the IB4 plant lectin revealed that by 7 days post-rhizotomy very few Trk-negative axons had regenerated beyond the lesion site in either genotype, and by 28 days none had traversed the PNS:CNS interface (Fig 3.1d), indicating that the regeneration-suppressing effects of p75NTR are limited to Trk-expressing neurons. A possible mechanism underlying the regenerative response of Trk-expressing axons in p75-/- mice is elevated neurotrophin production compared to wild-type mice. We therefore analyzed NGF and NT-3 production in injured sciatic nerves from each genotype. We chose a three-day timepoint since axons in injured dorsal roots will have reached the dorsal root entry zone by three days post-rhizotomy. Western blot analysis of NGF and NT-3 revealed that they were present in comparable amounts in knockout and wild-type mice (Fig 2.1f). Therefore, elevated neurotrophin levels are not responsible for successful regeneration in p75-/- mice.  2.3.2 Regeneration is accompanied by functional recovery in p75-/- mice We next asked whether anatomical regeneration in p75-/- mice was accompanied by return of function. We assessed forepaw responsiveness to progressive punctate force  59  (using the Ugo Basile Dynamic Plantar Aesthesiometer) in intact mice and those, which received septuple dorsal rhizotomy four weeks earlier (Fig 2.2a). In intact animals, withdrawal thresholds were significantly greater in p75-/- mice (p=0.04, n=10 p75+/+ and 11 p75-/- mice), as has been reported previously (Bergmann et al., 1997). The majority of rhizotomized p75+/+ mice did not respond to progressive punctate force by the cut-off (2.3g). p75-/- mice responded at a significantly lower threshold (p=0.003). We also assessed response latency to a cooling stimulus (topical acetone, Fig 2.2b). While there was a slight pre-operative difference between genotypes (p=0.17), rhizotomized p75-/- mice responded with a significantly shorter latency than did p75+/+ mice (p<0.001), of which the majority failed to respond before the 60 second cut-off. In two other groups of mice, we stimulated nociceptive terminals in the palmar forepaw with a 5ml subcutaneous injection of 2% formalin. Formalin injection typically leads to a biphasic nocifensive response (5-10 minute first phase, 5-10 minute interphase and 60-90 minute second phase (Sawynok and Liu, 2004). The small volume and titre used here produced only a first phase, which was equivalent in uninjured p75+/+ and p75-/mice (Fig 2.2c). While spontaneous licking behaviour occurred in septuply rhizotomized mice (~10-50s per 10 minute interval), in wild-type mice the formalin-evoked response was eliminated (n=5). In contrast, in septuply rhizotomized p75-/- mice, there was a delayed and prolonged formalin response beginning between 10 and 20 minutes postinjection and lasting for the remainder of the testing period (n=7, p=0.002). The apparent amplification of the second phase of the formalin response in regenerated p75-/- mice is consistent with increased NGF availability: exogenous NGF amplifies the second phase in rats (Kerr et al., 1999).  60  To determine whether return of nociception in p75-/- mice was correlated with primary afferent activation of second-order spinal cord neurons, we examined expression of the transcription factor Fos following subcutaneous formalin injection (Hunt et al., 1987). Fos upregulation occurs as a result of high-intensity noxious primary afferent stimulation and is a reliable indicator of connectivity between nociceptive sensory axons and dorsal horn neurons. In intact formalin-injected p75+/+ and p75-/- mice, Fosimmunoreactive nuclei were present throughout the ipsilateral dorsal horn but most numerous in laminae I and II (n=4/genotype) (Fig 2.2d, e). Fos expression in formalininjected p75+/+ mice 28 days following rhizotomy was nearly abolished. Fos-positive nuclei in the p75-/- dorsal horn following formalin injection were significantly more abundant following the equivalent recovery time (n=5/group; p<0.001), although their distribution was mainly restricted to superficial laminae. These results indicate that regenerating axons re-connected with spinal targets.  2.3.3 Successful regeneration in p75-/- mice depends on endogenous neurotrophins In a direct test of the neurotrophin-dependence of successful regeneration in p75-/mice, we applied NGF-sequestering TrkA-Fc, or NT-3-sequestering TrkC-Fc to the spinal cord concomitant with dorsal rhizotomy. At low (100 µg/ml) or high (1 mg/ml) concentrations, TrkC-Fc attenuated regeneration of large-diameter axons in p75-/- mice (low dose, n=4, p=0.01; high dose, n=6, p<0.001) (Fig 2.3a, b), in agreement with endogenous TrkC receptor distribution on myelinated sensory neurons (McMahon et al., 1994; Wright and Snider,1995). TrkA is also expressed in a subset of myelinated axons  61  (Karchewski et al., 1999), and regeneration was accordingly reduced in mice treated with low or high doses of TrkA-Fc (low dose, n=5, p=0.001; high dose, n=5, p=0.003). WGA-labeled p75-/- axon regeneration was reduced with a high dose of TrkC-Fc (Fig 2.3A, B; n=6, p=0.006), most likely reflecting NT-3 sensitivity of some nociceptive axons (Karchewski et al., 1999; McMahon et al., 1994). Regeneration was also significantly reduced with both doses of TrkA-Fc (low dose, n=5, p=0.011; high dose, n=5, p<0.001), indicating that NGF is primarily responsible for regeneration of this population of axons in the absence of treatment (Karchewski et al., 1999; McMahon et al., 1994). These effects of NT-3- and NGF sequestration demonstrate that spontaneous regeneration in p75-/- mice is neurotrophin-dependent. Since the amount of NGF and NT-3 protein was equivalent between genotypes (Fig 2.1F), these data provide evidence for either increased sensitivity of p75-/- axons to neurotrophins, or increased effective neurotrophin availability to axons in the absence of p75NTR. The remaining experiments were aimed at distinguishing between these two possibilities.  2.3.4 Regeneration of primary afferent axons in p75-/- mice is only marginally augmented with the addition of neurotrophic factors Intrathecal delivery of neurotrophins results in improved regeneration into the CNS following rhizotomy (Ramer et al., 2000). To determine whether axons in p75-/mice were more sensitive to neurotrophins, we first asked whether exogenous neurotrophin-mediated regeneration could be further enhanced in the absence of p75NTR. Immediate treatment with NT-3 in p75+/+ mice resulted in significant regeneration of CTB-labelled axons across the PNS:CNS interface, but did not further increase axonal  62  regeneration in p75-/- mice (Fig 2.3c). In both p75+/+ mice and p75-/- mice, CTB-labelled axons did not significantly respond to NGF treatment. Since NT-3 did not augment regeneration in p75-/- mice, this implies either that the absence of p75NTR renders largediameter axons unresponsive to NT-3, or more plausibly (given the regeneration-reducing effect of TrkC-Fc treatment) that NT-3 is already present in saturating amounts. NT-3 treatment augmented regeneration of (presumably large-diameter) WGAlabelled axons in p75+/+ mice, but not in p75-/- mice (Fig 2.3c). NGF treatment substantially augmented WGA-labeled (presumably small-calibre) axonal growth in both p75+/+ and p75-/- mice, but to a lesser extent in the latter group. These results suggest that although endogenous NGF is responsible for regeneration of WGA-binding axons across the PNS:CNS interface in p75-/- mice (demonstrated by the effects of TrkA-Fc treatment), p75-/- axons are not more sensitive to NGF.  2.3.5 Absence of axonal p75NTR does not promote regeneration In p75-/- mice, the neurotrophin-binding domain of the receptor is absent from both SCs and neurons. This is potentially important to the interpretation of results in p75/-  mice since there is some suggestion of a ‘ligand-passing’ model in which axonal p75NTR  passes a neurotrophin dimer to a membrane-adjacent Trk monomer (Wehrman et al., 2007; He and Garcia, 2004). To determine the influence of axonal p75NTR on regeneration of sensory axons into the CNS, we transplanted dissociated, purified eGFPexpressing p75+/+ or p75-/- dorsal root ganglion (DRG) neurons into rhizotomized rat DRGs. We have used this technique previously to show that axons extending from transplanted neurons behave in the same way as endogenous axons in that they fail to  63  cross the dorsal root entry zone (DREZ) following rhizotomy, and that NT-3 can promote their regeneration into the spinal cord (McPhail et al., 2005). Previous experiments examining the phenotypic distribution of dissociated neurons in culture have shown that it closely reflects the distribution in vivo (Gavazzi et al., 1999), and in cultures of neurons prepared identically to dissociated neurons used for injection, we found no difference in size-distribution or the proportion of neurons expressing TrkA between genotypes (Fig 2.4a). Furthermore, the proportion of TrkA-positive neurons (~30%) roughly corresponded to that found in adult mouse DRGs in vivo (43%) (Molliver and Snider, 1997). Irrespective of genotype, by two weeks post-rhizotomy, eGFP-positive axons had arrived at, but had not penetrated, the astroglial scar at the DREZ (Fig 2.4a-c). Therefore, it is the absence of p75NTR from SCs, not axons, that allows for spontaneous regeneration in p75-/- mice. To corroborate the finding that the absence of p75NTR from SCs but not axons enhances regeneration, we transplanted pre-degenerated nerve grafts from p75-/- mice into a 1-2 mm-long cavity in the thoracic dorsal columns of p75+/+ mice. In this case, p75NTR is expressed on NT-3-responsive ascending mechanoreceptive and proprioceptive sensory axons, but is absent from the graft: in essence, this represents the converse experiment to that described above. As has been described in rats (Oudega et al., 1994), very few CTBlabeled sciatic nerve axons had penetrated p75+/+ grafts by four weeks post-injury (Fig 2.4d-f). In contrast, significantly more CTB-labeled axons had invaded p75-/- grafts (n=5 per genotype, p=0.04). It is noteworthy, though unsurprising, that regeneration into p75-/grafts was less robust than that across the PNS:CNS interface in p75-/- mice: in the spinal cord, injured axons are not directly exposed to a SC-containing environment and so do  64  not benefit to the same extent from SC-derived factors as those injured within the dorsal root.  2.3.6 p75-/- Schwann cells enhance neurotrophin-dependent regeneration in vitro The in vivo effects of p75NTR deletion suggested that endogenous SC-derived neurotrophins effect spontaneous regeneration into the spinal cord. To test this hypothesis, we cultured adult eGFP-expressing p75+/+ DRG neurons on purified, confluent sciatic SC cultures from p75+/+ or p75-/- mice. P75-/- SC cultures bore no obvious differences in morphology or density from p75+/+ cultures (Fig 2.5c). Neurons on p75-/- SCs had longer neurites, and branching was twice that which occurred on p75+/+ SCs (Fig 2.5a, b; n=4 cultures/group; p=0.005). The addition of TrkA-Fc and TrkC-Fc to the media reduced neurite outgrowth on p75-/- SCs to that on p75+/+ SCs (Fig 2.5a, b; n=4 cultures). Neurite outgrowth in wild-type cultures was unaffected by Trk-Fc treatment, in agreement with previous in vivo results using function-blocking antibodies against NGF and NT-3 (Diamond et al., 1987; Diamond et al., 1992). These results indicate that enhanced outgrowth on p75-/- SCs, like successful regeneration in vivo, is neurotrophindependent. To further establish the relative contribution of neuronal versus glial p75NTR on neurite outgrowth, we cultured p75-/- neurons on wild-type SCs in the presence or absence of Trk-Fc (n=4 cultures). Neurite number did not differ between neuronal genotypes on p75+/+ SCs (1.6±0.4 for p75+/+ neurons versus 1.5±0.6 for p75-/- neurons). Trk-Fc treatment also did not significantly reduce neurite number for p75-/- neurons on p75+/+ SCs (0.9±0.2). These data confirm those from DRG transplantation experiments  65  described above, that the absence of p75NTR from neurons has no bearing on successful regeneration.  2.4 Discussion p75NTR is a point of convergence for multiple signaling processes in the nervous system, many of which are functional rivals. It acts as a receptor for neurotrophins to regulate survival, neurite outgrowth and myelination; for pro-neurotrophins to mediate apoptosis; and for myelin-associated inhibitory proteins (MAIPs) to restrict plasticity and regeneration (Barker 2004; Gentry et al., 2004). We now show a regenerationprohibitive role for SC-expressed p75NTR. The underlying mechanism involves competition for endogenous neurotrophins between reactive SCs and axonal Trk receptors (Fig 2.6). This represents an important departure from an established model of p75NTR-neurotrophin-Trk interactions in nerve regeneration, in which SC p75NTR was thought to ‘present’ neurotrophins to Trks (Johnson, Jr. et al., 1988). The role of neurotrophins in peripheral nerve regeneration, and the validity of the presentation model, has come into question before. While exogenous neurotrophins clearly enhance regeneration of sensory axons (particularly evident from studies using dorsal rhizotomy as a model (Ramer et al., 2000) there appears to be little requirement for endogenous neurotrophins in naturally-occurring regeneration in the PNS. For example, axotomized TrkA- and TrkC- expressing nociceptive and mechanosensitive axons reinnervate their peripheral targets normally in the presence of antibodies which block neurotrophin-mediated collateral sprouting (Diamond et al., 1987; Diamond et al., 1992).  66  The findings of the present study show that, far from presenting neurotrophins to regenerating axons, Schwann cell-expressed p75NTR actually reduces their availability. P75NTR-neurotrophin-Trk interactions are probably quite different on axons. There is now good structural evidence for a presentation or ‘ligand-passing’ model in which an axonal p75NTR dimer binds a neurotrophin dimer in such a way as to leave a Trk-binding region exposed (Wehrman et al., 2007; He and Garcia, 2004). P75NTR then passes neurotrophin to an adjacent Trk monomer, at which point a second Trk monomer is recruited to initiate signaling. Given the close association between receptors in lipid rafts required for this interaction on the axonal membrane (Higuchi et al., 2003), a similar ligand passing mechanism is not likely to occur between more widely-separated Schwann cell p75NTR and axonal Trk receptors. Other previously demonstrated roles of p75NTR may also contribute to spontaneous functional regeneration in p75-/- mice. One of these involves its participation in SC apoptosis. Following axotomy, there is an induction of Schwann cell proliferation followed by apoptosis (Syroid et al., 2000), the latter being mediated by autocrine NGF- or pro-NGF signaling through p75NTR. In the absence of p75NTR, SC death following peripheral nerve injury is significantly reduced and regeneration is improved (Boyd and Gordon, 2001; Hirata et al., 2001; Petratos et al., 2003; SoiluHanninen et al., 1999). Regeneration of DRG axons in p75-/- mice in vivo may therefore be supported by both increased numbers of neurotrophin-expressing SCs and a lack of competitive neurotrophin binding by SC-expressed p75NTR. However, the dissociated DRG/SC co-culture work presented here (neurite outgrowth was greater on p75-/- than on p75+/+ confluent Schwann cell cultures) argues that, at least in vitro, increased numbers of  67  Schwann cells alone are not responsible for the enhanced neurite outgrowth conferred by an absence of p75NTR. Axonal p75NTR also mediates inhibitory myelin signaling (Wang et al., 2002a): MAIPs (Nogo, myelin-associated protein and oligodendrocyte myelin glycoprotein) bind a receptor complex including the Nogo receptor (NgR), LINGO-1 and p75NTR, all of which are expressed by dorsal root ganglion (DRG) neurons (Mi et al., 2004; Wang et al., 2002b; Wang et al., 2002a). However, primary afferents do not regenerate following dorsal column injury in p75-/- mice (Song et al., 2004). Although this stands in contrast with successful regeneration following DRI, it is explained by the spatial distribution of supportive or inhibitory glial elements surrounding regenerating axons in each model, and the role that axonal or glial p75NTR takes on in each environment. The astroglial scar, for example, represents the most immediate impediment to regeneration following both rhizotomy and spinal cord injury. Given that p75-/- axons do not traverse the PNS:CNS interface in a wild-type environment (present results) or the injury site following direct cord injury in p75-/- mice (Song et al., 2004), it is clear that the absence of axonal p75NTR confers no regenerative advantage at the scar. The key to successful regeneration in rhizotomized p75-/- mice is the inability of p75-/- SCs to act as a neurotrophin sink: just as with exogenous neurotrophin treatment in wild-type animals (Ramer et al., 2000), endogenous SC-derived NGF and NT-3 prompt regeneration into the CNS. The glial environment of axons injured within the p75-/- spinal cord lacks neurotrophin-secreting SCs and so regeneration across the scar fails. In the dorsal rhizotomy model, scartraversing axons are subsequently faced with degenerating myelin, which is more refractory to neurotrophin treatment (Ramer et al., 2001). In this environment, the  68  MAIP-signaling role of axonal p75NTR may become important, as in its absence rhizotomized axons regenerate through myelin debris at a constant rate. The balance between regeneration-promoting and regeneration-inhibiting influences in the CNS is a fine one. The role of p75NTR as the fulcrum has primarily been considered in light of its expression in neurons, where it is paired with Trks and MAIP receptors. The present results show that SC p75NTR is equally important as it restricts regenerative growth of injured axons by reducing neurotrophin availability. Since SC apoptosis is also reduced in p75-/- mice (Soilu-Hanninen et al., 1999), the implication is that p75NTR-neutralizing strategies, or in the case of cell transplant therapies, the use of p75NTR-negative, neurotrophin-producing cells is likely to lead to improved outcome following nervous system trauma.  69  Fig. 2.1. p75NTR prevents spontaneous regeneration into the CNS. A, p75NTR is stronglyupregulated in the injured dorsal root (the uninjured root is indicated with an arrow). B, By 28 days following rhizotomy, CTB-labeled DRG axons extended medially into the dorsal columns and reached superficial laminae of the dorsal horn (arrows) in p75-/- mice (bottom) but not in p75+/+ mice (top). C, By 28 days following dorsal rhizotomy, WGAlabeled axons had likewise regenerated into the dorsal columns and into the superficial laminae of the dorsal horn (arrows) in p75-/- mice (bottom) but not p75+/+ mice (top). Scale bar in A: 150 µm; B and C: 50µm. D, IB4-labeled non-peptidergic primary afferents do not regenerate into the cord in either genotype. In all photomicrographs, arrowheads indicate the PNS:CNS interface. E, Quantification of axonal growth reavealed significant differences (asterisks) between p75+/+ and p75-/- mice (representative images of regeneration at 7 days following injury are shown in Fig 2.3). F, Western blots and analyses illustrating equivalent neurotrophin protein levels in sciatic nerves from p75+/+ and p75-/- mice 3d following injury.  70  71  Fig. 2.2. Functional recovery in rhizotomized p75-/- mice. A, Response threshold and B, response latency to progressive punctate force and acetone application, respectively. In both cases, there were significant differences between genotypes (pre: preoperative; post: four weeks post-operative). C, Paw licking duration in early (0-10min) and late (1060min) phases of the formalin response injection was equivalent in uninjured p75+/+ and p75-/- mice. Only an early phase response occurred in both strains. Twenty-eight days following deafferentation, formalin responses were absent from p75+/+ mice, but present (although delayed) in p75-/- mice. D, E, Fos was upregulated in neuronal nuclei in the spinal cord dorsal horn ipsilateral to forepaw formalin injection in uninjured p75+/+ and p75-/- mice (top row in E). Four weeks after septuple dorsal rhizotomy, formalin-induced Fos immunoreactivity was negligible in p75+/+ mice (bottom left). Fos-positive nuclei in the dorsal horn of p75-/- mice (bottom right) were significantly more numerous (asterisk in D). Scale bar: 100µm.  72  73  Fig. 2.3. Regeneration in p75-/- mice is neurotrophin-dependent. A, B, Low, and to a greater extent, high doses of TrkC-Fc prevented regeneration of CTB-labeled primary afferent axons in p75-/- mice (arrows). Likewise, low and high doses of TrkA-Fc also inhibited CTB-labeled axon regeneration. All treatment groups had significantly less regeneration of CTB-labeled axons than vehicle-treated p75-/- mice. Regeneration of WGA-labeled sensory axons (arrows) was not diminished in p75-/- mice treated with a low dose of TrkC-Fc, but was significantly below vehicle levels in those treated with a high dose. p75-/- mice treated with low and high doses of TrkA-Fc showed a significant reduction in WGA-labeled axonal regeneration compared to those with vehicle treatment. Scale bar: 25µm. C, Regeneration of primary afferent axons in p75-/- mice is only marginally augmented with the addition of neurotrophic factors. CTB-labeled axon growth 7 days post-injury was enhanced with NT-3 treatment in p75+/+ mice (n=4) but not in p75-/- mice (n=4) (vehicle treatment: n=6). NGF treatment failed to improve regeneration of CTB-labeled axons in both p75+/+ mice and p75-/- mice. NT-3 treatment significantly enhanced WGA-labeled axonal growth in p75+/+ mice, but did not further enhance axonal growth in p75-/- mice. NGF treatment promoted regeneration of WGAlabeled axons in both p75+/+ mice and, to a lesser extent, in p75-/- mice. Asterisks: significant differences between p75+/+ and p75-/- mice; daggers: differences between neurotrophin-treated and vehicle-treated groups.  74  75  Fig. 2.4. p75NTR expression by the axonal environment, not the axon, limits regeneration. A, Experimental model and phenotypic analysis of eGFP-expressing neurons from p75+/+ and p75-/- mice. There was no significant difference in size-frequency distribution or TrkA immunoreactivity of neurons prepared as for injection. B, Axons from adult eGFPexpressing mouse DRG neurons transplanted into a rhizotomized rat DRG grew up to, but not beyond the PNS:CNS interface (dotted lines) irrespective of p75NTR expression. Scale bar: 100µm. C, D, In p75+/+ mice receiving p75+/+ peripheral nerve allografts, CTB-labelled axons approached, but most stopped at, the graft:host interface (defined by laminin-1 expression in the graft). In wild-type mice receiving p75-/- grafts, a significantly larger proportion of axons (arrows in inset) penetrated the graft. Scale bar: 50µm.  76  77  Fig. 2.5. Enhanced regeneration in a p75-/- environment is neurotrophin-dependent in vitro. A, Dissociated adult eGFP-expressing p75+/+ DRG neurons plated on p75-/- SCs send out more neurites and possess more filopodia (visualized with TrkA immunocytochemistry, arrows in middle row), than do those on confluent p75+/+ SC cultures. Trk-Fc abolished the growth-enhancing effects of p75-/- SCs. Scale bars: 50µm (top and bottom panels); 25µm (middle panel). B, Quantification of neurite-bearing neurons and number of neurites per neuron. C, S100b-labeled Schwann cell confluent cultures from p75+/+ mice and p75-/- mice were identical. Scale bar: 50µm.  78  79  Fig. 2.6. A, Regeneration is limited in wild-type mice due to neurotrophin-sequestering Schwann cell p75NTR. B, Increased neurotrophin availability in exon III mutants allows for successful regeneration.  80  2.5. Bibliography Barker PA (2004) p75NTR is positively promiscuous: novel partners and new insights. Neuron 42: 529-533. Bergmann I, Priestley JV, McMahon SB, Brocker EB, Toyka KV, Koltzenburg M (1997) Analysis of cutaneous sensory neurons in transgenic mice lacking the low affinity neurotrophin receptor p75. Eur J Neurosci 9: 18-28. Boyd JG, Gordon T (2001) The neurotrophin receptors, trkB and p75, differentially regulate motor axonal regeneration. J Neurobiol 49: 314-325. Chan JR, Cosgaya JM, Wu YJ, Shooter EM (2001) Neurotrophins are key mediators of the myelination program in the peripheral nervous system. Proc Natl Acad Sci USA 98: 14661-14668. 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EMBO J 22: 1790-1800. Hirata H, Hibasami H, Yoshida T et al. (2001) Nerve growth factor signaling of p75 induces differentiation and ceramide-mediated apoptosis in Schwann cells cultured from degenerating nerves. Glia 36: 245-258.  81  Hunt SP, Pini A, Evan G (1987) Induction of c-fos-like protein in spinal cord neurons following sensory stimulation. Nature 328: 632-634. Johnson EM, Jr., Taniuchi M, DiStefano PS (1988) Expression and possible function of nerve growth factor receptors on Schwann cells. Trends Neurosci 11: 299-304. Karchewski LA, Kim FA, Johnston J, McKnight RM, Verge VM (1999) Anatomical evidence supporting the potential for modulation by multiple neurotrophins in the majority of adult lumbar sensory neurons. J Comp Neurol 413: 327-341. Kerr BJ, Bradbury EJ, Bennett DL et al. (1999) Brain-derived neurotrophic factor modulates nociceptive sensory inputs and NMDA-evoked responses in the rat s pinal cord. J Neurosci 19: 5138-5148. Krekoski CA, Parhad IM, Clark AW (1996) Attenuation and recovery of nerve growth factor receptor mRNA in dorsal root ganglion neurons following axotomy. J Neurosci Res 43: 1-11. McMahon SB, Armanini MP, Ling LH, Phillips HS (1994) Expression and coexpression of Trk receptors in subpopulations of adult primary sensory neurons projecting to identified peripheral targets. Neuron 12: 1161-1171. McPhail LT, Plunet WT, Das P, Ramer MS (2005) The astrocytic barrier to axonal regeneration at the dorsal root entry zone is induced by rhizotomy. Eur J Neurosci 21: 267-270. Mi S, Lee X, Shao Z et al. (2004) LINGO-1 is a component of the Nogo-66 receptor/p75 signaling complex. Nat Neurosci 7: 221-228. Molliver DC, Snider WD (1997) Nerve growth factor receptor TrkA is down-regulated during postnatal development by a subset of dorsal root ganglion neurons. J Comp Neurol 381: 428-438. Oudega M, Varon S, Hagg T (1994) Regeneration of adult rat sensory axons into intraspinal nerve grafts: promoting effects of conditioning lesion and graft predegeneration. Exp Neurol 129: 194-206. Petratos S, Butzkueven H, Shipham K et al. (2003) Schwann cell apoptosis in the postnatal axotomized sciatic nerve is mediated via NGF through the low-affinity neurotrophin receptor. J Neuropathol Exp Neurol 62: 398-411. Raivich G, Kreutzberg GW (1987) Expression of growth factor receptors in injured nervous tissue. I. Axotomy leads to a shift in the cellular distribution of specific beta-nerve growth factor binding in the injured and regenerating PNS. J Neurocytol 16: 689-700. Ramer LM, McPhail LT, Borisoff JF et al. (2007) Endogenous TrkB ligands suppress functional mechanosensory plasticity in the deafferented spinal cord. J Neurosci 27: 5812-5822. Ramer MS, Duraisingam I, Priestley JV, McMahon SB (2001) Two-tiered inhibition of axon regeneration at the dorsal root entry zone. J Neurosci 21: 2651-2660. Ramer MS, Priestley JV, McMahon SB (2000) Functional regeneration of sensory axons into the adult spinal cord. Nature 403: 312-316. Ramon y Cajal S (1991) Cajal's degeneration & regeneration of the nervous system. Oxford: Oxford University Press. Sawynok J, Liu XJ (2004) The formalin test: characteristics and usefulness of the model. Reviews in Analgesia 7: 145-163.  82  Scott AL, Borisoff JF, Ramer MS (2005) Deafferentation and neurotrophin-mediated intraspinal sprouting: a central role for the p75 neurotrophin receptor. Eur J Neurosci 21: 81-92. Soilu-Hanninen M, Ekert P, Bucci T, Syroid D, Bartlett PF, Kilpatrick TJ (1999) Nerve growth factor signaling through p75 induces apoptosis in Schwann cells via a Bcl2-independent pathway. J Neurosci 19: 4828-4838. Song XY, Zhong JH, Wang X, Zhou XF (2004) Suppression of p75NTR does not promote regeneration of injured spinal cord in mice. J Neurosci 24: 542-546. Streppel M, Azzolin N, Dohm S et al. (2002) Focal application of neutralizing antibodies to soluble neurotrophic factors reduces collateral axonal branching after peripheral nerve lesion. Eur J Neurosci 15: 1327-1342. Swett JE, Woolf CJ (1985) The somatotopic organization of primary afferent terminals in the superficial laminae of the dorsal horn of the rat spinal cord. J Comp Neurol 231: 66-77. Syroid DE, Maycox PJ, Soilu-Hanninen M et al. (2000) Induction of postnatal schwann cell death by the low-affinity neurotrophin receptor in vitro and after axotomy. J Neurosci 20: 5741-5747. Taniuchi M, Clark HB, Johnson EM, Jr. (1986) Induction of nerve growth factor receptor in Schwann cells after axotomy. Proc Natl Acad Sci USA 83: 4094-4098. Tong YG, Wang HF, Ju G, Grant G, Hokfelt T, Zhang X (1999) Increased uptake and transport of cholera toxin B-subunit in dorsal root ganglion neurons after peripheral axotomy: possible implications for sensory sprouting. J Comp Neurol 404: 143-158. Wang KC, Kim JA, Sivasankaran R, Segal R, He Z (2002a) P75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature 420: 74-78. Wang KC, Koprivica V, Kim JA et al. (2002b) Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 417: 941-944. Wehrman T, He X, Raab B, Dukipatti A, Blau H, Garcia KC (2007) Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors. Neuron 53: 25-38. Wright DE, Snider WD (1995) Neurotrophin receptor mRNA expression defines distinct populations of neurons in rat dorsal root ganglia. J Comp Neurol 351: 329-338. Yamauchi J, Chan JR, Shooter EM (2004) Neurotrophins regulate Schwann cell migration by activating divergent signaling pathways dependent on Rho GTPases. Proc Natl Acad Sci USA 101: 8774-8779. Zhou XF, Rush RA, McLachlan EM (1996) Differential expression of the p75 nerve growth factor receptor in glia and neurons of the rat dorsal root ganglia after peripheral nerve transection. J Neurosci 16: 2901-2911.  83  CHAPTER THREE  Deafferentation and neurotrophin-mediated intraspinal sprouting: a central role for the p75 neurotrophin receptor3  3  A version of this chapter has been published. Scott, A.L.M, Borisoff, J.F. and Ramer, M.S. (2005) Deafferentation and neurotrophin-mediated intraspinal sprouting: a central role for the p75 neurotrophin receptor. European Journal of Neuroscience 21: 8192. 84  3.1 Introduction Injury to the centrally projecting axons of primary afferent neurons (dorsal rhizotomy) results in permanent loss of sensation in the affected dermatomes and can be associated with chronic phantom-like pain (Berman et al., 1998). The loss of sensory function after dorsal rhizotomy is attributable to the inability of dorsal root axons to spontaneously regenerate into the spinal cord. One anatomical consequence of dorsal rhizotomy is the intraspinal sprouting of undamaged axons, a phenomenon that has been correlated not only with functional recovery (Helgren & Goldberger, 1993; Darian-Smith & Brown, 2000) but also with the development of hyperalgesia and allodynia (Romero et al., 2000, 2001; Obata et al., 2004). The extent of rhizotomy-induced sprouting differs among axonal phenotypes (Polistina et al., 1990; Zhang et al., 1993; Wong et al., 2000; Jeffery & Fitzgerald, 2001; Darian-Smith, 2004). In particular, peptidergic primary sensory axons containing calcitonin gene-related peptide (CGRP) and substance P have been shown to sprout more vigorously than non-peptidergic sensory axons in the dorsal horn after dorsal rhizotomy (Polistina et al., 1990; Belyantseva & Lewin, 1999; DarianSmith, 2004). Similarly, descending serotonergic and dopaminergic populations sprout to a greater extent than noradrenergic axons (Wang et al., 1991; Zhang et al., 1993). The variable tendency of axonal populations to sprout in response to spinal deafferentation is probably attributable to their differing responses to spinally produced growth-promoting factors and inhibitory molecules (Schwab, 2002; Furue et al., 2004). Neurotrophins (NTs) may enhance injury-induced plasticity of undamaged primary afferents (Romero et al., 2000; Darian-Smith, 2004) and descending supraspinal tracts (Schnell et al., 1994; Zhou & Shine, 2003; Zhou et al., 2003). The effects of NTs are  85  largely mediated by their tropomyosin-related kinase (Trk) receptors (Wright & Snider, 1995; King et al., 1999; Chao, 2003) but they also bind to the p75NTR receptor, which is widely expressed within the central nervous system and peripheral nervous system (Chao, 2003). A complete understanding of the role of p75NTR in spinal plasticity is hampered by its multiple functions: it can modify NT-Trk affinities and specificities (Mahadeo et al., 1994; Vesa et al., 2000; Esposito et al., 2001) potentially leading to enhanced neurite outgrowth; it constitutively activates RhoA, potentially leading to diminished neurite outgrowth (Yamashita et al., 1999; Yamashita & Tohyama, 2003) and it interacts with the Nogo receptor (NgR), which binds myelin-associated growth-inhibiting proteins such as myelin-asso-ciated glycoprotein, oligodendrocyte myelin glycoprotein and NogoA (Wang et al., 2002). Importantly, the interaction of NgR with p75NTR has been found to augment RhoA activation (Yamashita & Tohyama, 2003), whereas NT interaction with p75NTR has been shown to suppress this activation (Yamashita et al., 1999). P75NTR may therefore be pivotal in central nervous system plasticity. In the present study, to better understand the role of p75NTR, we examined the intraspinal sprouting response of primary afferents and descending monoaminergic fibres after dorsal rhizotomy in p75NTR mutant mice in the presence and absence of exogenous NTs.  3.2 Materials and methods Animal surgery and tissue preparation. For this study, we used the previously characterized p75NTR hypomorphic mouse (p75– /–, Ngfr  tm1Jae,version 1  ; Jackson Laborat-  ory/JAX Mice, Bar Harbor, ME, USA) (Lee et al., 1992). By way of a functional deletion, these mice possess a splice variant of p75NTR , which lacks the extracellular NT-  86  binding domain. Age-matched, 8–10-week-old wild-type littermates were used for comparison. Under general anesthesia (ketamine hydrochloride, 80–100 mg/kg i.p.; Bimeda-MTC, Cambridge, ON, Canada and xylazine hydrochloride, 5–10 mg/kg i.p.; Bayer Inc., Etobicoke, ON, Canada), the dorsolateral cervical spinal cord and dorsal roots from C4 to T2 were exposed by dorsal laminectomy. The dura mater was opened with iris scissors and all seven exposed roots were crushed (three times, 10 s each) with fine forceps (no. 5). Fibrin glue (3 lL), prepared as a combination of equal parts of thrombin (25 U/mL in 45 mmCaCl2), fibrinogen (100 mg/mL in distilled water) and fibronectin (8 mg/mL in distilled water) (all from Sigma, St Louis, MO, USA), was then applied to the dorsolateral spinal cord at the site of injury. The fibrin glue served to prevent cerebrospinal fluid leakage by sealing the incision in the dura mater and as a delivery vehicle for NTs [nerve growth factor (NGF) and NT-3, gifts from Genentech, San Fransisco, CA, USA]. NTs were prepared in rat serum albumin (1 mg ⁄ mL in sterile phosphate-buffered saline) and mixed with the fibrin glue to produce a final concentration of 100 µg/mL. Vehicle-treated animals received fibrin glue alone (p75+/+, n=6; p75–/–, n=6) and NT-treated animals received fibrin glue with NGF (p75+/+, n=4; p75–/–, n=4) or fibrin glue with NT-3 (p75+⁄+, n=4; p75–⁄–, n=4). Survival times were 1 week (all treatments) or 4 weeks (vehicle only). Fibrin glue has previously been used to deliver NTs (Iwaya et al., 1999), although the precise rate of delivery remains unknown. All surgical procedures were performed under the guidelines of the University of British Columbia and the Canadian Council for Animal Care. Immunohistochemistry. Animals were killed with an overdose of chloral hydrate (100 mg ⁄ kg i.p.) and perfused transcardially with 4% paraformaldehyde. Spinal cords with  87  attached DRGs (from C6 to T1) were removed and postfixed overnight. The tissue was cryoprotected in 20% sucrose in 0.1 mphosphate buffer for 24 h at 4 C, frozen and stored at -80 °C. Spinal cords were cut transversely (16µm) on a cryostat and sections were processed immunohistochemically for serotonin (5-HT; 1:16,000, host rabbit; Immunostar, Hudson, WI, USA), tyrosine hydroxylase (TH; 1:200, host sheep; Chemicon, Temecula, CA, USA) and CGRP (1:4000, host rabbit; Sigma). After incubation in 10% normal donkey serum (in 0.1 mphosphate-buffered saline, 0.2% Triton X-100 and 0.1% sodium azide) for 1 h, primary antibodies were applied overnight. The sections were washed and incubated in secondary antibodies all raised in donkey and conjugated to cyanine (1:200; Jackson ImmunoResearch, West Grove, PA, USA) or Alexa 488 (1: 400; Molecular Probes, Eugene, OR, USA) for 2 h. After a final wash, sections were coverslipped with Immunomount (Fisher Scientific, Pittsburgh, PA, USA).  Image analysis. Digital images of the spinal cord dorsal horn were captured with an Axioplan 2 microscope (Zeiss, Jena, Germany) using a 20· objective, a digital camera (Q Imaging, Burnaby, BC, Canada) and Northern Eclipse software (Empix Imaging Inc., Mississauga, ON, Canada). Image analysis was performed with Sigma Scan Pro 4 software (SPSS, Chicago, IL, USA). Axon density was measured and expressed as a function of depth in the dorsal horn as described previously (Ramer et al., 2001, 2004; Gaudet et al., 2004). Briefly, we first processed images with an omnidirectional edgedetection filter, which had the effect of normalizing non-specific background signal and of rendering all axonal profiles equal in thickness. We then applied a threshold to the image, which gave all axonal pixels equal weight irrespective of their original immunoreactivity. The axon density measurements reported reflect a proportion of the 88  area of the dorsal gray matter occupied by immunoreactive axons (regardless of their brightness) as a function of depth. CGRP-positive axon density was measured in segments C6–C8 and 5-HT-and TH-positive axon density was measured in segments C6– T1. This meant that roots were intact one to two levels rostral and caudal to the segments analysed, minimizing the influence of adjacent uninjured regions. We began our measurements blind with respect to treatment but it became obvious which mice were which due to the reduced size of the dorsal columns. Total blindness was therefore precluded. Statistics. All statistical analysis was performed with a two-way analysis of variance on ranks and a pairwise multiple comparison (Holm-Sidak test) (significance set at P=0.05). Error bars are indicative of the SEM.  3.3 Results The axonal populations studied here were chosen because previous reports have demonstrated that they undergo sprouting to varying extents after dorsal rhizotomy and because no cell somata in the dorsal horn express any of these antigens, facilitating analysis and interpretation. We present the effects of dorsal rhizotomy and the effects of NTs in the ipsilateral dorsal horn in Figs 3.1–3.7. For clarity, separate axonal density depth profiles are presented that compare ipsilateral dorsal horns of p75+⁄+ and p75–⁄– mice (panels c, f and i in all cases). Additional density–depth profiles compare the axonal response to rhizotomy 7 and 28 days after rhizotomy with intact animals (panels j and k in Figs 3.1, 3.3 and 3.5) or the effects of NGF and NT-3 with vehicle treatment (panels j and k in Figs 3.2, 3.4 and  89  3.6). For comparison with intact and 28-day vehicle-treated animals, and with NGF-and NT-3-treated animals, the data from 7-day vehicle-treated mice are represented twice, once in time-course figures (Figs 3.1, 3.3 and 3.5) and once in NT figures (Figs 3.2, 3.4 and 3.6). A summary of changes in axon density with rhizotomy or rhizotomy plus NTs is presented in Fig. 3.7, which represents the mean axonal densities throughout the analysed region of the dorsal horn. Figure 3.7 also provides a summary of contralateral changes which were analysed in the same way as the ipsilateral changes presented in Figs 3.1–3.6. 3.3.1 Primary afferent axons The distribution of CGRP-positive afferents in p75+/+ and p75-/- mice (Fig. 3.1a and b) was consistent with previous observations showing termination patterns in superficial laminae (I and II outer) and laminae V and VI (Belyantseva & Lewin, 1999; Hannila & Kawaja, 2003). However, in p75-/- mice, Lissauer’s tract was smaller and the axon density within laminae II outer was less than that in p75+/+ mice (Fig. 3.1c). This result is consistent with a developmental loss of peptidergic primary afferent neurons in p75-/- mice (Dreetz-Gjerstal et al., 2002). To determine the response of intact primary afferents originating from adjacent uninjured regions of the spinal cord, the density of CGRP-immunoreactive axons was measured centrally (at levels C6–C8) within the deafferented dorsal horn (C4–T2 septuple rhizotomy). Quantitative analysis of p75+/+ mice 1 week after rhizotomy revealed that the presence of CGRP-positive fibres was virtually abolished in the region ipsilateral to the injury (Fig. 3.1d and j). However, in p75-/- mice, the density of these fibres was significantly higher within lamina I and outer lamina II of the denervated  90  segments (Fig. 3.1e and f). As peptidergic innervation of the dorsal horn was similar between p75+/+ and p75-/- mice (Fig. 3.1c), this suggests an enhanced sprouting response of these fibres to spinal deafferentation in p75-/- mice. We examined axon density of primary afferent projections to the dorsal horn 1 month after dorsal rhizotomy in order to determine whether the sprouting phenomenon is persistent and whether there are still marked differences between p75+/+ and p75-/- mice. CGRP-positive axon density ipsilateral to rhizotomy was significantly higher at 28 than at 7 days post-injury in both p75+/+ and p75-/- mice (Fig. 3.1g, h, j and k). Axon density in p75-/- mice was greater than that in p75+/+ mice at both time-points (Fig. 3.1f and i), indicating that the absence of p75NTR enhances intraspinal sprouting of CGRP-positive axons. Nerve growth factor treatment significantly increased the density of CGRPimmunoreactive axons ipsilateral to rhizotomy in p75+/+ but not p75-/- mice (Fig. 3.2a–f). NT-3 had little or no effect on CGRP-positive axon density in p75+/+ mice (Fig. 3.2g and j). The densities of CGRP-positive axons were significantly higher in NT-3-treated p75-/mice than p75+/+ mice treated with either NT-3 or NGF (Fig. 3.2g–k). 3.3.2 Serotonergic axons Serotonin-positive axon density in uninjured p75+/+ mice was greatest in the superficial laminae of the dorsal horn (Fig. 3.3a and c). While the pattern of serotonergic innervation of the dorsal horn was similar in p75-/- mice (Fig. 3.3b), the overall density was less than that observed in 7 days after rhizotomy (Fig. 3.3e, h and j) and higher than in p75+/+mice (Fig. 3.3c). In p75+/+ mice 1 week after rhizotomy, the 5- HT-positive axon density in the dorsal horn was significantly elevated above that observed in intact animals  91  (Fig. 3.3a, d and j). The same was true for rhizotomized p75-/- mice 1 week after injury (Fig 3.3 b, e and k). The magnitude of the increase in 5-HT-positive axon density above that observed in intact animals was much greater than in p75-/- mice (compare Fig. 3.3c and f), indicating an increased serotonergic sprouting response in the absence of fulllength p75NTR. In the ipsilateral dorsal horn of p75+/+ mice rhizotomized for 28 days, 5-HTpositive axon density was the same as in those rhizotomized for 7 days (Fig. 3.3d, g and j). In p75-/- mice, serotonergic axon density was significantly higher than in p75+/+ mice 28 days post-injury (Fig. 3.3i). Nerve growth factor treatment had little effect on the rhizotomy-induced increase in 5-HT-positive axon density in p75+/+ mice (Fig. 3.4a, d and j). In p75-/- mice, NGF treatment resulted in a large increase in serotonergic axon density in the ipsilateral dorsal horn, accompanied by an increased axonal density in deeper laminae (Fig. 3.4b, e, f and k). Given the lack of TrkA expression by bulbospinal serotonergic neurons (King et al., 1999), this effect of NGF is likely to be indirect. In p75+/+ mice, NT-3 treatment resulted in an increase in the serotonergic axon density in the ipsilateral dorsal horns over that observed in vehicle-treated rhizotomized p75+/+ mice (Fig. 3.4a, g and j). NT-3 treatment had a large effect on the serotonergic axon density ipsilateral to rhizotomy in p75-/- mice (Fig. 3.4b, h and k), particularly in deeper laminae (Fig. 3.4i). Unlike TrkA, TrkC is expressed on spinally projecting serotonergic axons (King et al., 1999) and so the NT-3-mediated effects are more likely to be direct.  92  3.3.3 Tyrosine hydroxylase - expressing axons Tyrosine hydroxylase-positive fibres (dopaminergic and noradrenergic) were most heavily concentrated in laminae I and III–IV in both genotypes (Fig. 3.5a and b) but, as with serotonergic axons, the peak TH-positive axon density was reduced in p75-/- mice (Fig. 3.5c). In p75+/+ mice, there was a small but significant increase in the density of THpositive axons 1 week after rhizotomy compared with uninjured p75+/+ mice (Fig. 3.5a, d and j). An increase in TH-positive axon density was also observed in p75-/- mice, although the magnitude of the increase above that in uninjured mice was much greater (Fig. 3.5c, e, f and k). In both genotypes TH-positive axon density continued to increase for 4 weeks (Fig. 3.5g–k). Tyrosine hydroxylase-positive axon density increased with NGF treatment after rhizotomy in both genotypes (Fig. 3.6a–f, j and k) but, again, the increase observed in p75-/- mice was significantly greater than that which occurred in p75+/+ mice (Fig. 3.6f). Spinally projecting noradrenergic and dopaminergic axons are not known to express TrkA (King et al., 1999) and so again these NGF-mediated effects are probably indirect. Neurotrophin-3 treatment augmented the rhizotomy-induced increase in THpositive axon density in both the p75+/+ and p75-/- mice (Fig. 3.6g–k). In p75-/- mice, THpositive axon density was higher than that observed in NT-3-treated p75+/+ mice (Fig. 3.6g–i). Figure 3.7 shows integrated (0–400 µm deep in the dorsal horn) axon densities from all experiments. In general, contralateral responses to rhizotomy and NTs were similar to ipsilateral responses but smaller in magnitude. The exception to this  93  generalization involved TH-expressing axons, where ipsilateral and contralateral changes were equivalent. Innervation of the dorsal horn by primary afferent and descending monoaminergic axons was reduced in uninjured p75-/- mice but rhizotomy-induced sprouting was greater. NGF treatment had a smaller effect on primary afferent sprouting but a larger effect on descending monoaminergic sprouting in p75-/- than p75+/+ mice. In p75-/- mice, NT-3 treatment enhanced the sprouting response of all axonal populations to a greater degree than in p75+/+ mice. Differences between genotypes with and without NT treatment support the notion that the presence of p75NTR plays a role in the development and plasticity of ascending and descending axonal populations within the spinal cord.  3.4 Discussion Functional recovery after spinal cord injury or deafferentation may be more readily attainable through eliciting intraspinal sprouting of spared axons than through achieving regeneration of injured axons (reviewed in Chen et al., 2002; Schwab, 2002; Weidner & Tuszynski, 2002; Edgerton et al., 2004). The ability of various phenotypes of spinal axons to undergo collateral sprouting is dependent upon their intrinsic growth potential and their responsiveness to extrinsic cues. Neurotrophic factors and inhibitory myelin proteins, which act to enhance (Chao, 2003) or diminish (Hunt et al., 2002) the plasticity of spinal axons, respectively, signal via p75NTR (Kaplan & Miller, 2000; Hempstead, 2002). Here we found that, in the absence of the NT-binding domain of p75NTR, rhizotomy-and NT-induced increases in intraspinal sprouting were augmented, illustrating that the expression of full-length p75NTR plays a central role in the modulation of central nervous system plasticity by environmental cues.  94  It is known that CGRP-expressing primary afferent neurons, serotonergic neurons in the Raphe nuclei, noradrenergic neurons of the locus ceruleus and dopaminergic neurons of the hypothalamus all express p75NTR in several species, including humans (Sobreviela et al., 1994; Berg-von der Emde et al., 1995; Wright & Snider, 1995; Chen et al., 1996; Yamuy et al., 2000), implicating a possible direct role for p75NTR in suppressing spinal plasticity. TH-expressing axons in the substantia nigra pars compacta, some of which also project spinally, do not express p75NTR (Chaisuksunt et al., 2003) so, if these axons sprout after rhizotomy and ⁄or neurotrophic factor treatment, the underlying mechanism is likely to be p75NTR independent. Neurotrophins and their corresponding Trk receptors are important regulators of cell survival, proliferation and neurite outgrowth (reviewed in Bibel & Barde, 2000; Huang & Reichardt, 2003). Developmental deficiencies in DRG neurite outgrowth in p75-/- embryos, along with substantial (50%) losses of sensory neurons of the DRG (Lee et al., 1992, 1994; Kawaja, 1998), illustrate that p75NTR is important for neuronal survival during development. Here we found that the density of 5-HT-, TH-and, to a lesser extent, CGRP-positive axons was lower in uninjured p75-/- mice than in p75+/+ mice. The small decrease in CGRP-positive axon density in the dorsal horn of uninjured mice indicates that the central projections of primary afferent neurons compensate for a significant reduction in neuronal number through increasing their central arborization. The present results represent the first report of decreased spinal innervation by descending monoaminergic axons in p75-/- mice. While these axonal phenotypes are known to express p75NTR (Sobreviela et al., 1994; Berg-von der Emde et al., 1995; Chen et al., 1996; Yamuy et al., 2000), it remains unknown whether the decrease in spinal axon  95  density is due to a developmental decrease in neuronal number and ⁄or to a decrease in spinal branching. Despite the decreased spinal innervation by CGRP-, 5-HT-and TH-positive axons in intact p75-/- mice, the axonal density of all of these subpopulations was increased after rhizotomy compared with p75+/+ mice. This discussion will therefore focus first on the potential cause of intraspinal sprouting after dorsal rhizotomy and then on the enhanced sprouting in the absence of full-length p75NTR. We attribute the latter phenomenon to one or more of the following: (i) a lack of competition between Trks and p75NTR for NTs in p75-/- mice; (ii) altered affinities and specificities between NTs and Trk receptors; (iii) altered interactions between intracellular signaling cascades initiated by Trks and p75NTR and (iv) a differential ability of inhibitory myelin-derived proteins to suppress plasticity in the presence and absence of p75NTR. These are summarized in Fig. 3.8.  3.4.1 Spinally-derived neurotrophins may initiate intraspinal sprouting Brain-derived neurotrophic factor (BDNF) and NT-3 are upregulated in spinal gray matter after cervical dorsal rhizotomy (Johnson et al., 2000). As descending serotonergic, dopaminergic and noradrenergic neurons express TrkB and TrkC (King et al., 1999; Loudes et al., 1999) and can be encouraged to regenerate with BDNF and NT-3 treatment after spinal cord injury (Bregman et al., 1997), these factors are likely to contribute to intraspinal sprouting of monoaminergic axons. In rats, we have found that application of intrathecal K252-a (which inhibits Trk signaling) does indeed reduce serotonergic sprouting after dorsal rhizotomy (unpublished observations).  96  3.4.2 Altered neurotrophin-tropomyosin-related kinase affinity ⁄ specificity and intraspinal sprouting In p75-/- mice, we found that sprouting of all axonal populations examined was increased relative to their wild-type counterparts. This may have been due, in part, to altered affinities and specificities of Trk receptors for spinally produced BDNF and NT-3 (1 in Fig. 3.8). In vitro studies have demonstrated that, when p75NTR is coexpressed with TrkA, NGF-TrkA binding occurs with high affinity and specificity (Mahadeo et al., 1994; NTR  Esposito et al., 2001). Likewise, p75  increases the specificity of TrkB-BDNF binding  (Bibel et al., 1999). TrkC, however, can be activated by any of NT-3, BDNF and NT-4 ⁄ 5 when p75NTR is present (Vesa et al., 2000). In the absence of p75NTR, NGF binds to TrkA with a lower affinity (Mahadeo et al., 1994; Esposito et al., 2001) and NT-3 gains the ability to signal through all Trks (Vesa et al., 2000; Mischel et al., 2001). According to this scenario, in p75+/+ mice, spinally produced BDNF would activate both TrkB and TrkC and NT-3 would activate TrkC only while, in p75-/- mice, TrkB and TrkC would be activated by both NTs. This additional interaction between NT-3 and TrkB in the absence of p75NTR may underlie the enhanced rhizotomy-induced sprouting of monoaminergic (TrkB and TrkC-expressing) axons observed here. Changing NT-Trk specificities may also partially account for differences in CGRP-positive axon densities between genotypes after dorsal rhizotomy. In vehicletreated p75+/+ mice, very little CGRP-positive sprouting occurred and this was probably due to the relative insensitivity of TrkA-expressing primary afferents to spinally produced BDNF and NT-3. In p75-/- mice, in which spinally produced NT-3 is expected to activate all Trks (Vesa et al., 2000; Mischel et al., 2001), CGRP-positive axon density  97  was increased. The results from mice treated with exogenous NT-3 also support differences in plasticity due to altered NT-Trk affinities and specificities in the absence of full-length p75NTR. NT-3 treatment not only increased the density of monoaminergic axons but also CGRP-positive axons in p75-/- mice over that of p75+/+ mice. In NGF-treated p75+/+ mice, but not p75-/- mice (in which NGF-TrkA affinity is decreased), we observed an increase in CGRP-positive axon density over rhizotomy plus vehicle treatment. We were surprised to find that, in p75-/- mice, NGF increased the intraspinal density of both serotonergic and TH-expressing axons. The lack of TrkA expression in descending monoaminergic axons (King et al., 1999) suggests that this effect is indirect. One possible mechanism involves NGF-mediated upregulation of BDNF in primary afferent neurons, which is anterogradely transported to and released in the spinal cord (Michael et al., 1997; Lever et al., 2001, 2003; Karchewski et al., 2002). It is reasonable to speculate that this primary afferent-derived BDNF may contribute to intraspinal monoaminergic sprouting.  3.4.3 Intracellular tropomyosin-related kinase and p75 signaling cascades and intraspinal sprouting An important consideration is the influence, which NTs may have on axonal outgrowth via p75NTR, independent of NT receptor complexes. P75NTR has been shown to constitutively activate the small GTPase RhoA (4 in Fig. 3.8) (Yamashita et al., 1999; Yamashita & Tohyama, 2003; Gehler et al., 2004), which can cause growth cone collapse and the subsequent suppression of axonal outgrowth (Borisoff et al., 2003; Dubreuil et al., 2003; Ramer et al., unpublished results). Upon binding to p75NTR, NTs depress  98  RhoA activation and increases neurite outgrowth (Yamashita et al., 1999; Gehler et al., 2004). This may account, in part, for NT-mediated increases in axonal density in the spinal cord of p75+/+ mice, but not in p75–/– mice where the NT-binding portion of the receptor is deleted. However, NT-Trk binding can lead to Rho-GTP hydrolysis independent of p75NTR via a GTPase-activating protein (Grit), also leading to enhanced neurite outgrowth (Nakamura et al., 2002). Thus, the relationship between Trk and p75NTR signaling may contribute to the observed increase in responsiveness of p75-/axons to rhizotomy and NTs.  3.4.4 Myelin-derived inhibitory protein signaling and intraspinal sprouting Inhibitory proteins expressed by oligodendrocytes also modulate RhoA activation via p75NTR (6 in Fig. 3.8). These proteins, including myelin-associated glycoprotein, NogoA and oligodendrocyte myelin glycoprotein, signal through a common extracellular receptor, the NgR, expressed on multiple populations of neurons (reviewed in Hunt et al., 2002; Schwab, 2002). NgR, p75NTR (Wang et al., 2002) and LINGO-1 (Mi et al., 2004) interact to create a receptor complex that augments the activation of RhoA by p75NTR (Yamashita et al., 2002; Yamashita & Tohyama, 2003). The absence of the extracellular portion of p75NTR in hypomorphic mice is likely to render it incapable of NgR binding, accounting for the reduction of Rho activation observed in the spinal cord of p75–/– mice after injury (Dubreuil et al., 2003). The subsequent reduction in myelin-mediated RhoA activation would therefore be expected to contribute to the substantive degree of plasticity observed in the p75–/– spinal cord. Myelin-associated glycoprotein is known to inhibit neurite elongation through the NgR and p75NTR (Wong et al., 2002). Myelin-associated glycoprotein is also known to 99  suppress NT-mediated elevation of neuronal cAMP, inhibiting neurite outgrowth (Cai et al., 1999; Gao et al., 2003). Injecting a non-hydrolysable form of cAMP into the DRG increases regeneration in the spinal cord after dorsal column lesions (Qiu et al., 2002) and the phosphodiesterase inhibitor rolipram, whose effect is to elevate cAMP, enhances regeneration after spinal hemisection (Nikulina et al., 2004; Pearse et al., 2004). In p75-/mice, myelin-associated glycoprotein is unable to suppress cAMP, increasing the ability of axons to sprout (6 in Fig. 3.8). In summary, these results demonstrate that p75NTR plays a pivotal role in the modulation of collateral sprouting, both during development and after spinal cord injury. Intact p75-/- mice have a reduction in the number of primary afferent neurons and decreased monoaminergic innervation of the spinal dorsal horn. The density of monoaminergic axons and peptidergic primary afferent neurons in the spinal cord was increased ipsilateral to rhizotomy above that observed in wild-type mice. Where neurotrophic factor treatment increased the density of axons in wild-type mice, this response was further enhanced in p75-/- mice. The conclusion of the present work is that, while the full spectrum of p75NTR-mediated effects on axonal plasticity remains undefined, p75NTR determines the sprouting potential of intraspinal axons after dorsal rhizotomy. The underlying mechanism is consistent with the role of p75NTR in NT-Trk affinity and specificity and in transducing inhibitory myelin signals.  100  Fig. 3.1. Time-course of primary afferent axon [calcitonin gene-related peptide (CGRP)expressing] sprouting in the dorsal horn after rhizotomy in p75+⁄+ and p75–⁄– mice. CGRP immunoreactivity in intact p75+ ⁄ + (a) and p75– ⁄ – (b) mice. (c) The depth profile reveals a small but significant decrease in axon density in p75– ⁄ – compared with p75+ ⁄ + mice. CGRP-positive axons 7 days after septuple rhizotomy in p75+ ⁄ + (d) and p75– ⁄ – (e) mice. (f) Axon density is elevated in p75– ⁄ – mice compared with p75+ ⁄ + mice. CGRP-positive axons 28 days after septuple rhizotomy in p75+ ⁄ + (g) and p75– ⁄ – (h) mice. (i) Axon density remains elevated in p75– ⁄ – compared with p75+ ⁄ + mice. Depth profiles comparing axon density in intact mice and those which had received septuple rhizotomies 7 or 28 days earlier in p75+ ⁄ + (j) and p75– ⁄ – (k) mice. Scale bar: 100 µm.  101  102  Fig. 3.2. Effects of neurotrophins on primary afferent axon [calcitonin gene-related peptide (CGRP)-expressing] sprouting in the dorsal horn 7 days after rhizotomy in p75+ ⁄ + and p75– ⁄ – mice. CGRP immunoreactivity in vehicle-treated p75+ ⁄ + (a) and p75– ⁄ – (b) mice. (c) The depth profile reveals a small but significant increase in axon density in p75-/- compared with p75+ ⁄ + mice. CGRP-positive axons in nerve growth factor (NGF)treated p75+ ⁄ + (d) and p75– ⁄ – (e) mice. (f) Axon density is less in p75– ⁄ – than in p75+ ⁄ + mice. CGRP-positive axons in neurotrophin-3 (NT-3) -treated p75+ ⁄ + (g) and p75– ⁄ – (h) mice. (i) Axon density is significantly elevated in p75– ⁄ – compared with p75+ ⁄ + mice. Depth profiles comparing axon density in p75+ ⁄ + (j) and p75– ⁄ – (k) mice treated with vehicle, NGF or NT-3. Scale bar: 100 µm.  103  104  Fig. 3.3. Time-course of serotonergic axon [serotonin (5-HT)-expressing] sprouting in the dorsal horn after rhizotomy in p75+ ⁄ + and p75– ⁄ – mice. 5-HT immunoreactivity in intact p75+ ⁄ + (a) and p75– ⁄ – (b) mice. (c) The depth profile reveals a small but significant decrease in axon density in p75– ⁄ – compared with p75+ ⁄ + mice. 5-HT-positive axons 7 days after septuple rhizotomy in p75+ ⁄ + (d) and p75– ⁄ – (e) mice. (f) Axon density is the same in p75– ⁄ – and p75+ ⁄ + mice. 5-HT-positive axons 28 days after septuple rhizotomy in p75+ ⁄ + (g) and p75– ⁄ – (h) mice. (i) At this time-point, axon density elevated in p75– ⁄ – compared with p75+ ⁄ + mice. Depth profiles comparing axon density in intact mice and those which had received septuple rhizotomies 7 or 28 days earlier in p75+ ⁄ + (j) and p75– ⁄ –  (k) mice. Scale bar: 100 µm.  105  106  Fig. 3.4. Effects of neurotrophins on serotonergic axon [serotonin (5-HT)-expressing] sprouting in the dorsal horn 7 days after rhizotomy in p75+ ⁄ + and p75– ⁄ – mice. 5-HT immunoreactivity in vehicle-treated p75+ ⁄ + (a) and p75– ⁄ – (b) mice. (c) Axon density is the same in p75– ⁄ – and p75+ ⁄ + mice. 5-HT-positive axons in nerve growth factor (NGF)treated p75+ ⁄ + (d) and p75– ⁄ – (e) mice. (f) Axon density is greater in NGF-treated p75– ⁄ – than in p75+ ⁄ + mice. 5-HT-positive axons in NT-3-treated p75+ ⁄ + (g) and p75– ⁄ – (h) mice. (i) Axon density is significantly elevated in both genotypes after NT-3 treatment but is higher in p75– ⁄ – than in p75+ ⁄ + mice. Depth profiles comparing axon density in p75+ ⁄ + (j) and p75– ⁄ – (k) mice treated with vehicle, NGF or NT-3. Scale bar: 100 µm.  107  108  Fig. 3.5. Time-course of tyrosine hydroxylase (TH)-expressing axon sprouting in the dorsal horn after rhizotomy in p75+ ⁄ + and p75– ⁄ – mice. TH immunoreactivity in intact p75+ ⁄ + (a) and p75– ⁄ – (b) mice. (c) The depth profile reveals a small but significant decrease in axon density in p75– ⁄ – compared with p75+ ⁄ + mice. TH-positive axons 7 days after septuple rhizotomy in p75+ ⁄ + (d) and p75– ⁄ – (e) mice. (f) Axon density is elevated in p75– ⁄ – compared with p75+ ⁄ + mice. Calcitonin gene-related peptide-positive axons 28 days after septuple rhizotomy in p75+ ⁄ + (g) and p75– ⁄ – (h) mice. (i) Axon density increases in both genotypes but remains elevated in p75– ⁄ – compared with p75+ ⁄ + mice. Depth profiles comparing axon density in intact mice and those which had received septuple rhizotomies 7 or 28 days earlier in p75+ ⁄ + (j) and p75– ⁄ – (k) mice. Scale bar: 100 µm.  109  110  Fig. 3.6. Effects of neurotrophins on tyrosine hydroxylase (TH)-positive axonal sprouting in the dorsal horn 7 days after rhizotomy in p75+ ⁄ + and p75–⁄– mice. TH immunoreactivity in vehicle-treated p75+ ⁄ + (a) and p75– ⁄ – (b) mice. (c) Axon density is elevated in p75– ⁄ – compared with p75+ ⁄ + mice. Serotonin (5-HT)-positive axons in nerve growth factor (NGF)-treated p75+ ⁄ + (d) and p75– ⁄ – (e) mice. (f) Axon density is greater in NGF-treated p75– ⁄ – than in p75+ ⁄ + mice. 5-HT-positive axons in NT-3-treated p75+ ⁄ + (g) and p75– ⁄ – (h) mice. (i) Axon density is significantly elevated in both genotypes after NT-3 treatment but is higher in p75– ⁄ – than in p75+ ⁄ + mice. Depth profiles comparing axon density in p75+ ⁄ + (j) and p75– ⁄ – (k) mice treated with vehicle, NGF or NT-3. Scale bar: 100 µm.  111  112  Fig. 3.7. Summaries of deafferentation and neuro-trophin-mediated changes in axon density after septuple dorsal rhizotomy. *Significant differences between p75+⁄+ mice and p75-/- mice. † Significant differences from intact animals (first and third columns) or from vehicle-treated animals (second and fourth columns). Significance determined using a one-way anova, P < 0.05. CGRP, calcitonin gene-related peptide; 5-HT, serotonin; TH, tyrosine hydroxylase.  113  NTR  Fig. 3.8. Positive and negative influences on intraspinal plasticity mediated by p75  .  (1) The presence (top) or absence (bottom) of the neuro-trophin-binding domain of NTR  p75  determines tropomyosin-related kinase (Trk)-neurotrophin ligand specificities ⁄ NTR  affinities (specificities only are indicated). (2) In p75+ ⁄ + mice, p75  and Trks may  compete for ligand. 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Neurosci. Res. 74, 221–226. Zhou L, Baumgartner BJ, Hill-Felberg SJ, McGowen LR, Shine HD (2003) Neurotrophin-3 expressed in situ induces axonal plasticity in the adult injured spinal cord. J. Neurosci. 23, 1424–1431.  120  CHAPTER FOUR  Differential effects of endogenous neurotrophins on dorsal rhizotomy-induced dendritic plasticity reveal a novel interaction between NGF and BDNF signaling4  4  A version of this chapter has been submitted for publication. Scott, A.L.M and Ramer, M.S. Differential effects of endogenous neurotrophins on dorsal rhizotomy-induced dendritic plasticity reveal a novel interaction between NGF and BDNF signaling. 121  4.1 Introduction Following spinal cord injury (SCI), spontaneous recovery is largely correlated with the re-modeling of uninjured neuronal processes, rather than the regeneration of original projections. The relationship between spontaneous axonal sprouting and the return of function following SCI is well documented (Kaplan and Miller, 2000; Huang and Reichardt, 2003; Teng and Hempstead, 2004); however, less is known about modifications to the dendritic architecture. Reports of SCI-induced dendritic reorganization have been limited to spinal motoneurons (Gazula et al., 2004; Bose et al., 2005; Fenrich et al., 2007), and pyramidal neurons of the motor cortex (Kim et al., 2006; Kim et al., 2008). Outside the motor neuraxis, demonstration of sensory dendritic plasticity within the spinal cord comes from deafferentation studies (dorsal root injury). These highlight morphological changes in the spinal dorsal horn including those to spinocervical tract neurons (Smith, 1974; Brown et al., 1979; Sedivec et al., 1986) and in Clarke’s nucleus (Sugimoto and Gobel, 1984). Here, we use this model to further characterize injury-induced dendritic changes in a spinal neuropil that is also involved in sensory processing. Given the reported benefits of synaptic plasticity following CNS injury, many therapeutic approaches to SCI have been developed to maximize these spontaneous responses. Neurotrophins, including nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT4/5) and brain-derived neurotrophic factor (BDNF), belong to a class of proteins known to influence the growth of axonal (Cohen-Cory and Fraser, 1995) and dendritric processes (McAllister et al., 1995), and as such, are attractive candidates for therapy. The effects of BDNF, in particular, have been studied extensively in several  122  brain regions and include enhanced dendritic growth and differentiation of cortical, neostriatal, hippocampal and cerebellar neurons following injury (Lessmann et al., 2003). BDNF induces these morphological changes through several modes of action, including neuronal depolarization (Jin et al., 2003), activation of secondary effectors (Dijkhuizen and Ghosh, 2005), and destabilization of dendritic arbors and spines (Horch et al., 1999). BDNF-induced changes to dendritic organization are mediated by its interaction with its high affinity receptor, tropomyosin-related tyrosine kinase receptor B (TrkB), or the pan neurotrophin receptor, p75NTR (Yacoubian and Lo, 2000). During early dendritic formation, both TrkB and p75NTR are involved in the elongation and branching of dendritic processes (Gascon et al., 2005). However, at later stages, the growth-promoting role of p75NTR switches to that of an inhibitor reducing both the length and complexity of mature dendritic trees in the hippocampus (Zagrebelsky et al., 2005). One proposed mechanism for p75NTR-mediated growth inhibition is the role of p75NTR as a co-receptor for myelin-associated inhibitory proteins (Wang et al., 2002a). This interaction results in the activation of RhoA, a small GTPase that stiffens the cytoskeleton and leads to inhibition of neurite growth (Yamashita et al., 1999; Yamashita and Tohyama, 2003). Taken together, these findings point to p75NTR as a limiting factor to compensatory dendritic growth within the spinal cord following injury. In this study, we examined dendritic plasticity within the adult spinal cord following dorsal root injury, evaluated its dependency on neurotrophin signaling, and determined the relative influence of p75NTR. We conclude that injury-induced dendritic sprouting is restricted in the spinal cord, but this restriction is overcome with the application of exogenous BDNF. Surprisingly, p75NTR appears to have a minimal  123  inhibitory role on dendritic plasticity, which suggests that another molecule is responsible for limiting endogenous BDNF signaling. We propose that the truncated form of the TrkB receptor, TrkBT1, is responsible, and demonstrate a compelling correlation between its expression and the inhibition of dendritic sprouting. Interestingly, we also demonstrate that endogenous NGF prevents dendritic sprouting, and may do so by regulating TrkBT1 expression.  4.2 Materials and methods Genotyping. We used the p75 (exon III) hypomorphic mouse (p75-/-), which does not possess the extracellular binding domain for neurotrophic factors. The mice were offspring of the commercially available p75+/- breeders (Ngfrtm1Jae, version 1; Jackson Laboratory/ JAX Mice; Bar Harbor, Maine), and genotyped in triplicate. Age-matched, 824 weeks, C57Bl/6 wild-type littermates (p75+/+) were used as controls. Each sample of DNA was extracted from the pinna and prepared for polymerase chain reaction with a REDExtract-N-Amp Tissue PCR kit (Sigma-Aldrich, St. Louis, MO). Primer sequences to identify a neo generic sequence and Ngfr sequence (oIMR0013, oIMR0014, oIMR0710, oIMR0711; JAX® Mice, Bar Habour, ME) were produced by Integrated DNA technologies (Coralville, IA, USA). The PCR products were separated by electrophoresis on a 2% agarose gel and visualized with Ethidium Bromide under Ultraviolet light exposure.  Surgical Procedures. All surgical procedures were performed under the guidelines of the University of British Columbia Animal Care Committee and the Canadian Council of  124  Animal Care. Adult p75+/+ or p75-/- mice, ranging in age from 8-24 weeks, were anesthetized with a mixture of ketamine hydrochloride (80-100 mg/kg, i.p.; BimedaMTC; Cambridge, ON) and xylazine hydrochloride (5-10 mg/kg, i.p.; Bayer Inc; Etobicoke, ON), and administered a dose of buprenorphine (0.08 mg/kg, i.m.) for pain control. Each animal underwent a unilateral dorsal laminectomy, which exposed the dorsolateral cervical spinal segments C4 – T2. Within this region the dura mater was opened and all seven dorsal roots were repeatedly crushed with fine forceps (#5), as previously described (Scott et al., 2005). Following surgery, the animals were monitored daily for clinical signs of distress/pain and maintenance of body weight. Soluble BDNF (100 µg/ml; a generous gift from Regeneron via Prof. Wolfram Tetzlaff) or tropomyosin-related receptor kinase (Trk) fragments, TrkA-Fc, TrkB-Fc or TrkC-Fc (1 mg/ml; Neuromics, Edina, MN) were topically applied to the surface of the spinal cord in a fibrin glue matrix (3 µl) immediately following rhizotomy. Fibrin glue was prepared as a combination of equal parts thrombin (25U/ml in 45mM CaCl2), fibrinogin (100mg/ml in distilled water) and fibronectin (8mg/ml in distilled water) (all from Sigma; Saint Louis, Missouri), as described by (Iwaya et al., 1999). Both p75+/+ and p75-/- mice were included in each treatment group: fibrin glue alone (p75+/+, n = 5; p75-/-, n = 4), BDNF (p75+/+, n = 4; p75-/-, n = 6), or TrkA-Fc (p75+/+, n = 4; p75-/-, n = 4), TrkBFc (p75+/+, n = 5; p75-/-, n = 4), or TrkC-Fc (p75+/+, n = 4; p75-/-, n = 6).  Immunohistochemistry. Seven days post-injury the animals were given an interperitoneal injection of chloral hydrate (100 mg/kg) and perfused with 4% paraformaldehyde, and spinal cords (from C6 to T1) were dissected out and postfixed overnight. Spinal cords  125  were cryoprotected in 20% sucrose in 0.1 M phosphate buffer in 4°C for 24 hours, then stored at -80°C. Transverse cryosections (16 µm) of the spinal cords were incubated for one hour in 10% normal donkey serum (in 0.1 M PBS, 0.2% Triton X-100, and 0.1% sodium azide), and overnight with primary antibodies including: mitogen-associated peptide-2 (MAP2; 1:5000, host chicken; Abcam, Cambridge, MA); serotonin-transporter (SERT; 1:1200, host rabbit; Immunostar, Hudson, WI); nerve growth factor (NGF; 1:1000, host goat; Neuromics, Edina, MN); truncated TrkB (TrkBT1 (c13); 1:200, host rabbit; Santa Cruz Biotechnology Inc., Santa Cruz, CA); and p75NTR (1:500; host goat; Neuromics). Sections were then washed in PBS and incubated for 2 hours in secondary antibodies conjugated to either cyanine (CyTM3; 1:200; Jackson ImmunoResearch; West Grove, PA) or Alexa 488 (1:400; Molecular Probes; Eugene, OR). Following three 15minute washes in PBS, Immunomount (Fisher Scientific; Pittsburgh, PA) was applied to the slides for coverslipping.  Image Analysis. Three random images of each spinal dorsal horn segment were visualized with an Axioplan 2 microscope (Zeiss, Jena, Germany), and captured in grayscale with a digital camera (Q Imaging, Burnaby, BC), and Northern Eclipse software (Empix Imaging Inc., Mississauga, ON). All settings for image capture, including exposure time, were kept constant across all sections for each fluorochrome. Each picture was converted to a monochrome image with Sigma Scan Pro 4 software (SPSS; Chicago, IL), using a threshold manually set for fiber detection with each immuno-label. A line detection filter, Laplace Version 2, was used to identify only neuronal processes and eliminate cell bodies from density measurements. Fiber densities  126  were calculated within the dorsal horn and lateral spinal columns of four spinal segments (C6-T1) for each animal. Dorsal horn measurements were taken from a 20,000 µm2 area within the dorsal horn. Each pixel density within these areas were averaged across the dorsal horn and all spinal segments in each animal, and expressed as a density of SERTpositive axons. In the white matter, the density of MAP2-labelled dendrites was measured in a similar manner and determined for the ipsilateral lateral spinal nucleus (LSN). This area was easily defined within the spinal cord and chosen because of its involvement in normal sensory processing (these neurons lose synaptic input from primary afferents following deafferentation). Due to the high dendritic density within the grey matter, the analysis of dendritic changes was limited to the white matter. The immunoreactivity of the TrkBT1 receptor was also measured in this way. However, due to the diffuse expression of this receptor, the grey scale digital images were not subjected to a line detection filter.  Western Blot. The spinal cords of p75+/+ and p75-/- adult mice were freshly dissected out one week following dorsal rhizotomy. The spinal cords were separated into the ipsilateral and contralateral sides, and the dorsal and ventral roots were removed. The tissue from the ipsilateral spinal cord was then homogenized in 0.01M Tris(hydroxymethyl)aminomethane with protease inhibitors (Gibco). Protein concentration was determined with BCA protein tritration, and 30µg of protein per lane were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred overnight to a PVDF transfer membrane (Amersham Corp, Arlington Heights, IL). The protein extract from each individual spinal cord of each genotype  127  (p75+/+, n=3; p75-/-, n=3) was run in separate lanes. The membranes were then blocked in 5% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO), and incubated overnight with anti-TrkB (1:500; host rabbit), which binds to the extracellular portion of the receptor (h-181), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:500, host mouse) (both from Santa Cruz Biotechnology Inc., Santa Cruz, CA) as a loading control. The membranes were then washed in 0.05M Tris, 0.15M NaCl, and 0.1% Tween 20, and incubated with affinity purified rabbit or mouse horseradish peroxidaseconjugated secondary antibodies (1:5000; Jackson Immunoresearch Laboratories Inc., West Grove, PA) for 2 hours at room temperature. The membranes were washed again, and treated for 15 minutes with the horseradish peroxidase substrate, 3,3’,5,5’tetramethylbenzidine (TMB; Promega, Madison, WI), for colorimetric detection of the protein. The reagent was then removed and the membrane was washed in distilled water and scanned. Protein density was calculated from digital images with Sigma Scan Pro 4 Software (Chicago, IL), and normalized to both the pixel density of the background exposure and the loading control. Protein concentrations extracted from rhizotomized spinal cords were expressed as a proprtional change in concentration compared to the naïve spinal cords of each genotype.  Statistics. All statistical analyses within were performed with One-Way ANOVAs with pairwise multiple comparisons (Holm Sidak method) (significance set at P < 0.05). Error bars are indicative of the standard error of the mean. In each figure the phi symbol (φ) signifies significant differences between p75+/+ and p75-/- groups; and the cross (+) was used to indicate significant differences between the vehicle and treatment groups.  128  4.3 Results 4.3.1 Exogenous BDNF induces sprouting of intraspinal dendritic processes The lateral spinal nucleus (LSN) is located within the lateral columns of the spinal cord, adjacent to the superficial dorsal horn (Fig. 4.1a, top left). Neurons within the LSN are innervated directly by primary afferents from the dorsal horn (Menetrey et al., 1980). Removal of these connections by dorsal rhizotomy did not generate dendritic sprouting within the LSN in the ipsilateral side of the spinal cord of p75+/+ mice (Fig. 4.1a). Although we cannot assess subtle changes to the dendritic morphology with this analysis, these data suggest that compensatory sprouting may be inhibited within the deafferented spinal cord. In the postnatal CNS, dendritic processes in many regions have been shown to have a preferential responsiveness to BDNF over NGF or NT-3 (McAllister et al., 1997; Horch et al., 1999). Given this, we hypothesized that with the administration of BDNF we could induce dendritic sprouting within the spinal cord. Significant dendritic sprouting occurred in the LSN of wild-type mice: in response to exogenous BDNF, dendritic process density increased more than two-fold (Fig. 4.1a). This finding indicates that spinal dendritic processes retain their responsiveness to BDNF in adulthood. Since endogenous BDNF expression is upregulated in the deafferented dorsal horn (Johnson et al., 2000; Ramer et al., 2007), dendritic BDNF-dependent sprouting must be actively restricted.  129  4.3.2 P75NTR does not account for the inhibition of dendritic plasticity following injury In the spinal cord, p75NTR expression is present primarily in the grey matter in uninjured mice, due to the relatively high expression of this receptor in primary afferents (Wright and Snider, 1995). Following deafferentation, p75NTR was reduced in the grey matter, but upregulated in the peripheral dorsal root, and to a lesser extent, the white matter, including the LSN (Fig. 5.1b, top left). Upregulation of p75NTR in these areas following injury has been shown to occur almost exclusively in glia (King et al., 2000; Syroid et al., 2000). Given that p75NTR has been implicated in inhibitory myelin signaling (Wang et al., 2002a), and restricting deafferentation-induced axonal sprouting (Scott et al., 2005), we asked whether p75NTR influenced dendritic sprouting. Dendritic densities in the LSN of p75-/- mice were equivalent to p75+/+ mice in both the uninjured state and following rhizotomy (Fig. 4.1b). Therefore, p75NTR expression cannot account for the restriction to dendritic plasticity in the deafferented spinal cord. Dendritic processes in adult p75-/- mice were also responsive to exogenous BDNF treatment (Fig. 4.1b). In fact, MAP2-positive dendritic density was more than doubled in these mice with BDNF treatment (Fig. 4.1b), equivalent to what was observed in p75+/+ mice. We can therefore conclude that signaling via p75NTR (either in response to BDNF or myelin-associated proteins) does not have significant effects on dendritic sprouting in this model.  130  4.3.3 The expression of TrkBT1 is increased in the deafferented spinal cord The expression of the truncated form of the TrkB receptor (TrkBT1) increases following SCI (King et al., 2000), and has been shown to interfere with BDNF-mediated neuronal growth (Biffo et al., 1995; Fryer et al., 1997). We therefore hypothesized that TrkBT1 may suppress plasticity of dendrites in response to deafferentation-induced BDNF upregulation (Johnson et al., 2000; Ramer et al., 2007). The expression of the 95kDa TrkBT1 receptor was significantly increased in the spinal cord following rhizotomy (Fig. 4.2a). Protein concentrations of TrkBT1 were similar in p75+/+ and p75-/mice, suggesting that p75NTR is not required for the regulation of TrkB expression. To determine whether smaller changes in TrkBT1 between genotypes might have been diluted out in the Western blot analysis of entire hemicord homogenates, we also determined the localization of TrkBT1 immunohistochemically, and analyzed its density in the LSN. Interestingly, there was higher expression of TrkBT1 in the LSN of p75-/mice (Fig. 4.2b), indicative of potential compensatory adaptations in this knockout previously shown for other Trk receptors (Hannila et al., 2004).  4.3.4. Endogenous NGF regulates the expression of the truncated TrkB receptor In order to test the influence of TrkBT1 expression on dendritic sprouting, we sought a method to prevent its upregulation. It has been previously shown that NGF positively regulates the expression of TrkBT1 (Kumar et al., 1993), so we examined the relationship between NGF and TrkBT1 in our model system. Under normal conditions, levels of NGF in the spinal cord of adult mice are low, and restricted to the intermediate grey matter (Fig. 4.5a). Following transection or clip  131  compression injuries to the spinal cord, NGF expression is significantly increased in neuronal and non-neuronal cell types (Brown et al., 2004). Accordingly, we observed increases in NGF immunoreactivity following deafferentation, presenting as a punctate distribution within the ipsilateral grey matter, and more diffusely in white matter (Fig. 4.5b). The pre- and post-injury expression pattern was similar in p75-/- mice (Fig. 4.5b). Inhibition of endogenous NGF via TrkA-Fc treatment resulted in dramatic reductions in TrkBT1 expression in both p75+/+ and p75-/- mice (Fig. 4.2b). These findings suggest that NGF plays a regulatory role in the expression of TrkBT1 following injury, and antagonism of this neurotrophin prevents the upregulation of TrkBT1 in the LSN. Expression of TrkBT1 is increased in astrocytes following spinal cord injury (Widenfalk et al., 2001). Dorsal rhizotomy is known to induce proliferation and migration of astrocytes in the spinal cord (Kozlova, 2003), which may also contribute to the increase of TrkBT1 expression.  4.3.5 Endogenous NGF limits dendritic sprouting in the lateral spinal nucleus Removal of NGF by TrkA-Fc enhanced dendritic sprouting in the ipsilateral LSN of p75+/+ mice (Fig. 4.3a) when compared to vehicle (Fig. 4.3b, solid line), strikingly reminiscent of BDNF treatment. This suggests that the prevention of NGF signaling, and the subsequent upregulation of TrkBT1, disinhibited dendritic plasticity within the spinal cord following deafferentation. TrkA-Fc treatment similarly enhanced dendritic sprouting in p75-/- mice (Fig. 4.3b). In light of the responsiveness of spinal dendrites to exogenous BDNF, and the fact that BDNF is upregulated following spinal deafferentation (Johnson et al., 2000; Ramer  132  et al., 2007) we wanted to determine the role of endogenous BDNF in the maintanence and stability of dendritic density following dorsal root injury. TrkB-Fc had no effect on dendritic sprouting in either genotype (Fig. 4.3b), suggesting that endogenous BDNF may already be sequestered in the injured spinal cord by TrkBT1. In addition to BDNF, NT-3 is also widely expressed throughout the CNS (Friedman et al., 1995), and has been shown to influence dendritic growth during development through its high affinity receptor, TrkC (McAllister et al., 1995). In contrast to what was observed with TrkB-Fc treatment, administration of TrkC-Fc resulted in modest increases in dendritic sprouting within the LSN of p75+/+ mice (Fig. 4.3). There was no difference in dendritic density between p75-/- mice treated with vehicle or TrkCFc (Fig. 4.3).  4.3.6 Deafferentation-induced serotonergic sprouting is enhanced by TrkA-Fc Dorsal root injury induces sprouting of serotonergic axons in rats (Ramer et al., 2007) and mice (Scott et al., 2005). We have recently shown that serotonergic sprouting is prevented with TrkB-Fc treatment, and thus is a BDNF-dependent consequence of deafferentation (Ramer et al., 2007). If TrkBT1 restricts BDNF availability, then we reasoned that TrkA-Fc treatment (which prevents its upregulation) would increase the density of serotonergic axons in the deafferented spinal cord. Indeed, the density of axonal processes positive for the serotonin transporter (SERT) roughly tripled in both p75+/+ and p75-/- mice (Fig. 4.4). These data provide strong evidence for a negative interaction between NGF and BDNF signaling in the deafferented spinal cord.  133  4.4 Discussion The focus on injury-induced plasticity in the CNS has been mainly restricted to axons. Any subsequent change in function, whether beneficial or detrimental, necessarily relies not only on the ability of axons to transmit signals, but also on the ability of target neurons to receive and integrate those signals. Therefore, examination of changes in dendritic structure, and eventually of their interaction with sprouting axons, is necessary in order to understand the relationship between structure and function in the damaged CNS. Here we provide evidence for neurotrophin-mediated dendritic sprouting following spinal deafferentation, and further demonstrate a novel interplay between endogenous NGF that acts to restrict plasticity via TrkBT1, and endogenous BDNF that acts to stimulate it.  4.4.1 Injury-induced dendritic plasticity in the CNS The structural plasticity of dendritic processes has been primarily examined in cortical injury models and is induced by several experimental manipulations including ischemia (Biernaskie and Corbett, 2001), seizures (Shapiro et al., 2007) direct trauma (Jones and Schallert, 1992) and deafferentation (Jones and Thomas, 1962). Injuryinduced dendritic changes in the spinal cord have been noted, but limited mainly to those of spinal motoneurons. In response to SCI, these changes include an elongation of primary dendrites, an increase in dendritic diameter (Bose et al., 2005), a reduction in distal branch number (Gazula et al., 2004), and the formation of new axons from their distal ends (Fenrich et al., 2007).  134  Following spinal deafferentation changes have been described for other populations of intraspinal dendrites, the foremost being those in the spinocervical nucleus (SCT) within the dorsal horn. SCT neurons exhibit enhanced dendritic diameters, and modest increases in dendritic branching and expansion into distal layers of the dorsal horn (Brown, 1983; Sedivec et al., 1986). In our study, the neuronal population examined is also involved with sensory processing. Although we did not examine subtle changes in diameter, length, or branching that might have occurred, we did not observe any changes to dendritic density following the loss of sensory input in wild-type or p75-/- mice. These results are supported by similar findings of stable MAP2 density in the mouse LSN following deafferentation (Hampton et al., 2007).  4.4.2 Regulation of dendritic plasticity by neurotrophins The interplay between growth-promoting and growth-inhibiting factors determines the degree of intraspinal plasticity in response to injury. Neurotrophins stimulate growth by initiating several intracellular signaling cascades via their respective Trk receptors (Kaplan and Miller, 2000). The extracellular immunoglobulin-like domains of Trk receptors dictate the receptors’ specificities, such that NGF preferentially binds to TrkA, NT-3 to TrkC, and BDNF and NT4/5 to TrkB. Within the developing and adult CNS, the regulation of dendritic growth and maintenance by endogenous neurotrophins largely depends on the differential expression patterns of both neurotrophins and Trk receptors. For example, NGF expression is restricted to specific regions within the CNS (Maisonpierre et al., 1990), and has a limited influence on dendritic growth that is confined to these regions (McAllister et al., 1995; Salama-Cohen et al., 2005). On the  135  other hand, NT-3 is the most widely and abundantly expressed neurotrophin during early development, but is downregulated over time and replaced by BDNF (Maisonpierre et al., 1990). While dendrites retain some responsiveness to NT-3, BDNF principally contributes to the later phase of dendritic growth and maturation via TrkB (Gascon et al., 2005; Gao et al., 2009). Accordingly, dendrites within the adult spinal cord also retained BDNF-responsiveness and responded significantly to exogenous BDNF treatment. The increase in sprouting was equivalent in p75-/- animals, suggesting that p75-/- neurons are just as responsive to BDNF, and there is no influence of p75NTR in BDNF-mediated dendritic sprouting. Neurotrophins are recognized as having differential effects on dendritic plasticity, and can act in opposition to promote or restrict growth. In the cortex, for example, blocking endogenous NT-3 with TrkC-Fc enhanced dendritic growth in layer IV but prevented it in layer VI. In contrast, TrkB-Fc promoted dendritic sprouting in layer VI, yet inhibited it in layer IV (McAllister et al., 1995; McAllister et al., 1997). Thus, endogenous NT-3 promotes dendritic growth in layer VI, while endogenous BDNF inhibits it, and the opposite is true for dendritic growth in layer IV. We also observed differential responses of dendritic processes within the mature spinal cord to BDNF and NT-3, such that exogenous BDNF substantially enhanced dendritic sprouting and endogenous NT-3 acted to limit it.  4.4.3 TrkB isoforms and dendritic sprouting in the deafferented spinal cord The equivalent response of intraspinal dendrites to exogenous BDNF in p75+/+ and p75-/- mice implied that growth was not mediated by p75NTR, but instead by TrkB. In  136  the CNS, the TrkB receptor is present in multiple isoforms, one containing the intracellular kinase domain (TrkB) and 3 isoforms lacking intracellular domains (TrkBT1, TrkBT2 and TrkBT4), all of which bind BDNF with high affinity and selectivity (Biffo et al., 1995). In the adult spinal cord TrkBT1 is the dominant isoform and is expressed in both neurons and glial cells, while TrkB is expressed at relatively low levels and restricted to neuronal processes (Ernfors et al., 1993; Liebl et al., 2001). Following SCI, the expression of TrkB remains unchanged, but TrkBT1 is massively upregulated by three weeks post-injury in non-neuronal cell types, such as astrocytes and leptomeningeal cells (Frisen et al., 1992; Frisen et al., 1993; King et al., 2000; Liebl et al., 2001). Here, following spinal deafferentation, TrkBT1 was also upregulated in the spinal cord (Foschini et al., 1994). The lack of a kinase domain presumably limits the direct contribution of TrkBT1 to BDNF signaling on neurons, but this receptor still affects BDNF signaling indirectly. When co-expressed with TrkB in neurons, TrkBT1 down-regulates the cell surface expression of TrkB, and prevents its autophosphorylation by BDNF (Haapasalo et al., 2001; Haapasalo et al., 2002). Additionally, TrkBT1 on non-neuronal cells can inhibit BDNF-induced neurite outgrowth. Binding of BDNF to TrkBT1 results in a rapid internalization of the ligand and a reduction in its availability to growing neurites (Fryer et al., 1997). This TrkBT1-mediated inhibition is only overcome by high concentrations of BDNF (Biffo et al., 1995). Given the elevated expression level of TrkBT1 at the astrocytic barrier following SCI, TrkBT1 is believed to contribute to failed axonal regeneration around the lesion by competing for available BDNF (King et al., 2000; Liebl et al., 2001).  137  One factor shown to regulate the expression of TrkBT1 is NGF. Treatment of cultured astrocytes with NGF results in significant increases in the transcription rate of TrkBT1 mRNA (Kumar et al., 1993), an effect that must be mediated by its interaction with the TrkA receptor given our findings in p75-/- mice. Although the presence of TrkA in astrocytes has been debated, TrkA expression in astrocytes reportedly occurs in the hippocampus (McCarthy et al., 2002; Tonchev et al., 2008) and in the spinal cord following experimental autoimmune encephalomyelitis (Oderfeld-Nowak et al., 2001) and deafferentation (Foschini et al., 1994). From studies of SCI, we know that NGF expression is upregulated in the injured spinal cord in non-neuronal cells, including microglia, astrocytes, and leptomeningial cells (Krenz and Weaver, 2000; Brown et al., 2004). Here, we have shown that following deafferentation NGF is also upregulated in the intermediate grey matter, as well as diffusely throughout the white matter. We have also demonstrated that antagonizing endogenous NGF prevents TrkBT1 upregulation, and that this reduction of TrkBT1 expression correlated with the dendritic response to TrkA-Fc in the LSN.  4.4.4 P75NTR does not suppress dendritic plasticity following spinal deafferentation Given the inhibitory influence of p75NTR on sprouting of axons in the deafferented spinal cord (Hannila and Kawaja, 2005; Scott et al., 2005), we were surprised to find that p75NTR did not inhibit dendritic sprouting. In a previous study, an inhibitory influence of p75NTR on dendritic growth was demonstrated in the hippocampus. Zagrebelsky and colleagues (2005) showed that hippocampal neurons in both complete (exon IV) and hypomorphic (exon III) p75-/- mice had greater dendritic complexity (branching and spine  138  densities) compared to neurons in wild-type animals. They also demonstrated that conditional overexpression of p75NTR in postnatal neurons led to the reduction of apical dendritic branches and number of dendritic spines, suggesting that p75NTR can act acutely to restrict dendritic growth. Here we find that rhizotomy does not induce sprouting of dendritic processes in the absence of p75NTR.  4.5 Conclusions Although classically thought of as growth-promoting, neurotrophins can have a detrimental effect on growth and promote the development of undesirable consequences. For example, endogenous NGF induces the growth of pain-sensing afferents as well as the onset of autonomic dysreflexia following spinal cord injury (Krenz et al., 1999; Krenz and Weaver, 2000; Marsh et al., 2002). Endogenous NGF also restricts the growth of dendritic processes in the LSN following spinal deafferentation (present results), and preventing NGF signaling is likely to alter transmission of nociceptive signals to the brain. Precisely how this affects behavioural function following rhizotomy is difficult to predict from anatomical studies alone, but depends on the use made of the additional dendritic membrane by primary afferents, intrinsic interneurons, and descending painmodulating systems. As such, the robust effect of NGF antagonism on serotonergic axon sprouting, along with its inhibitory effect on peptidergic nociceptor sprouting, may well provide an anatomical substrate for an effective therapy following spinal cord injury.  139  Fig. 4.1. Dendritic density in the lateral spinal nucleus (lsn), outlined by dotted line (a, top left panel). Density of MAP2-positive fibers in p75+/+ mice was not significantly increased following rhizotomy, but was dramatically enhanced in the LSN of rhizotomized p75+/+ mice with BDNF treatment (a). p75NTR is diffusely expressed in the LSN following dorsal rhizotomy (b, top left panel). In the absence of p75NTR (p75-/mice), the dendritic density of MAP2-positive fibers were equivalent to p75+/+ values following rhizotomy, and significantly enhanced with BDNF treatment (b). Cross (+) denotes significant differences from vehicle treatment. Error bars: +/- SEM; Scale bars: 25 µm.  140  141  Fig. 4.2. Injury-induced TrkBT1 (95 kDa) expression was significantly upregulated 1.5 fold following injury. All injured samples were normalized to uninjured levels in both p75+/+ and p75-/- mice (a). TrkA-Fc treatment inhibited TrkBT1 expression of both genotypes in the lateral spinal nucleus (LSN; b). TrkBT1 expression was higher in p75-/(b). Cross (+) denotes significant differences from vehicle treatment; phi (φ) denotes significant differences from wild-type. Error bars: +/- SEM; Scale bar: 25 µm.  142  143  Fig. 4.3. TrkA-Fc treatment (a, top panels) substantially promoted the increase of MAP2positive dendrite density in both p75+/+ and p75-/- mice following injury. TrkB-Fc treatment did not change dendritic density within the LSN of p75+/+ or p75-/- mice (a, middle panels), while TrkC-Fc treatment (a, bottom panels) promoted sprouting in only p75+/+ mice. Dendritic densities are shown in relation to mean vehicle levels (solid line +/- SEM, dotted lines). Cross (+) denotes significant differences from vehicle treatment. Error bars: +/- SEM; Scale bar: 25 µm.  144  145  Fig. 4.4. Deafferentation-induced serotonergic sprouting is enhanced by TrkA-Fc treatment. In both p75+/+ (a) and p75-/- (b) mice, the density of SERT-positive axons was significantly increased (+) in TrkA-Fc-treated animals than in vehicle-treated animals. Micrographs are inverted and taken from the region indicated (*) in the schematic. Error bars: +/- SEM; Scale bar: 25 µm.  146  147  Figure 4.5. Nerve growth factor (NGF) expression is upregulated following spinal deafferentation. Injury-induced increases in NGF expression occured in the white matter and the intermediate laminae in both p75+/+ and p75-/- mice. Scale bar: 50 µm. This figure is a supplemental figure in the manuscript.  148  4.6 Bibiography Biernaskie J, Corbett D (2001) Enriched rehabilitative training promotes improved forelimb motor function and enhanced dendritic growth after focal ischemic injury. J Neurosci 21:5272-5280. Biffo S, Offenhauser N, Carter BD, Barde YA (1995) Selective binding and internalisation by truncated receptors restrict the availability of BDNF during development. 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Nat Neurosci 6:461-467. Yamashita T, Tucker KL, Barde YA (1999) Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth. Neuron 24:585-593. Zagrebelsky M, Holz A, Dechant G, Barde YA, Bonhoeffer T, Korte M (2005) The p75 neurotrophin receptor negatively modulates dendrite complexity and spine density in hippocampal neurons. J Neurosci 25:9989-9999.  152  CHAPTER FIVE  Discussion  153  5.1 Summary of thesis P75NTR has been implicated in many effects depending on its ligand or co-receptor interactions including the prevention of axonal growth by intracellular factors (Rho-GDI and Sall2), the alteration of neurotrophin-Trk binding properties, the reduction of neurotrophin availability by glial p75NTR, the interaction with myelin-associated inhibitory proteins, and secondary damage due to apoptosis. Taken together with the results presented here, many of these roles of p75NTR are incompatible with axonal regeneration and compensatory sprouting within the injured spinal cord. Although I could not address the influences of all the possible interactions, I was able to determine some of the primary contributors to p75NTR-mediated inhibition of axonal regeneration and sprouting in the CNS following rhizotomy. Following dorsal rhizotomy, sensory axons normally fail to regenerate across the DREZ. In Chapter 3, I demonstrated that in the absence of p75NTR, axonal regeneration across the DREZ is dis-inhibited and is followed by functional recovery. By using neuronal/Schwann cell co-cultures in vitro, complementary transplantation studies in vivo, and pharmacological reagents, I was able to conclude that successful axonal regeneration across the DREZ in p75-/- mice was neurotrophin-dependent and specifically reliant on the removal of p75NTR from Schwann cells. Following dorsal rhizotomy, spinal deafferentation is also associated with spontaneous intraspinal sprouting of specific neural populations. Treatments that maximize this spontaneous response may help to compensate for the absence of regeneration and re-establish lost connections. In Chapter 4, I discovered that the spontaneous sprouting of spared primary afferents and bulbospinal projections was  154  significantly greater in p75-/- mice than in p75+/+ mice following dorsal rhizotomy. This difference was apparent within one week following rhizotomy, as well as during periods of significant myelin degeneration (4 weeks post-injury). Exogenous neurotrophins also increased the sprouting of these populations within the spinal cord, but when applied in combination with p75NTR antagonism, sprouting was enhanced beyond either manipulation alone. These data suggested that increasing neurotrophin availability as well as preventing p75NTR signaling is an effective way to increase intraspinal axonal sprouting. Finally, in Chapter 5, I examined whether dendritic plasticity was influenced by either injury or neurotrophin availability in the spinal cord following dorsal rhizotomy. My findings showed that dendritic density does not change spontaneously following rhizotomy, but can be increased by the administration of exogenous BDNF. So, like axons in the adult spinal cord, dendrites retain neurotrophin responsiveness. However, unlike axonal sprouting, dendritic sprouting was prevented in both p75-/- and p75+/+ mice. This suggested that neurotrophin-mediated dendritic growth is limited by another factor following rhizotomy. TrkBT1, similar to p75NTR, is upregulated following CNS injury and has been shown to interfere with neurotrophin-mediated signaling. In subsequent experiments, I showed that a reduction of TrkBT1 expression in the spinal cord following dorsal rhizotomy was correlated with significant increases in dendritic density. Given this, I proposed that the expression of TrkBT1 limits BDNF-mediated dendritic sprouting following dorsal rhizotomy.  155  5.2 The multifaceted role of p75NTR in axonal regeneration 5.2.1 Glial p75NTR inhibits neurotrophin-mediated regeneration across DREZ Previous work has clearly demonstrated the capacity of exogenous neurotrophins to promote functional regeneration of sensory axons across the DREZ following dorsal rhizotomy (Ramer et al., 2000; Ramer et al., 2001). Following dorsal rhizotomy, degenerating Schwann cells proliferate within the dorsal root and upregulate RAGs including neurotrophins (Funakoshi et al., 1993; Soilu-Hanninen et al., 1999). However, the Schwann cells also significantly upregulate p75NTR expression (Syroid et al., 2000; Provenzano et al., 2008). Given the successful axonal regeneration across the DREZ in p75-/- mice, but not in p75+/+ mice, I hypothesized that endogenous p75NTR interfered with this local supply of neurotrophins and prevented neurotrophin-mediated re-growth. The prevention of spontaneous regeneration in p75-/- mice following the reduction of neurotrophin availability via Trk-Fc confirmed that regeneration in these mice is neurotrophin-dependent and normally inhibited by p75NTR. Earlier models of peripheral nerve regeneration postulated that the upregulation of Schwann cell p75NTR contributed to axonal regeneration by ‘presenting’ neurotrophins to Trk receptors on regenerating axons (Johnson et al., 1988). In order to effectively ‘present’ neurotrophins to Trk receptors on axons, glial-expressed p75NTR would have to either interact directly with the receptor to alter the binding characteristics of Trk receptors or change the conformation of neurotrophins in a way that would enhance its association to Trks. Although these receptors may interact intracellularly receptors (Jung et al., 2003; Bibel et al., 1999), direct interaction between Trks and p75NTR does not occur between the extracellular domains (Wehrman et al., 2007). Additionally, Trks and p75NTR  156  both bind with a 2:2 stiochiometry, and have mutually exclusive binding sites on neurotrophins with opposite orientations (Gong et al., 2008). Together, these suggest that extracellular p75NTR does not enhance neurotrophin binding to Trks when the receptors are expressed on different cells, but rather competes with extracellular Trks for available ligands. Observations of enhanced neurite outgrowth in vitro and axonal regeneration in vivo in the presence of p75-/- Schwann cells compared to the reduction in axonal growth among p75+/+ Schwann cells supports this assertion.  5.2.2 Axonal p75NTR promotes Trk-mediated axonal regeneration In contrast to the glial expression of p75NTR, it is likely that neuronal p75NTR assists rather than interferes with neurotrophin-Trk signaling. Intracellular p75NTR interaction with Trk receptors creates higher affinity binding sites for neurotrophins (Mahadeo et al., 1994; Esposito et al., 2001). Moreover, in the absence of p75NTR, association rates of neurotrophins to Trk receptors are decreased and neurons are less sensitive to neurotrophins (Mahadeo et al., 1994). Following injury in the PNS, neurotrophins are not needed for spontaneous regeneration to occur (Diamond et al., 1987; Diamond et al., 1992), however, they do increase regenerative sprouting and the extent of recovery (Verge et al., 1996; Bregman et al., 1997; Weidner et al., 1999; Bregman et al., 2002; English et al., 2005). Thus, the lack of p75NTR expression by axons may reduce Trk-mediated regeneration when neurotrophins are readily available. This difference in neurotrophin-sensitivity in p75-/- mice may also be magnified by the large reductions in the expression of all Trk receptors following the disconnection of DRG neurons from their peripheral targets (Bergman et al., 1996). In line with this, neurite  157  outgrowth (both distance and branching) on p75-/- Schwann cells was significantly enhanced only when the neurons expressed p75NTR, but not if they were also derived from p75-/- mice (A.L.S., unpublished observations). This suggests that the capacity of p75-/- axons to take advantage of increases in neurotrophin availability was reduced in comparison to p75+/+ axons. The absence of axonal p75NTR may also reduce the rate in which regeneration occurs in the PNS. Within the first few days post-injury, axonal regeneration from the proximal stump occurs in a slow and staggered manner through the lesion site (Holmquist et al., 1993). Although PNS neurons increase their expression of RAGs, by elevating neuronal neurotrophin expression further during this period, with electrical stimulation for example, the rate in which this process occurs is signficantly increased (Al-Majed et al., 2000a; Al-Majed et al., 2000b; English et al., 2007). This neuronal souce of neurotrophins is important during the earlier time points of axonal regeneration, since neurotrophin expression by Schwann cells is delayed in comparison and only beneficial to regenerating axons following their alignment in the endoneurial tubes (Witzel et al., 2005; Gordon, 2009). So, before Schwann cells play a large role, if axons were less sensitive to the lower levels of neuronal neurotrophins available at earlier time points, as they are in the absence of p75NTR, axonal regeneration across the lesion site could be delayed. Indeed, the distance regenerating axons reach within a sciatic nerve 7 days following injury is reduced in p75-/- mice compared to p75+/+ mice (Song et al., 2009). Axonal regeneration in the dorsal root or across the DREZ following rhizotomy, however, does not appear to be influenced by the neural expression of p75NTR. In general, injury to centrally-projecting sensory axons has little effect on RAG expression within  158  their somata in the DRG (Chong et al., 1994; Bradbury et al., 2000). This includes minor changes to Trk receptor expression (Ernfors et al., 1993). Thus, differences in the neuronal expression of p75NTR may not be a contributing factor. Following dorsal rhizotomy, the relative density of regenerating axons across the lesion site within the dorsal roots is not different between p75+/+ and p75-/- mice (A.L.S., unpublished observations). In addition, there were no observed differences in axonal regeneration noted between e-GFP p75+/+ and p75-/- regenerating axons within the p75+/+ host dorsal root, and all failed to cross the DREZ. Thus, the absence of p75NTR expression on Schwann cells appears to be the primary determinant for regeneration across the DREZ, and the contribution of the neural interactions of p75NTR with Trk receptors, myelinreceptors (NgR and Lingo-1), or intracellular proteins (Rho-GDI and Sall2) seems relatively minimal. This has been further confirmed within the injured spinal cord. Ascending sensory axons in the dorsal columns failed to regenerate following SCI in both p75+/+ and p75-/- mice (Song et al., 2004; Zheng et al., 2005).  5.2.3 p75NTR regulates cellular death following axonal injury The reduction of neurotrophin availability via Schwann cell p75NTR may not only occur by the sequestration of neurotrophins, but also by the induction Schwann cell apoptosis following injury. In p75-/- mice, Schwann cell death is significantly reduced compared to p75+/+ mice by 21 days following peripheral nerve injury (Ferri and Bisby, 1999). Since regenerating axons within the dorsal root reach the DREZ within the first 37 days post-injury, this is not likely to have significant effects on the success of axonal regeneration in the dorsal root or across the DREZ. On the other hand, transplantation of  159  Schwann cells into the spinal cord is routinely studied as one of the potential treatment strategies for SCI (Fortun et al., 2009). One major problem with this treatment is the prevalence of Schwann cell death post-transplantation: only 22% of transplanted Schwann cells survive beyond one week (Hill et al., 2006; Hill et al., 2007). It is possible that the increased survival of Schwann cells in the p75-/- transplant accounted for the enhanced axonal regeneration within this graft. However, enhanced CNS axonal regeneration into p75NTR-expressing Schwann cell transplants engineered to produce mutated neurotrophins capable of binding Trk receptors but not p75NTR, suggests that sequestration of neurotrophins by p75NTR is sufficient to inhibit regeneration (Golden et al., 2007). In contrast to the role of p75NTR in glial cell apoptosis, neuronal expression of p75NTR may be important for cellular survival. In p75-/- mice, there are developmental losses of neurons that normally express p75NTR (Lee et al., 1992). Following SCI, neuronal death at the level of the lesion is also elevated in p75-/- mice, which is associated with a reduction in locomotor recovery (Chu et al., 2007). Dorsal rhizotomy does not induce neuronal death in the DRG, but does cause a transient increases neuronal death at 2 days post-injury in the dorsal horn (Chew et al., 2008). An examination of the number of neurons in the dorsal horn of p75+/+ and p75-/- mice present at 28 days following rhizotomy, revealed equivalent neuron densities within the two genotypes (A.L.S., unpublished observations). These results suggest that, with this injury model, it is unlikely that neuronal death contributed to the results in either p75+/+ or p75-/- mice.  160  5.2.4 p75NTR controls neurotrophin-mediated myelination Given the expression of p75NTR on both neurons and glia, it is not surprising that this receptor is also involved in myelination. Neurotrophins differentially regulate myelination depending on the neuronal receptors expressed: NGF promotes myelination via TrkA (Chan et al., 2004); NT-3 prevents myelination via TrkC; and BDNF both promotes myelination via p75NTR, and inhibits myelination via TrkB (Cosgaya et al., 2002; Xiao et al., 2009). BDNF-mediated myelination via p75NTR is dependent on its intracellular interaction with a scaffolding protein, Par-3, on the surface of Schwann cells (Chan et al., 2006). Preventing neurotrophin binding to p75NTR causes significant reductions in myelin thickness, as observed in p75-/- mice (Cosgaya et al., 2002). Re-myelination of regenerating neurons is important for their stabilization, survival, and proper conduction (Riethmacher et al., 1997; Wilkins et al., 2003). Recent studies have correlated myelination deficits present in p75-/- mice to reductions in axon regeneration of motoneurons in both the facial and sciatic nerve, and functional recovery following a crush injury (Song et al., 2006; Song et al., 2009; Tomita et al., 2007). However, it may be argued that by delaying re-myelination and the expression of MAIPs present in myelin, axonal regeneration could be improved. In line with this, earlier studies showed that the re-innervation of peripheral muscles is enhanced in p75-/- mice compared to p75+/+ mice following sciatic nerve injury (Boyd and Gordon, 2001) and motor axon regeneration in the facial nerve is substantially higher, not lower, in p75-/- mice when compared to p75+/+ mice following injury (Ferri et al., 1998). Following dorsal rhizotomy in p75-/- mice, delayed or poor re-myelination did not appear to restrict axonal  161  regeneration of sensory neurons: for those normally myelinated (large-diameter) or nonmyelinated (smaller-diameter), axonal regeneration across the DREZ was greater in p75-/mice than p75+/+ mice.  5.3 Differential regulation of intraspinal plasticity via p75NTR 5.3.1 MAIPs inhibit intraspinal axonal sprouting via p75NTR As previously discussed in Chapter 1, MAIPs inhibit intraspinal sprouting following injury in the spinal cord. Following dorsal rhizotomy in particular, axonal sprouting is enhanced following antagonism of MAIP signaling (MacDermid et al., 2005). In addition to the p75NTR-Lingo1-NgR complex, MAIPs can signal through a different receptor complex consisting of NgR, Lingo1 and TROY, another member of the TNF receptor family (Park et al., 2005; Shao et al., 2005). However, TROY expression in the spinal cord is even more limited than p75NTR. Although TROY is expressed in a large proportion of DRG neurons (Park et al., 2005), it is absent from all descending projections in the spinal cord (Barrette et al., 2007). This implies that TROY expression may be consequential to the sprouting of sensory afferents, but not to bulbospinal populations. P75NTR, on the other hand, is expressed by many bulbospinal populations (Barrette et al., 2007) including those examined in this dissertation.  5.3.2 p75NTR influences neurotrophin-responsiveness of spared axons In addition to the myelin receptors, the co-expression of neural p75NTR with Trk receptors can also influence the sprouting of spared axons in the injured CNS. Due to the lack of high affinity receptor sites generated by the interaction between p75NTR and Trk  162  receptors, NGF sensitivity of TrkA-expressing neurons (such as CGRP-positive neurons) is reduced in the absence of p75NTR (Dechant and Barde, 1997). Indeed, CGRP-positive primary afferents in p75-/- mice were relatively unresponsive to exogenous NGF, unlike their p75+/+ counterparts. Exogenous NT-3, on the other hand, increased the sprouting of this population in p75-/- mice, but not p75+/+ mice. NT-3 acts to decrease the association of NGF with TrkA in the presence of p75NTR; but in the absence of p75NTR, NT-3 binds all Trk receptors, including TrkA (Vesa et al., 2000; Mischel et al., 2001). So, in p75-/mice, I would predict that NGF signaling via TrkA is inhibited, and NT-3 signaling via TrkA is enhanced. Since bulbospinal projections do not express TrkA, these relationships did not influence the sprouting of these populations (King et al., 1999).  5.3.3 Glial p75NTR sequestration of neurotrophins in the CNS Following injury to the spinal cord, p75NTR expression is significantly upregulated at the lesion within the CNS by oligodendrocytes and invading Schwann cells (King et al., 2000; Casha et al., 2001; Beattie et al., 2002). In comparison to injury in the PNS, neurotrophin availability is much more restricted in the CNS. Although the small number of invading Schwann cells may provide a modicum of additional support, it is likely that endogenous neurotrophins are sequestered by glial p75NTR following SCI. However, with a dorsal rhizotomy model, Schwann cells do not invade the CNS and p75NTR expression in CNS glia is relatively limited (A.L.S., unpublished observations). This suggests that the competition for available neurotrophins via glial p75NTR is likely to play a smaller role in preventing spontaneous growth in the CNS following dorsal rhizotomy, but may be a significant factor following SCI.  163  5.3.4 p75NTR induces oligodendrocyte cell death The induction of apoptosis is deleterious to recovery following SCI: preventing oligodendrocyte death following SCI reduces secondary de-myelination and the expansion of the glial scar, which results in functional recovery (McTigue and Tripathie, 2008). The induction of oligodendrocyte apoptosis following SCI is induced by the interaction of p75NTR with either pro-NGF (Beattie et al., 2002), or mature NGF (Casaccia-Bonnefil et al., 1996). The interaction of pro-NGF with p75NTR, however, appears to be of greater concern. Pro-NGF is much more highly expressed in the CNS than NGF, is fifty-fold more active than NGF, and specific blockade of pro-NGF completely prevents oligodendrocyte death in vitro (Beattie et al., 2002). If the production of pro-NGF following SCI in vivo is restricted by minocycline, oligodendrocyte death is reduced and functional recovery is enhanced (Yune et al., 2007). In the results presented here, secondary damage due to oligodendrocyte death may have been reduced in the p75-/mice, since oligodendrocyte death is reduced by 15% in these mice following SCI (Beattie et al., 2002). Like SCI, dorsal rhizotomy leads to cellular death in the spinal cord. Although oligodendrocyte death has not been examined specifically in this model, non-neuronal apoptotic cells are prevalent in the dorsal column white matter following dorsal rhizotomy (Chew et al., 2008).  164  5.3.5 Intraspinal plasticity of dendrites is not governed by p75NTR In contrast to the p75NTR-mediated effects on axonal sprouting, p75NTR does not affect intraspinal dendritic sprouting following injury. Dendritic density was equivalent in p75+/+ and p75-/- mice (in the absence or presence of exogenous BDNF) following dorsal rhizotomy, suggesting that another factor restricted neurotrophin-dependent dendritic sprouting. As demonstrated in Chapter 4, the expression of the truncated isoform TrkBT1 highly correlated to the inhibition of dendritic sprouting. Consistent with neurotrophinsensitivity during development (Gascon et al., 2005; Gao et al., 2009), adult spinal dendrites were responsive to BDNF. Following dorsal rhizotomy, spontaneous sprouting of LSN dendrites was normally absent, but induced by exogenous BDNF. However, by antagonizing NGF and subsequently reducing the expression of TrkBT1, dendritic sprouting in the LSN was dis-inhibited. The effect of TrkBT1 on dendritic growth is likely due to its expression in CNS glia, given the significant upregulation of TrkBT1 in glial cells post-injury and its ability to sequester BDNF (Fryer et al., 1997; King et al., 2000). Thus, it is possible, though not yet confirmed, that glial TrkBT1 antagonizes BDNF signaling within the spinal cord by restricting its availability following dorsal rhizotomy, similar to role of glial p75NTR in the dorsal root.  5.4 Final comments The p75NTR receptor is a convergent point for many signaling interactions that have a role in inhibiting and enhancing axonal regeneration and intraspinal sprouting.  165  The results presented here have shed light on the contribution of p75NTR to the inhibitory nature of the CNS. However, the multiple roles of p75NTR present a challenge in distinguishing which of these influences functional outcome following injury. Within the confines of the methods used here, I have concluded that the Schwann cell expression of p75NTR is a primary factor in the inhibition of neurotrophin-mediated CNS axonal regeneration following injury. This finding directly opposes earlier testiments of the ‘ligand passing’ role of this receptor on Schwann cells and highlights the overall inhibition to axonal regeneration via p75NTR. As a result, p75NTR antagoism has implications to the uses of Schwann cell transplantation approaches following SCI, as well as non-surgical treatments of bracial plexus injuries. The data presented in Chapter 3 was the first to show that intraspinal sprouting of uninjured axons following injury is also inhibited by p75NTR. Here, it is likely that signaling from CNS myelin via p75NTR plays a larger role in governing axonal growth, and sequestration of neurotrophins a smaller role than in axonal regeneration. Given the additive effect of exogenous neurotrophin treatment and p75NTR antagonism, this is a promising combinatorial approach for the promotion of neuroplasticity following injury. 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