Osteopontin Expression During the Acute Immune Response Mediates Reactive Synaptogenesis ...

337 Pages·2016·7.35 MB·English

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Theses and Dissertations Graduate School
2013
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Julie Chan
Virginia Commonwealth University
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Osteopontin Expression During the Acute Immune Response Mediates Reactive
Synaptogenesis and Adaptive Outcome

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of
Philosophy at Virginia Commonwealth University

by

Julie Lynn Chan
B.S. Neurobiology, University of California, Irvine, 2005

Director: Linda L. Phillips, Ph.D.
Professor, Department of Anatomy and Neurobiology

Virginia Commonwealth University
Richmond, Virginia
August 2013
Acknowledgement

First and foremost, I would like to thank my advisor, Dr. Linda L. Phillips, for her
continuous support, expertise, and guidance. I cannot thank her enough for being a superb role
model, providing me with lifelong skills and invaluable mentorship. I would also like to thank
my PhD advisory committee: Dr. Michelle L. Block, Dr. Kurt F. Hauser, Dr. Dong Sun, Dr.
Thomas M. Reeves, and Dr. John T. Povlishock whose enthusiasm for my project and
willingness to assist in my endeavors are immeasurable. Dr. Reeves has been a wealth of
knowledge and insight, providing both technical and academic advice throughout the years. I am
also forever grateful to the Department of Anatomy and Neurobiology and our Chairman, Dr.
Povlishock, who has provided me with excellent guidance and numerous national and
international opportunities to pursue my ambitions as a physician-scientist in the laboratory and
clinic. In addition, I am thankful to Dr. Gordon L. Archer and Sandra Sorrell for their support
and guidance in the MD-PhD Program.

I am also eternally grateful to ‘The Ladies’: Lesley K. Harris, Raiford T. Black, Nancy N.
Lee, and Terry L. Smith for their tireless efforts, technical expertise, and source of social
camaraderie over the years. I would also like to thank Dr. Adele E. Doperalski and Justin
Brooks for their advice, friendship, and humor which kept me motivated throughout my PhD.
Finally, my utmost appreciation goes to my mom and brother for their constant input and
encouragement, and I dedicate these collective efforts to my Dad, who has been, and will always
be, a major source of inspiration.
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Table of Contents

List of Figures ............................................................................................................................... vi
List of Tables ................................................................................................................................ ix
List of Abbreviations ..................................................................................................................... x
Abstract ........................................................................................................................................ xv
Chapter 1: Introduction .............................................................................................................. 1
Traumatic Brain Injury ...................................................................................................... 2
Impact, Epidemiology, and Classification ............................................................. 2
Pathophysiology ..................................................................................................... 5
Neuroexcitation Following Traumatic Brain Injury .............................................. 6
Traumatic Brain Injury Inflammatory Response ................................................... 7
Traumatic Brain Injury Induces Neuronal Cell Death ......................................... 11
Traumatic Brain Injury-Induced Axonal Injury ................................................... 12
Neuroplasticity and Recovery from Traumatic Brain Injury ........................................... 14
Deafferentation-Induced Synaptogenesis ............................................................ 14
Inflammation, Neuroglia, and Synaptic Plasticity ............................................... 19
Extracellular Matrix and Neuroplasticity ......................................................................... 23
Proteoglycans and Cell Adhesion Molecules Influence Synapse Formation ...... 25
Matrix Metalloproteinases and Synaptogenesis ................................................. 28
Stromelysin-1 (Matrix Metalloproteinase-3) and Synaptogenesis ...................... 30
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Gelatinases (Matrix Metalloproteinase-2, -9) and Synaptogenesis ...................... 34
The Hippocampus: Structure and Value as a Model of Synaptic Plasticity after Traumatic
Brain Injury .......................................................................................................... 37
Structure and Function of the Hippocampus ....................................................... 37
Hippocampal Plasticity: Neurogenesis and Synaptogenesis ................................ 43
Osteopontin ...................................................................................................................... 45
Discovery, Structure, and Role in Inflammation ................................................. 45
Inflammatory Role in the Central Nervous System ............................................. 51
Role in Neurodegenerative Disease ..................................................................... 53
Role Following Central Nervous System Injury ……………………………….. 56
Interaction with Extracellular Matrix Proteins and Role in Synaptic Plasticity .. 60
Response after Traumatic Brain Injury ................................................................ 63
Exploration of Osteopontin Role in Synaptogenesis after Traumatic Brain Injury ......... 64
Traumatic Brain Injury Models of Adaptive and Maladaptive Plasticity ............ 64
Osteopontin Knockout Mice ................................................................................ 66
Summary and Experimental Hypotheses ......................................................................... 68
Chapter 2: Osteopontin Response Following Traumatic Brain Injury ................................ 71
Abstract ............................................................................................................................ 72
Introduction ...................................................................................................................... 73
Methods ............................................................................................................................ 79
Results .............................................................................................................................. 92
Discussion ...................................................................................................................... 117
Summary ........................................................................................................................ 133
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Chapter 3: Osteopontin Knockout Mice and Synaptogenesis ............................................. 135
Abstract .......................................................................................................................... 136
Introduction .................................................................................................................... 138
Methods .......................................................................................................................... 144
Results ............................................................................................................................ 153
Discussion ...................................................................................................................... 185
Summary ........................................................................................................................ 206
Chapter 4: General Discussion ............................................................................................... 207
Summary of Results ....................................................................................................... 208
Osteopontin and the Inflammatory Response ................................................................ 211
Osteopontin-Microglial Interaction During the Immune Response ............................... 215
Osteopontin and Synaptic Plasticity .............................................................................. 216
Osteopontin as a Promising Target of Therapeutic Intervention ................................... 224
Future Directions ........................................................................................................... 226
Final Remarks ................................................................................................................ 228
List of References ...................................................................................................................... 230
Appendices ................................................................................................................................. 291
Appendix A Affymetrix Rat Genome Microarray Screening ...................................... 291
Appendix B Novel Object Recognition Paradigm ....................................................... 293
Appendix C Osteopontin Knockout Mouse Characterization ..................................... 298
Appendix D Fluid Percussion Injury of Olfactory Bulb .............................................. 303
Appendix E Delayed Osteopontin and CD44 Localization to Reactive Astrocytes .... 314
Vita ............................................................................................................................................. 317
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List of Figures

Chapter 1
1.1 Stages of Reactive Synaptogenesis ....................................................................................... 16
1.2 Laminar Structures of the Hippocampus .............................................................................. 38
1.3 Schematic of the Trisynaptic Circuit within the Hippocampus ............................................ 41
1.4 Structural Features of Osteopontin ....................................................................................... 47
Chapter 2
2.1 MMP/OPN Proteolysis Schematic ........................................................................................ 77
2.2 Rat Unilateral Entorhinal Cortex Lesion Stereotaxic Locations ........................................... 81
2.3 TBI+BEC Injury Locations and Fluid Percussion Injury Device ......................................... 84
2.4 Hippocampal and Dentate Molecular Layer OPN Protein Expression Following Adaptive
UEC ...................................................................................................................................... 93
2.5 IHC of OPN in the Dentate Gyrus ........................................................................................ 95
2.6 IHC of OPN and Glial Cells in the Dentate Gyrus Following UEC ..................................... 98
2.7 IHC of MAP1B in the Dentate Gyrus ................................................................................. 101
2.8 OPN Transcript Expression and Microglial Localization Following UEC ........................ 104
2.9 Hippocampal OPN Protein Expression Following Maladaptive TBI+BEC ....................... 107
2.10 IHC of OPN and Glial Cells in the Dentate Gyrus Following TBI+BEC ........................ 110
2.11 Minocycline Attenuates Hippocampal UEC-Induced OPN Response ............................. 113

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2.12 Minocycline Alters Migration of OPN Positive Microglia and Pattern of Presynaptic
Terminal Removal in Deafferented Molecular Layer Following UEC .............................. 115
2.13 Minocycline Immunosuppression Attenuates Hippocampal MMP-9 Activity and OPN
Proteolytic Fragment Generation Following UEC ............................................................. 118
Chapter 3
3.1 Lipocalin 2 Schematic Representation of Tertiary Structure and Proposed Binding
Mechanisms ......................................................................................................................... 142
3.2 Novel Object Recognition Cognitive Assessment Task Schematic ................................... 151
3.3 OPN KO Mouse Genotype Verification ............................................................................. 154
3.4 MAP1B Protein Expression Following UEC ..................................................................... 157
3.5 IHC of MAP1B in WT and OPN KO Mice Following UEC ............................................. 160
3.6 N-cadherin Protein Expression Following UEC ................................................................. 163
3.7 IHC of N-cadherin in WT and OPN KO Mice Following UEC ......................................... 165
3.8 Synapsin 1 Protein Expression Following UEC ................................................................. 168
3.9 IHC of Synapsin 1 in WT and OPN KO Mice Following UEC ......................................... 171
3.10 OPN KO Mice Exhibit Cognitive Deficits in the NOR Test ............................................ 174
3.11 Microglial Reactivity in WT and OPN KO Mice Following UEC ................................... 178
3.12 OPN KO Attenuates Hippocampal MMP-9 Activity Following UEC ............................. 181
3.13 UEC Induces Protein Expression in LCN2 ...................................................................... 183
3.14 IHC of LCN2 in WT and OPN KO Mice Following UEC ............................................... 186
Chapter 4
4.1 Cytokine-Astrokine Signaling During Degeneration .......................................................... 218
4.2 Cytokine-Astrokine Signaling During Regeneration .......................................................... 220
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Appendix B
B-1 Specific Novel Object Recognition Apparatus .................................................................. 296
Appendix C
C-1 Hippocampal OPN Protein Expression Following UEC in WT and OPN KO Mice ........ 301
Appendix D
D-1 Olfactory Bulb OPN Protein Expression Following Central Fluid Percussion Injury ...... 306
D-2 Olfactory Bulb MMP Activity Following Central Fluid Percussion Injury ...................... 308
D-3 IHC of OPN and Glial Cell Types in the Olfactory Bulb at 3d Following Central Fluid
Percussion Injury ............................................................................................................... 310
D-4 IHC of Synaptophysin and GFAP 3d Following Central Fluid Percussion Injury ............ 312
Appendix E
E-1 IHC of OPN Colocalized with IBA1 or GFAP 15d Following UEC ................................ 315
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