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Title: Matrix metalloproteinases as modulators of retinal dendritic remodeling and axonal regeneration in the injured zebrafish retinotectal system. Primary insights into an antagonistic interplay between dendritic and axonal regrowth upon optic nerve damage
Other Titles: Matrix metalloproteinases als modulators van retinale dendritische hermodellering en axonal regeneratie in het beschadigd zebravis retinotectaal circuit. Primaire inzichten in een antagonistische interactie tussen dendritische en axonale hergroei na aanbreng van een optisch zenuwletsel ,,
Authors: Lemmens, Kim
Issue Date: 20-Sep-2016
Abstract: Dysfunction of the central nervous system (CNS) after injury or in neurodegenerative diseases increasingly impairs life quality in our aging society. Also optic neuropathies, like glaucoma, are becoming increasingly prevalent in our elderly population, affecting over 60 million people worldwide. They are characterized by retinal ganglion cell (RGC) axonal degeneration and death, ultimately resulting in irreversible blindness because adult mammals lack the capacity to repair/regrow damaged axons in the CNS. Although combinatorial approaches indicate that it might be possible to repair the injured mammalian optic nerve (ON), they have not yet induced substantial visual recovery, nor are they, up to now, suitable for clinical applications. Therefore, comparative studies focus on the spontaneously regenerating adult CNS of zebrafish, which proves to be a valuable research model to investigate regenerative strategies of damaged neurons and to identify novel pro-regenerative molecules. Notably, a recent transcriptome profiling study performed on regenerating adult zebrafish eyes at various time-points after optic nerve crush (ONC), pinpointed several potential pro-regenerative molecules, under which four matrix metalloproteinases (mmp-2, -9, -13a, -14). While MMPs have been implicated in axonal outgrowth during CNS development and are known to be upregulated during vertebrate CNS repair, their impact on in vivo CNS regeneration remains undocumented. Within this context, this PhD project then also intended to unravel the role of Mmp-2, -9, -13a and -14 in ON regeneration in adult zebrafish subjected to ONC.
The first part of this thesis was dedicated to an in-depth characterization of RGC survival, axonal regrowth/extension and primary visual recovery in the zebrafish ONC model.
Next, we obtained first in vivo insights in a contributory role for MMPs in RGC axonal regrowth as retinal broad-spectrum MMP inhibition seriously attenuated optic tectum reinnervation by regenerating RGC axons without influencing RGC survival. Moreover, combined use of Western Blotting and immunostainings revealed a phase-dependent expression pattern for each MMP in the retina after ONC, primarily implying Mmp-2 and -13a in RGC axonal regrowth.
As such, via utilization of several loss-of-function approaches to interfere with the functioning of one specific MMP, e.g. pharmaceutical MMP inhibition, morpholino and genome editing technology, we were able to identify Mmp-2 and -13a as in vivo regulators of RGC axonal regeneration in adult zebrafish after ONC. Notably, since zebrafish lack an inhibitory environment, these data suggest a novel, neuron intrinsic, function for multiple MMPs in RGC axonal regrowth in addition to their role in breaking down environmental barriers as deduced from mammalian studies. The future identification of the underlying MMP substrates by means of quantitative proteomics and subsequent validation of promising candidate molecules in fish and mice might then also unravel new pro-regenerative molecules for the injured mammalian CNS.
While being an important part of the neuronal circuitry, the response of dendrites upon induction of axonal regeneration in mammals is almost unattended. Notably, the few publications that do discuss this issue, indicate that dendritic morphology is seriously influenced by the pro-regenerative molecule used to trigger axonal regrowth, ranging from a reduction to an increase in size and complexity. As the eventual recovery of sight after ON injury will equally depend on the proper restoration of dendritic structure, we then also characterized the inherent response of dendrites during successful RGC axonal regeneration in adult zebrafish. Remarkably, both temporal expression analyses of synaptic and dendritic markers, as well as morphometric analysis of inner plexiform layer thickness, indicated that regeneration-competent injured neurons repeat the developmental order of neurite growth, where axogenesis is primary to dendritogenesis.
Finally, a last part of this work focused on the elucidation of a potential role of MMPs in retinal dendritic remodeling in adult zebrafish after ONC. Both retinal broad- and narrow-spectrum Mmp(-2) inhibition seemingly prevented RGC dendrites from shrinking upon ON injury. Intriguingly, in both conditions, a disturbance of dendritic retraction occurred concurrently with a reduced RGC axonal regrowth, thus indicative of a potential antagonistic interplay between dendritic remodeling and axonal regrowth after ONC. In addition, due the consecutive progress of axonal and dendritic growth during development and regeneration, and based on a publication discussing TRAK1/2 driven polarized mitochondrial transport in neurons respectively regulating axonal and dendritic outgrowth, we hypothesized that an energetic trade-off might lie at the base of this apparent inter-dependency. As such, future experiments should interfere with mitochondrial trafficking in zebrafish RGC dendrites and analyze the effect on RGC axonal regeneration. If these data would result in a proof-of-concept, i.e. dendritic remodeling serving as fuel for axonal regeneration, we envision a major shift in the research focus of the neuroregenerative research field and the potential uncovering of various novel therapeutic targets.
Table of Contents: TABLE OF CONTENTS
TABLE OF CONTENTS V
LIST OF ABBREVIATIONS XI
NOMENCLATURE GUIDELINES XIV
CONTEXT AND RESEARCH FOCUS XV

CHAPTER 1: GENERAL INTRODUCTION 1
1 Failure of axonal regeneration in the adult mammalian central nervous system (CNS): the main known barriers 3
1.1 A reduced intrinsic growth capacity 3
1.2 A non-permissive growth environment 4
1.3 Insufficient neurotrophic support 6
2 The visual system 8
2.1 The visual system as a research model 8
2.2 Anatomy of the mammalian eye and retina 9
2.3 The mammalian retinofugal projection 12
3 Inducing optic nerve regeneration in adult mammals: how far are we? 14
3.1 Inducing RGC survival by an increase of neurotrophic support 15
3.2 Enhancement of the intrinsic growth potential of adult RGCs 16
3.2.1 Triggering developmental regulators of RGC intrinsic growth capacity 16
3.2.2 Inducing intraocular inflammation 17
3.2.3 Inhibiting cell-intrinsic suppressors of regeneration 19
3.3 Neutralization of the non-permissive environment 20
3.4 Combinatorial approaches to induce long-distance axonal regeneration 22
3.5 A role for Dendritic remodeling in optic nerve repair? 23
4 Zebrafish as a model to study CNS regeneration 25
4.1 The zebrafish visual system 26
4.1.1 The eye 26
4.1.2 The optic chiasm and optic tectum 28
4.2 Regeneration of the adult zebrafish retinotectal system 29
4.2.1 Neuronal regeneration in the adult zebrafish retina 29
4.2.2 Neuroprotection and axonal regeneration in the adult zebrafish retina 29
4.2.2.1 The zebrafish optic nerve crush (ONC) model 30
4.2.2.2 Mechanisms underlying successful ON regeneration in adult zebrafish 31
5 Matrix metalloproteinases 36
5.1 Gene evolution and classification 36
5.2 Structure and function 40
5.3 Regulation of MMP activity 41
5.4 MMPs in the CNS 43
5.4.1 MMP functioning in dendritic and axonal outgrowth and guidance 44
5.4.1.1 Axonal outgrowth and guidance 44
5.4.1.2 Dendritic outgrowth 46
5.4.2 MMP functioning in dendritic remodeling and axonal regrowth 48
5.4.2.1 Axonal regrowth 48
5.4.2.2 Dendritic remodeling 49

OBJECTIVES AND RATIONALE 51

CHAPTER 2: IN-DEPTH CHARACTERIZATION OF RGC AXONAL REGENERATION IN THE ZEBRAFISH ONC MODEL 53
1 Introduction 55
2 Material en methods 56
2.1 Zebrafish maintenance 56
2.2 Optic nerve crush (ONC) 56
2.3 immunohistochemistry 57
2.4 Western blotting (WB) 58
2.5 Antibody characterization 59
2.6 Visualization and quantification of optic tectum reinnervation 60
2.7 Dorsal light reflex 62
2.8 Statistical analysis 63
3 Results 65
3.1 Optic nerve crush in zebrafish does not induce retinal apoptosis 65
3.2 Detailed characterization of axonal regeneration in the zebrafish ONC model 66
3.2.1 Determination of the Gap-43 retinal spatiotemporal expression pattern 66
3.2.2 Analysis of OT reinnervation 67
3.3 Assesment of the Target contact and synaptic refinement phase in the zebrafish ONC model 70
3.3.1 Characterization of RGC synaptic target contact repair in the OT after ONC: Znp-1 spatiotemporal expression pattern 70
3.3.2 Mapping primary visional functional recovery: dorsal light reflex 72
4 Discussion 73

CHAPTER 3: MMPS AS PROMISING REGULATORS OF AXONAL REGROWTH IN THE INJURED ADULT ZEBRAFISH RETINOTECTAL SYSTEM 77
1 Introduction 79
2 Material and methods 81
2.1 Zebrafish maintenance 81
2.2 Optic nerve crush 81
2.3 Histology and immunohistochemistry 81
2.4 Western blotting 82
2.5 Antibody characterization 83
2.6 Retinal broad-spectrum MMP inhibition 85
2.7 Visualization and quantification of optic tectum reinnervation 86
2.8 Statistical analysis 86
3 Results 88
3.1 A role for MMPs in axonal regrowth during retinotectal regeneration 88
3.1.1 Inhibiting MMP functioning in the retina results in a reduced tectal reinnervation 88
3.1.2 RGC survival and retinal thickness are unaffected by repeated intravitreal GM6001 injections 90
3.2 Determination of the MMP spatiotemporal expression pattern after ONC 92
3.2.1 Increased expression of mature Mmp-13a in RGC somata and primary dendrites during axonal regrowth 92
3.2.2 Elevated presence of Mmp-2 in RGC somata and axons during axonal regeneration 93
3.2.3 Biphasic expression pattern of Mmp-14 in the zebrafish retina during optic nerve regeneration 96
3.2.4 Increased Mmp-9 expression in inner retinal synapses during retinotectal regeneration 98
4 Discussion 100



CHAPTER 4: IDENTIFICATION OF MMP-2 AND -13A AS IN VIVO ENHANCERS OF AXONAL REGROWTH IN ADULT ZEBRAFISH AFTER ONC 105
1 Introduction 107
2 Material and methods 108
2.1 Zebrafish maintenance 108
2.2 Optic nerve crush 108
2.3 immunohistochemistry 108
2.4 Western blotting (WB) 109
2.5 Antibody characterization 109
2.6 Retinal MMP-2 and MMP-9/-13a inhibition 110
2.7 TALENs-mediated MMP-2 ko zebrafish 111
2.7.1 Genotyping of Mmp-2 KO zebrafish 112
2.8 In vivo electroporation of morpholinos into the adult zebrafish retina 112
2.8.1 Evaluation of morpholino efficiency 114
2.9 Visualization and quantification of optic tectum reinnervation 115
2.10 Statistical analysis 115
3 results 116
3.1 A role for Mmp-2 in axonal regrowth during zebrafish retinotectal regeneration? 116
3.1.1 A role for Mmp-2 in RGC axonal regrowth after ONC: ABT-770 treatment 116
3.1.1.1 Inhibiting Mmp-2 functioning in the retina results in a reduced tectal reinnervation 116
3.1.1.2 RGC survival is unaffected by repeated intravitreal ABT-770 injections 118
3.1.2 A role for Mmp-2 in axonal regrowth during retinotectal regeneration: Mmp-2 KO fish 120
3.1.2.1 Overall Mmp-2 deficiency in adult fish results in reduced tectal reinnervation 120
3.1.2.2 RGC survival is unaffected in Mmp-2 KO fish after ONC 122
3.2 A role for Mmp-13a in axonal regrowth during zebrafish retinotectal regeneration? 123
3.2.1 Inhibiting Mmp-13a functioning in the retina through intravitreal MMP-9/-13 inhibitor injections does not diminish tectal reinnervation 123
3.2.2 Retinal Mmp-13a knockdown aggravates zebrafish retinotectal regeneration after ONC 126
3.2.2.1 Optimization of in vivo electroporation of morpholinos into the adult zebrafish retina 126
3.2.2.2 Analysis of Mmp-13a knockdown efficiency 128
3.2.2.3 Downregulation of retinal Mmp-13a expression results in a reduced tectal reinnervation 130
3.2.2.4 Analysis of RGC survival and neuronal proliferation in standard control and Mmp-13a MO electroporated fish 132
4 discussion 134

CHAPTER 5: IN-DEPTH CHARACTERIZATION OF RETINAL DENDRITIC REMODELING IN THE ZEBRAFISH ONC MODEL 141
1 Introduction 143
2 Material and methods 145
2.1 Zebrafish maintenance 145
2.2 Optic nerve crush (ONC) 145
2.3 Histology, immunohistochemistry and histomorphometric analysis 145
2.4 Western blotting (WB) 146
2.5 Antibody characterization 146
2.6 Statistical analysis 148
3 results 149
3.1 A fluctuating IPL thickness in the adult zebrafish retina after ONC 149
3.2 ONC induces A subsequent loss and repair of synaptic connectivity in the adult zebrafish retina 150
3.3 ON injury triggers dendritic remodeling in the adult zebrafish retina 152
4 Discussion 155

CHAPTER 6: DENDRITIC REMODELING AS FUEL FOR AXONAL REGENERATION IN THE INJURED RETINOTECTAL SYSTEM OF ADULT ZEBRAFISH? 163
1 Introduction 165
2 Material and methods 167
2.1 Zebrafish maintenance 167
2.2 Optic nerve crush 167
2.3 Retinal Broad-spectrum and MMP-2 inhibition 167
2.4 Histology and histomorphometric analysis 167
2.5 Western blotting (WB) 168
2.6 Antibody characterization 168
2.7 Statistical analysis 168
3 Results 169
3.1 Retinal broad-spectrum MMP inhibition prevents IPL thinning in adult zebrafish after ONC 169
3.2 Broad-spectrum MMP inhibition in the adult zebrafish retina blocks synaptic degeneration after ONC 170
3.3 A role for MMP-2 in retinal dendritic remodeling after ONC? 171
3.3.1 Retinal MMP-2 inhibition interferes with IPL thinning in adult zebrafish after ONC 172
3.3.2 A mild contribution of Mmp-2 to synaptic degeneration in the adult zebrafish retina after ONC 173
4 discussion 175

CHAPTER 7: GENERAL CONCLUSION & FUTURE PERSPECTIVES 181
1 Future MMP research in the injured retinotectal system of adult zebrafish: A reflection based on the current results 183
2 use of Contralateral, external sham-operated or naive fish as control for WB expression studies: reflections based on acquired experience 185
3 Use of pharmaceutical MMP inhibitors, MOrpholinos or KO lines as a loss-of-function approach in adult zebrafish: which way to go? 186
4 An evolutionary conserved MMP functioning in mice? 189
5 Proteomic identification of MMP regulated pathways 190
6 An energy-based Antagonistic relationship between dendritic remodeling and axonal regeneration in adult zebrafish? 191
7 Dendritic remodeling in mouse optic neuropathy models: what do we know and what does it teach us? 195
8 conclusion 196

SUMMARY 197
SAMENVATTING 199
REFERENCES 201
LIST OF PUBLICATIONS 223
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Animal Physiology and Neurobiology Section - miscellaneous

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