MATRIX METALLOPROTEINASE REQUIREMENTS FOR NEUROMUSCULAR SYNAPTOGENESIS
By
Mary Lynn Dear
Dissertation
Submitted to the Faculty of the
Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY in
Biological Sciences January 31, 2018 Nashville, Tennessee
Approved:
Todd Graham, Ph.D. Kendal Broadie, Ph.D.
Katherine Friedman, Ph.D. Barbara Fingleton, Ph.D. Jared Nordman, Ph.D.
Copyright © 2018 by Mary Lynn Dear
All Rights Reserved
ACKNOWLEDGEMENTS
I would like to acknowledge my advisor, Kendal Broadie, and the entire Broadie laboratory, both past and present, for their endless support, engaging conversations, thoughtful suggestions and constant encouragement. I would also like to thank my committee members both past and present for playing such a pivotal role in my graduate career and growth as a scientist. I thank the Department of Biological Sciences for fostering my graduate education. I thank the entire protease community for their continued support, helpful suggestions and collaborative efforts that helped my project move forward. I would like to acknowledge Dr. Andrea Page-McCaw and her entire laboratory for helpful suggestions, being engaged in my studies and providing many tools that were invaluable to my project. I thank my parents, Leisa Justus and Raymond Dear, my brother Jake Dear and my best friend Jenna Kaufman. Words cannot describe how grateful I am for your support and encouragement. Most importantly, I would like to thank my husband, Jeffrey Thomas, for always believing in me, your unwavering support and for helping me endure all the ups and downs that come with graduate school. I am forever grateful and could not have done this without you.
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ACKNOWLEDGEMENT OF FUNDING
This work was supported by the National Institutes of Health R01 MH096832 and R01 MH084989 to K.B.
PUBLICATIONS
Two classes of matrix metalloproteinases reciprocally regulate synaptogenesis. Dear ML, Dani N, Parkinson W, Zhou S, Broadie K. Development. 2016 Jan 1;143(1):75-87.
Synaptic roles for phosphomannomutase type 2 in a new Drosophila congenital disorder of glycosylation disease model. Parkinson WM, Dookwah M, Dear ML, Gatto CL, Aoki K, Tiemeyer M, Broadie K. Dis Model Mech. 2016 May 1;9(5):513-27.
Neuronal activity drives FMRP- and HSPG-dependent matrix metalloproteinase function required for rapid synaptogenesis. Dear ML, Shilts J, Broadie K. Sci. Signal. 2017 Nov 7;10:eaan3181.
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TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS...................................................................................................................iii ACKNOWLEDGEMENT OF FUNDING...............................................................................................iv PUBLICATIONS.................................................................................................................................iv LIST OF FIGURES AND TABLES.......................................................................................................viii LIST OF SUPPLEMENTARY MATERIAL...............................................................................................x LIST OF ABBREVIATIONS .................................................................................................................xi
Chapter I. Introduction................................................................................................................................ 1
The Extracellular Matrix ............................................................................................................. 1 Characterizing the Extracellular Matrix................................................................................... 1 Core Matrisome Components.................................................................................................... 5 Collagens.................................................................................................................................. 5 Proteoglycans .......................................................................................................................... 6 Glycoproteins........................................................................................................................... 8 Matrisome Associated Molecules .............................................................................................. 9 Extracellular Matrix Receptors................................................................................................ 9 Extracellular Matrix Modifying Enzymes............................................................................... 12 The Drosophila Neuromuscular Junction as a Genetic Model Synapse................................... 18 The NMJ System .................................................................................................................... 18 Structural Synaptogenesis..................................................................................................... 21 Functional Synaptogenesis.................................................................................................... 22 Wnt Trans-Synaptic Signaling................................................................................................ 25 Heparan Sulfate Proteoglycans in Synaptic Development....................................................... 32 Secreted HSPGs ..................................................................................................................... 32 Membrane-tethered HSPGs .................................................................................................. 34 HS Biosynthesis and HS-modifying Enzymes......................................................................... 35 Matrix Metalloproteinases in Neural Development ................................................................ 35 Thesis Outline........................................................................................................................... 44
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Part One................................................................................................................................. 44 Part Two................................................................................................................................. 44 References................................................................................................................................ 45
II. Two Classes of Matrix Metalloproteinases Reciprocally Regulate Synaptogenesis ................ 75
Summary................................................................................................................................... 76 Introduction.............................................................................................................................. 76 Materials and Methods ............................................................................................................ 78 Drosophila Stocks .................................................................................................................. 78 Antibody Production ............................................................................................................. 78 Western Blot Analyses........................................................................................................... 80 Immunocytochemistry Imaging............................................................................................. 80 Electrophysiology .................................................................................................................. 81 Electron Microscopy.............................................................................................................. 81 Statistical Measurements...................................................................................................... 81 Results ...................................................................................................................................... 82 Mmp1 and Mmp2 both regulate synapse morphogenesis................................................... 82 Mmp1 and Mmp2 both regulate differentiation of synapse function ................................. 85 Mmp1 and Mmp2 both regulate synapse molecular assembly............................................ 87 Mmp1, Mmp2 and Timp co-dependently localize to the NMJ synaptomatrix..................... 90 Mmp1 and Mmp2 restrict Wnt trans-synaptic signaling ...................................................... 93 Restoring Wnt co-receptor Dlp levels in Mmp mutants prevents synaptogenic defects..... 97 Discussion................................................................................................................................. 99 References.............................................................................................................................. 102
III. Neuronal Activity Drives FMRP- and HSPG-dependent Matrix Metalloproteinase Function
Required for Rapid Synaptogenesis ....................................................................................... 109 Summary................................................................................................................................. 110 Introduction............................................................................................................................ 110 Materials and Methods .......................................................................................................... 112 Drosophila Genetics............................................................................................................. 112 Activity Manipulations......................................................................................................... 113
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Immunocytochemistry Confocal Imaging ........................................................................... 113 Synaptic In Situ Zymography ............................................................................................... 114 Quantification and Statistical Analyses ............................................................................... 115 Results .................................................................................................................................... 116 Mmp1 is required for rapid activity-dependent synaptic bouton formation ..................... 116 Co-localized Mmp1 and Dlp exhibit positive activity-dependent regulation ..................... 119 Dlp localizes Mmp1 in synaptic domains and bidirectionally promotes Mmp1 intensity.. 123 Dlp is required for the rapid, activity-dependent increase of synaptic Mmp1................... 126 Synaptic Dlp bidirectionally determines proteolytic function at the NMJ.......................... 129 FMRP regulation of activity-dependent synaptic Mmp1 requires Dlp function................. 131 Discussion............................................................................................................................... 134 References.............................................................................................................................. 137
IV. Conclusions and Future Directions........................................................................................ 145
Regulation of Mmp and Timp Expression .............................................................................. 146 Mmp Regulation of Presynaptic and Postsynaptic Molecular Machinery............................. 153 Heparan Sulfate and Wingless Regulation of Mmp1 ............................................................. 158 Mmps and Activity-dependent Synaptogenesis .................................................................... 165 HSPGs and Activity-dependent Synaptogenesis .................................................................... 167 FMRP Regulation of Mmps and HSPGs .................................................................................. 168 Mmp Regulation of Other Trans-synaptic Signaling Pathways.............................................. 171 BMP trans-synaptic signaling .............................................................................................. 171 Jeb-Alk trans-synaptic signaling .......................................................................................... 175 Perspectives and Extended Future Directions ....................................................................... 178 References.............................................................................................................................. 181
Appendix A. Supplementary Material ........................................................................................................ 197
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LIST OF FIGURES AND TABLES
- Figures
- Page
1. Components of the ECM and ECM-interacting molecules ......................................................... 4 2. The Drosophila larval NMJ model synapse ............................................................................... 20 3. Cartoon schematic of a synapse at the Drosophila NMJ .......................................................... 24 4. Trans-synaptic signaling pathways driving NMJ synaptogenesis ............................................. 30 5. Cartoon schematic of the pre- and postsynaptic Wg-activated pathways at the NMJ............ 31 6. Mmps are members of the Metzincin superfamily of metalloproteinases.............................. 37 7. Drosophila Mmp structure and selected tools ......................................................................... 39 8. Overarching hypothesis ............................................................................................................ 43 9. Mmp1 and Mmp2 repress NMJ structural development......................................................... 84 10. Mmp1 and Mmp2 repress functional differentiation of the NMJ.......................................... 86 11. Mmp1 and Mmp2 modulate synaptic ultrastructural development ..................................... 89 12. Mmp1, Mmp2 and Timp exhibit co-dependent synaptic localization.................................... 92 13. Mmp1 and Mmp2 restrict Wnt trans-synaptic signal transduction ....................................... 95 14. Mmp1 and Mmp2 reciprocally regulate Wnt HSPG co-receptor Dlp..................................... 96 15. Restoring Dlp levels in Mmp mutants prevents defects in NMJ structure or function.......... 98 16. Mmp1 is required for rapid activity-dependent synaptic bouton formation....................... 118 17. Synaptic Mmp1 is rapidly increased following acute neuronal stimulation ........................ 121
18. Mꢀpꢁ aꢂd Dlp ꢃo-loꢃalizatioꢂ iꢂ syꢂaptiꢃ suꢄdoꢀaiꢂs is iꢂꢃꢅeased ꢄy aꢃute aꢃtiꢆity ........ 122 19. Syꢂaptiꢃ Dlp positiꢆely aꢂd ꢄidiꢅeꢃtioꢂally ꢅegulates seꢃꢅeted Mꢀpꢁ aꢄuꢂdaꢂꢃe.............. 125 20. Dlp is ꢅeꢇuiꢅed foꢅ the aꢃtiꢆity-depeꢂdeꢂt syꢂaptiꢃ Mꢀpꢁ ꢅegulatioꢂ................................ 128 21. Dlp positiꢆely aꢂd ꢄidiꢅeꢃtioꢂally ꢅegulates pꢅoteolytiꢃ aꢃtiꢆity at the syꢂapse .................. 130
22. FMRP regulation of the activity-dependent Mmp1 enhancement requires Dlp ................. 133 23. Mmps are expressed by both neuron and muscle ............................................................... 149 24. Mmp2 localizes to postsynaptic muscle nuclei..................................................................... 150 25. Mmp1 is abnormally sequestered at NMJs overexpressing Timp........................................ 151 26. Proximity ligation assay (PLA) detects protein-protein interactions.................................... 152 27. Glia-derived Mmp1 restricts NMJ growth ............................................................................ 155 28. Mmps differentially regulate GluRs at the NMJ ................................................................... 156
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29. Mmp1 and Mmp2 promote DLG at the NMJ........................................................................ 157 30. Reduced extracellular Mmp1 following Heparan Sulfate enzymatic digestion ................... 161 31. Mmp1 directly binds Heparan Sulfate .................................................................................. 162 32. Possible functions of Mmp-HSPG interactions ..................................................................... 163 33. Wg promotes Mmp1 at the NMJ .......................................................................................... 164 34. FMRP interacts with mmp1 but not mmp2 mRNA ............................................................... 170 35. Mmp1 restricts BMP trans-synaptic signaling ...................................................................... 174 36. Mmp2 promotes extracellular Jeb ligand and both Mmps restrict dpERK signaling ........... 177
- Tables
- Page
1. Raw data values for synaptic bouton number and ultrastructure ......................................... 215 2. Raw data values for functional neurotransmission and molecular assembly ........................ 217 3. Raw data values for selected IHC experiments ...................................................................... 219 4. Raw data values for in situ zymography assays...................................................................... 221 5. Raw data values for activity assays......................................................................................... 222
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LIST OF SUPPLEMENTARY MATERIAL
- Supplemental Material
- Page
1. Postsynaptic Timp expression suppresses NMJ morphology defects. ................................... 197 2. Mmp1 and Mmp2 regulate spontaneous SV fusion rate and response amplitude. .............. 198 3. Loss of Mmp1 and Mmp2 differentially regulate NMJ molecular assembly.......................... 199 4. Mmp2 negatively regulates GluRIIA-containing receptors..................................................... 200 5. Mmp1 and Mmp2 negatively regulate GluRIIB-containing receptors.................................... 201 6. Western blot characterization and specificity of Mmp2 and Timp antibodies. ..................... 202 7. Imaging characterization and specificity of Mmp2 and Timp antibodies .............................. 203 8. Characterization of Mmp1, Mmp2 and Timp localization at the NMJ ................................... 204 9. Temperature controls for dTRPA1 activity-induced synaptic bouton formation................... 205
10. Mꢀpꢈ is ꢂot ꢅeꢇuiꢅed foꢅ aꢃtiꢆity-depeꢂdeꢂt syꢂaptiꢃ ꢄoutoꢂ foꢅꢀatioꢂ.......................... 206
11. Mmp1 is rapidly and specifically increased following dTRPA1 neuronal stimulation.......... 207
12. Synaptic Mꢀpꢈ is ꢅapidly ꢅeduꢃed folloꢉiꢂg aꢃute ꢂeuꢅoꢂal stiꢀulatioꢂ ........................... 208 13. Syꢂaptiꢃ Dlp is ꢅapidly iꢂꢃꢅeased folloꢉiꢂg aꢃute ꢂeuꢅoꢂal stiꢀulatioꢂ .............................. 209
14. Synaptic Mmp1 and Dlp imaging controls at the NMJ terminal........................................... 210
15. Syꢂaptiꢃ Dlp ꢃhaꢂges ꢉith ꢄidiꢅeꢃtioꢂal dlp geꢂetiꢃ ꢀaꢂipulatioꢂs .................................... 211 16. Synaptic Mꢀpꢈ ꢃhaꢂges ꢉith ꢄidiꢅeꢃtioꢂal dlp geꢂetiꢃ ꢀaꢂipulatioꢂs............................... 212
17. Activity-dependent synaptic Dlp increase occurs in the absence of Mmp1 ........................ 213 18. Synaptic Dlp in FXS disease model is restored by single copy dlp co-removal..................... 214
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LIST OF ABBREVIATIONS
(ADAM) (ADAMTS) (Alk) a disintegrin and metalloprotease a disintegrin and metalloprotease with thrombospondin type I-like motifs Anaplastic lymphoma kinase
- Analysis of Variance
- (ANOVA)
(AP-1) (aPKC) (APP)
Activator protein 1 atypical Protein kinase C β-amyloid precursor protein
- Armadillo
- (Arm)
- (Arr)
- Arrow
- (ASD)
- autism spectrum disorder
- Active zone
- (AZ)
- (BBB)
- blood brain barrier
- (BL)
- basal lamina
- (BM)
- basement membrane
(BMP) (BMPR) (BONCAT) (Brp)
Bone morphogenic protein Bone morphogenic protein receptor bio-orthogonal non-canonical amino acid tagging Bruchpilot
- (BSA)
- Bovine Serum Albumin
(CaCl2) (CAM) (CamKII) (Cas9) (cat) calcium chloride cell adhesion molecule Calcium/Calmodulin-dependent protein kinase CRISPR associated protein 9 catalytic
(cGMP) (CNS) cyclic guanosine monophosphate central nervous system
(Cow) (CRISPR) (CS)
Carrier of Wingless Clustered Regularly Interspaced Short Palindromic Repeats Chondroitin sulfate
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- (CSP)
- Cysteine String Protein
(CSPG) (Dad)
Chondroitin sulfate proteoglycan Daughters against decapentaplegic Division-abnormally delayed double heterozygous
(Dally) (dblhet) (dblRNAi) (dCask) (DCV)
double UAS-mmp1RNAi; UAS-mmp2dsRNAi1794-1R-2
Calcium/Calmodulin Dependent Serine Protein Kinase dense-core vesicle
- (Dg)
- Dystroglycan
(DGC) (dGRIP) (dLAR) (DLG)
Dystrophin-associated glycoprotein complex glutamate receptor interacting protein Leukocyte common antigen-related Discs-large
- (Dlp)
- Dally-like protein
(DNA) (dpERK) (Dpp) deoxyribonucleic acid diphosphorylated extracellular signal-related kinase Decapentaplegic
(DQ-gelatin) (Drl) dye-quenched fluorogenic gelatin Derailed
(DROP-1) (DS)
Proteoglycan-1 Dermatan sulfate
(DSPG) (dTRPA1) (Dvl)
Dermatan sulfate proteoglycan Transient receptor potential cation channel A1 Dishevelled
- (Eag)
- Ether a go-go
(ECM) (EDTA) (EJC)
Extracellular matrix ethylenediaminetetraacetic acid excitatory junctional current excitatory junctional potential Embryonic lethal abnormal vision Extracellular signal-related kinase
(EJP) (Elav) (ERK)
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(Evi/Wls/Srt) (EXT1) (FACIT) (FAK)
Evenness Interrupted/Wntless/Sprinter Exostosin-1 Fibril-associated collagen with interrupted triple helix Focal adhesion kinase Fibroblast growth factor fluorescent in situ hybridization Four-jointed
(FGF) (FISH) (Fj) (FMR1) (FMRP) (FNI) fragile X mental retardation 1 Fragile X Mental Retardation Protein Frizzled nuclear import
- Faulty Attraction
- (Frac)
(Frz2) (FUNCAT) (FXS)
Frizzled-2 receptor fluorescent non-canonical amino acid tagging Fragile X syndrome
- (Fz)
- Frizzled
(GAG) (Gbb) (GF) glycosaminoglycan Glass Bottom Boat growth factor
- (GFP)
- Green fluorescent protein
- N-Acetylglucosamine
- (GlcNAc)
(GlcNS) (GluR) (GOF) (GPI) sulfated N-Acetylglucosamine Glutamate receptor gain-of-function glycosylphosphatidylinositol Glycogen Synthase Kinase 3 Beta heparitinase
(GSK-ꢊβ) (Hep) (HEPES) (Hh)
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Hedgehog
- (hpx)
- hemopexin
(HRP) (HS)
Horse Radish Peroxidase Heparan sulfate