THE ROLE OF RETROGRADE REPRESSION IN LIMITING AXONAL REGENERATION IN THE CENTRAL NERVOUS SYSTEM A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Master of Surgery In the Department of Surgery University of Saskatchewan Saskatoon By Adam S. Wu, M.D. Division of Neurosurgery, University of Saskatchewan Saskatoon, Saskatchewan, Canada Keywords: Retrograde repression, axon regeneration, central nervous system, GAP-43, FGF-2 © Copyright Adam Wu, April 2008. All rights reserved. PERMISSION TO USE In presenting this thesis/dissertation in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis/dissertation in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis/dissertation work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis/dissertation or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis/dissertation. Requests for permission to copy or to make other uses of materials in this thesis/dissertation in whole or part should be addressed to: Head of the Department of Surgery University of Saskatchewan Saskatoon, Saskatchewan [postcode] Canada OR Dean College of Graduate Studies and Research University of Saskatchewan 107 Administration Place Saskatoon, Saskatchewan S7N 5A2 Canada i ABSTRACT The regenerative capacity of mature mammalian CNS neurons after axonal injury is severely limited by a variety of mechanisms. Retrograde repression is the continuous inhibition of the expression of growth phenotypes by tonic signals produced by target tissues and transmitted to the neuron cell body via retrograde axonal transport. Loss of target contact through axonal injury is thought to interrupt this retrograde signal and allow the up-regulation of growth-associated proteins. Most CNS neurons, however, possess many widespread axon collaterals, such that retrograde repression is maintained by intact sustaining collaterals even if some axons are injured. In this project we investigated whether or not retrograde repression plays a role in limiting the expression of GAP-43 in transcallosal neurons. Because TCNs possess local axon collaterals to nearby cortex and project distal axons to homologous areas of contralateral cortex, we hypothesized that the simultaneous interruption of retrograde repressive signals from both ipsilateral and contralateral cortex would result in an up- regulation of GAP-43 expression in at least some TCNs. We found that a bilateral infusion of a function blocking antibody to FGF-2 into the parietal cortex of rats using implanted osmotic mini-pumps resulted in a significant increase in the level of expression of GAP-43 mRNA in TCNs identified by retrograde fluorescent labeling. In contrast, neither ipsilateral or contralateral antibody infusions alone increased GAP-43 expression significantly compared to controls. The level of expression of GAP-43 in TCNs did not significantly increase after stereotactic callosotomy alone, but callosotomized animals treated with an ipsilateral infusion of anti- FGF-2 had levels of increased GAP-43 expression equivalent to those seen in animals that had received bilateral antibody infusions. We conclude that FGF-2 provides a retrograde repressive signal for at least some mature mammalian TCNs, and that the expression of growth-associated proteins can be up-regulated in CNS neurons by simultaneously blocking retrograde repressive signals from all existing axon collaterals. The ability to alter the gene expression of mature CNS neurons in both normal and injured states through the targeted infusion of a pharmacological agent may have potential clinical implications in the future. ii ACKNOWLEDGMENTS To the members of my advisory committee for advice and supervision: Dr. Daryl R. Fourney, M.D., F.R.C.S.C., F.A.C.S. (Supervisor) Dr. David Schreyer, Ph.D. Dr. Stephen Hentschel, M.D., F.R.C.S.C. Dr. Roger Keith, M.D., F.R.C.S.C. Dr. Kotoo Meguro, M.D., F.R.C.S.C. To the following individuals for scientific, theoretical and technical advice and training: Dr. Valerie Verge, Ph.D. Jayne Johnston Ruiling Zhai To the following organizations for the provision of equipment, laboratory space and funding: Department of Surgery, University of Saskatchewan CAMECO MS Neuroscience Research Laboratory iii TABLE OF CONTENTS Page PERMISSION TO USE…………………………………………………………… i ABSTRACT……………………………………………………………………….. ii ACKNOWLEDGEMENTS……………………………………………………….. iii LIST OF TABLES………………………………………………………………… viii LIST OF FIGURES……………………………………………………………….. ix LIST OF ABBREVIATIONS……………………………………………………… x BACKGROUND 1.0 INTRODUCTION…………………………………………………….. 1 1.1 RESPONSES TO AXONAL INJURY: AN OVERVIEW OF NORMAL AXONAL REGENERATION………………………………… 2 1.2 GROWTH ASSOCIATED PROTEINS AND THE GAP HYPOTHESIS…………………………………………………………….. 4 1.3 AN OVERVIEW OF DEVELOPMENTAL AXON GROWTH AND PATHFINDING 1.3.1 The Initiation of Axon Outgrowth and the Establishment of Neuronal Polarity……………………………………………….. 5 1.3.2 Axon Pathfinding During Development…………………….. 6 1.3.3 The Development of Axon Collateral Branches…………….. 7 1.3.4 The Role of GAPs in Axon Outgrowth and Collateral Branching………………………………………………. 8 1.4 THE ROLE OF GAP-43 IN AXON GROWTH AND REGENERATION 1.4.1 Correlative Observations……………………………………. 9 1.4.2 Biochemical and Structural Properties……………………… 11 1.4.3 Genetic Knock-out Studies………………………………….. 12 1.5 THE RETROGRADE REPRESSION OF GAP EXPRESSION 1.5.1 GAP-43 Expression is inhibited by target-derived factors….. 13 1.5.2 Retrograde Repression May be Maintained in the CNS by Sustaining Collaterals………………………………………….. 15 1.6 HYPOTHESIS PART ONE…………………………………………... 17 iv 1.7 THE TRANSCALLOSAL NEURON AS AN EXPERIMENTAL MODEL……………………………………………………………………. 18 1.8 FGF-2 AS A POTENTIAL RETROGRADE INHIBITOR…………… 20 1.9 HYPOTHESIS PART TWO…………………………………………... 22 METHODS 2.0 OVERVIEW OF EXPERIMENTAL PLAN…………………………. 23 2.1 CONFIRMATION OF ACTIVITY AND SPECIFICITY OF THE ANTIBODY 2.1.1 Western Blot………………………………………………… 26 2.1.2 Cell Proliferation Assay…………………………………….. 26 2.2 SURGICAL PROCEDURES 2.2.1 Animal Care and Anaesthesia………………………………. 27 2.2.2 Labeling of Transcallosal Neurons with the Retrograde Fluorescent marker Fluorogold™………………………………….. 28 2.2.3 Preparation of the Osmotic Mini-pumps……………………. 30 2.2.4 Implantation of the Osmotic Mini-pumps…………………... 31 2.2.5 Stereotactic Callosotomy…………………………………… 33 2.2.6 Perfusion of Animals and Harvesting of Brain Tissue……… 36 2.3 HISTOLOGIC IDENTIFICATION OF TRANSCALLOSAL NEURONS………………………………………………………………… 37 2.4 IN SITU HYBRIDIZATION…………………………………………. 37 2.5 QUANTIFICATION OF GAP-43 mRNA EXPRESSION…………… 40 RESULTS AND ANALYSIS 3.0 DETERMINATION OF ENZYME ACTIVITY AND SPECIFICITY 3.0.1 Western Blot………………………………………………… 41 3.0.2 Cell Proliferation Assay…………………………………….. 42 3.1 QUALITATIVE OBSERVATIONS…………………………………. 43 3.2 SUMMARY OF RESULTS: MEAN LEVELS OF GAP-43 mRNA EXPRESSION 3.2.1 Control Group……………………………………………….. 48 3.2.2 Experimental Group 1: Bilateral and Unilateral v FGF-2 Blockade…………………………………………….……… 50 3.2.3 Experimental Group 2: Callosotomy With and Without Local FGF-2 Blockade…………………………………………….. 51 3.2.4 Between Group Comparisons……………………………….. 53 3.3 SUBPOPULATION ANALYSIS 3.3.1 Neuron Subpopulations with Different Levels of GAP-43 Expression………………………………………………... 53 3.3.2 Correlations of GAP-43 Expression with Cross-sectional Neuron Area………………………………………. 57 3.3.3 Correlations of GAP-43 mRNA Expression with the Distance of the Cell Body to the Injection Site………………… 58 3.3.4 Levels of GAP-43 Expression in Medial versus Lateral Neurons……………………………………………………. 59 3.3.5 Laminar Location of Transcallosal Neurons and Levels of GAP-43 Expression……………………………………... 60 DISCUSSION AND CONCLUSIONS 4.0 FGF-2 AS A RETROGRADE REPRESSOR OF GAP EXPRESSION IN TRANSCALLOSAL NEURONS……………………... 67 4.1 BILATERAL BLOCKADE OF FGF-2 IS NECESSARY FOR UP-REGULATION OF GAP-43 EXPRESSION TO OCCUR……… 68 4.2 THE INHIBITION OF AXON REGENERATION IN THE CNS 4.2.1 Retrograde Repression as a Signal for Continuing Target Contact ……………………………………………………………………... 72 4.2.2 Inhibition of Regeneration Mediated by CNS Glia………….. 73 4.3 EVOLUTIONARY CONSIDERATIONS……………………………. 75 4.4 THE REGULATION OF PLASTICITY 4.4.1 The Role of Retrograde Repression in Regulating Neuronal Plasticity………………….……………………………… 77 4.4.2 The Role of GAP-43 and FGF-2 in Neuronal Plasticity…….. 79 vi 4.5 FUTURE DIRECTIONS 4.5.1 The Rat Transcallosal Neuron as an Experimental Model and Proposed Refinements of the Experimental Technique……...... 82 4.5.2 The Effect of Time and Dosage on Neuronal Response to Bilateral FGF-2 Blockade……………………………. 86 4.5.3 Other GAPs and Other Proteins……………………………... 87 4.5.4 Subpopulation Investigation………………………………… 88 4.5.5 Axon Outgrowth after FGF-2 Blockade…………………..… 88 4.5.6 Transcallosal Neurons in Other Areas of Neocortex…………89 4.5.7 The Mechanism of Action of FGF-2…………………………90 4.5.8 The Effect of Bilateral FGF-2 Blockade on Non-transcallosal Neurons…………………………………………. 92 4.6 CONCLUDING