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TRPV4 in the CHOROID PLEXUS EPITHELIUM: PATHWAY ANALYSIS and IMPLICATIONS for CEREBROSPINAL FLUID PRODUCTION by Daniel Preston

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TRPV4 in the CHOROID PLEXUS EPITHELIUM: PATHWAY ANALYSIS and IMPLICATIONS for CEREBROSPINAL FLUID PRODUCTION by Daniel Preston TRPV4 IN THE CHOROID PLEXUS EPITHELIUM: PATHWAY ANALYSIS AND IMPLICATIONS FOR CEREBROSPINAL FLUID PRODUCTION by Daniel Preston A Thesis Submitted to the Faculty of Purdue University In Partial Fulfillment of the Requirements for the degree of Master of Science Department of Biology at IUPUI Indianapolis, Indiana December 2019 THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF COMMITTEE APPROVAL Dr. Bonnie Blazer-Yost, Chair Department of Biology Dr. Teri Belecky-Adams Department of Biology Dr. Nick Berbari Department of Biology Dr. James Clack Department of Biology, Indiana University-Purdue University Columbus Approved by: Dr. A.J. Baucum 2 To Stefanie, and to my Family, who have always supported me and will never read this thesis. And to my Cat, Loki, who never failed to sleep on my mouse when I was trying to write. 3 ACKNOWLEDGMENTS I would like to acknowledge my thesis committee for their support and guidance through the graduate program. In addition, I would like to thank Stefanie Simpson, Hillary Smith, Alex Hochstetler, Makenna Reed, Keith Gafunderi, Patrick Antonellis for their technical support, assistance with conducting experiments and contributions to data analysis. 4 TABLE OF CONTENTS LIST OF TABLES .......................................................................................................................... 8 LIST OF FIGURES ........................................................................................................................ 9 LIST OF ABBREVIATIONS ....................................................................................................... 11 ABSTRACT .................................................................................................................................. 13 INTRODUCTION .............................................................................................. 14 1.1 Hydrocephalus .................................................................................................................. 14 1.2 Treatment of Hydrocephalus ............................................................................................. 15 1.3 The Choroid Plexus ........................................................................................................... 16 1.4 Transient Receptor Potential Vanilloid 4 .......................................................................... 17 1.5 The WPK Rat Model of Hydrocephalus ........................................................................... 18 1.6 Porcine Choroid Plexus Cell Line..................................................................................... 19 1.7 Chloride Transporters ....................................................................................................... 19 1.8 Calcium-Activated Potassium Channels ........................................................................... 20 1.9 Sodium-Potassium-Chloride and Potassium-Chloride Cotransporters ............................. 21 1.10 STE20/SPS1-Related Proline-Alanine-Rich Protein Kinase .......................................... 22 1.11 Translational Potential .................................................................................................... 22 1.12 References ...................................................................................................................... 25 ACTIVATION OF TRPV4 STIMULATES ION FLUX IN PCP-R CELLS ..... 35 2.1 Preface ............................................................................................................................... 35 2.2 Activation of TRPV4 Stimulates Transepithelial Ion Flux in a Porcine CP Cell Line ..... 36 2.3 Abstract ............................................................................................................................. 37 2.4 Introduction ....................................................................................................................... 37 2.5 Materials and Methods ...................................................................................................... 39 2.6 Results ............................................................................................................................... 41 2.7 Discussion ......................................................................................................................... 43 2.8 Acknowledgements ........................................................................................................... 48 2.9 Grants ................................................................................................................................ 48 2.10 Disclosures ..................................................................................................................... 48 2.11 References ...................................................................................................................... 70 5 THE ROLE OF NKCC1 AND SPAK IN TRPV4-MEDIATED TRANSPORT 74 3.1 Preface ............................................................................................................................... 74 3.2 SPAK Inhibition Blocks TRPV4 Mediated Ion Flux in Choroid Plexus Epithelial Cells 74 3.3 Abstract ............................................................................................................................. 75 3.4 Introduction ....................................................................................................................... 75 3.5 Methods and Materials ...................................................................................................... 78 3.6 Results ............................................................................................................................... 80 3.7 Discussion ......................................................................................................................... 82 3.8 Acknowledgments............................................................................................................. 86 3.9 Grants ................................................................................................................................ 86 3.10 Disclosures ..................................................................................................................... 86 3.11 References .................................................................................................................... 105 CHLORIDE CHANNELS IN CPE TRANSEPITHELIAL TRANSPORT ..... 110 4.1 Introduction ..................................................................................................................... 110 4.2 Methods and Materials .................................................................................................... 112 4.3 Results ............................................................................................................................. 114 4.4 Discussion ....................................................................................................................... 116 4.5 References ....................................................................................................................... 143 INFLAMMATORY EFFECTS ON TRPV4 IN THE CHOROID PLEXUS ... 147 5.1 Preface ............................................................................................................................. 147 5.2 Inflammatory Effects on TRPV4 in the Choroid Plexus ................................................ 147 5.3 Abstract ........................................................................................................................... 148 5.4 Introduction ..................................................................................................................... 149 5.5 Materials and Methods .................................................................................................... 151 5.6 Results ............................................................................................................................. 154 5.7 Discussion ....................................................................................................................... 155 5.8 Acknowledgments........................................................................................................... 160 5.9 Grant Support .................................................................................................................. 160 5.10 Disclosures ................................................................................................................... 160 5.11 References .................................................................................................................... 189 SUMMARY ...................................................................................................... 194 6 6.1 Summary ......................................................................................................................... 194 6.2 Addressing the PCP-R Cell Polarity ............................................................................... 196 7 LIST OF TABLES Table 2.1 Primer Pairs Used for RT-PCR with Corresponding Product Sizes (bp). .................... 49 Table 3.1 Sus Scrofa Primers Used for RT-PCR with Corresponding Product Sizes (bp). .......... 87 Table 4.1 Sus Scrofa Primers Used for RT-PCR with Corresponding Product Sizes (bp). ........ 121 Table 5.1 Reported Cytokine Functions in the Inflammatory Pathway. ..................................... 161 Table 5.2 Sus Scrofa Primers Used for RT-PCR
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