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TABLE OF CONTENTS Page List of Figures ............................................................................................................ iii List of Tables ............................................................................................................. iv List of Abbreviations................................................................................................... v Chapter 1: Introduction and Statement of Problem .................................................... 1 cAMP signal transduction ................................................................................ 1 The Phosphodiesterase 8 (PDE8) Family ....................................................... 5 Statement of Problem ..................................................................................... 8 Chapter 2: Phosphodiesterase 8B is a Major Modulator of Adrenal Steroidogenesis ................................................................................................................................. 10 Summary ....................................................................................................... 10 Introduction ................................................................................................... 11 Materials and Methods .................................................................................. 15 Results .......................................................................................................... 21 Discussion ..................................................................................................... 41 Chapter 3: Characterization of a PDE8-selective Inhibitor (PF-04957325) .............. 47 Summary ....................................................................................................... 47 Introduction ................................................................................................... 48 Materials and Methods .................................................................................. 50 Results .......................................................................................................... 53 Discussion ..................................................................................................... 64 Chapter 4: The Role of PDE8B in the Central Nervous System .............................. 66 Summary ....................................................................................................... 66 Introduction ................................................................................................... 67 Materials and Methods .................................................................................. 69 Results .......................................................................................................... 75 Discussion ..................................................................................................... 90 Final Remarks and Future Directions....................................................................... 94 Bibliography ............................................................................................................. 98 Appendix A: Enzyme Kinetic Properties and Selective Inhibitors of PDEs ............. 111 Appendix B: Trilostane Control .............................................................................. 112 Appendix C: shRNA Plasmids Transfection Efficiency in Y-1 Cells ....................... 113 i Appendix D: PDE-selective Inhibitor (PF-04957325) Selectivity Data on PDE Isoforms ................................................................................................................. 115 Appendix E: The Novel Object Recognition Paradigm ........................................... 116 Appendix F: PDE8B KO Mice Had Same Mobility as WT Controls in Contextual Fear Conditioning ........................................................................................................... 118 Appendix G: PDE8B KO Mice Showed Normal Swimming Distance in the Morris Water Maze ........................................................................................................... 119 Appendix H: Training Significantly Decreased the Amount of Restraint-induced Corticosterone Release ......................................................................................... 120 Appendix I: Preliminary Results from the Adjusted Morris Water Maze ................. 121 ii LIST OF FIGURES Figure Number Page 1.1. The cAMP signaling pathway. ............................................................................. 3 1.2. Cyclic nucleotide phosphodiesterase (PDEs) families ........................................ 4 2.1. PDE8B is highly expressed in AZF cells ........................................................... 22 2.2 Full-length PDE8B mRNA transcript and PDE8B enzymatic activity are absent in PDE8B KO adrenal glands ...................................................................................... 24 2.3. PDE8B KO mice exhibit adrenal hypersensitivity in vivo ................................... 27 2.4. Weight of the adrenal glands of PDE8B KO mice compared to WT controls ... 28 2.5. PDE8B gene ablation increases mRNA expressions of p450s ......................... 30 2.6. PDE8 inhibition with inhibitors increases acute adrenal steroid production ...... 34 2.7. shRNA against PDE8B also increases acute adrenal steroid production in Y-1 cells.......................................................................................................................... 36 2.8. PDE8 inhibition increases basal PKA activity and also mRNAs of steroidogenic enzymes .................................................................................................................. 39 2.9. PDE8 inhibition increases the phosphorylation of HSL ..................................... 40 2.10. A model for modulation of PDEs ..................................................................... 46 3.1. Inhibition curve of PF-04957325 against PDE8B and PDE8A .......................... 55 3.2. PF-04975325 inhibition curve on recombinant PDE4D ..................................... 59 3.3. PF-04957325 potentiates pregnenolone secretion in Y-1 cells ......................... 62 3.4. 100 nM PF-04957325 selectively inhibits PDE8s in adrenal cells ..................... 63 4.1 Elevated plus maze ............................................................................................ 70 4.2. PDE8B expression in the mouse brain ............................................................. 77 4.3. IBMX-insensitive PDE activity found in the pellet of a PDE8B immunoprecipitate from striatal lysate .................................................................................................... 78 4.4. PDE8B KO mice exhibit anxiety-like behaviors ................................................. 81 4.5. PDE8B KO mice show enhanced freezing behavior in contextual fear conditioning .............................................................................................................. 83 4.6. PDE8B KO mice have increased generalized fear response after an adverse stimulus.................................................................................................................... 86 4.7. PDE8B KO mice did not show a significant improvement in performance in the Morris water maze ................................................................................................... 89 iii LIST OF TABLES Table Number Page 1. Inhibitor sensitivity of PDE8B ............................................................................... 49 2. Selectivity of PF-04957325 .................................................................................. 56 iv LIST OF ABBREVIATIONS 3HSD ------------------------ 3--hydroxysteroid dehydrogenase 5-DHEA ---------------------- 5-Dehydroepiandrosterone 5’-AMP ------------------------ adenosine monophosphate ACTH ------------------------- adrenocorticotropic hormone ADSD ------------------------- autosomal-dominant striatal degeneration AKAP ------------------------- A-kinase-anchoring protein ANP --------------------------- atrial natriuretic peptide ATP ---------------------------- adenosine 5’-triphosphate AZF ---------------------------- adrenal zona fasciculata BSA --------------------------- bovine serum albumin cAMP ------------------------- cyclic adenosine monophosphate cGMP ------------------------- cyclic guanosine monophosphate CNG channels -------------- cyclic nucleotide-gated (CNG) ion channels cpm ---------------------------- count per minute CRF --------------------------- corticotropin-releasing factor DMEM ------------------------ Dulbecco’s modified eagle medium DMSO ------------------------ dimethyl sulfoxide DTT ---------------------------- dithiothreitol EC50 --------------------------- half maximal effective concentration EDTA ------------------------- ethylenediamine-N,N,N’,N’-tetraacetic acid EGTA ------------------------- ethylene glycol (-aminoethyl ether)-N,N,N’,N’tetraacetic acid v ELISA ------------------------- enzyme-linked immunosorbent assay EPAC ------------------------- exchange protein activated by cAMP F12K -------------------------- F-12 Kaighn’s modification FBS ---------------------------- fetal bovine serum GFP --------------------------- cGMP regulated PDEs, Anabaena adenylyl cyclase, E. coli protein FhIA green fluorescent protein HBSS ------------------------- Hank’s balance salt solution HEPES ----------------------- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPA axis --------------------- hypothalamic-pituitary-adrenal axis GAF --------------------------- HSL ---------------------------- hormone sensitive lipase IBMX -------------------------- 3-isobutyl-1-methylxanthine IC50 ---------------------------- half maximal inhibitory concentration IPTG -------------------------- isopropyl -D-1-thiogalactopyranoside Km ------------------------------ Michaelis constant KO ----------------------------- knockout mouse mA ----------------------------- milliampere MC2R ------------------------- melanocortin 2 receptor mg ----------------------------- milligram mL ----------------------------- milliliter mM ---------------------------- millimolar mm ---------------------------- millimeter MOPS ------------------------- 3-(N-morpholino)propanesultonic acid vi mRNA ------------------------- messenger RNA nM ----------------------------- nanomolar p450 --------------------------- cytochrome P450 PAS --------------------------- Per, ARNT and Sim proteins PBS --------------------------- phosphate buffered saline PCR --------------------------- polymerase chain reaction PDEs -------------------------- cyclic nucleotide phosphodiesterases PFA ---------------------------- paraformaldehyde PKA --------------------------- protein kinase A pM ----------------------------- picomolar REC --------------------------- signal receiver RI ------------------------------ recognition index RNAi -------------------------- RNA interference RT-PCR ---------------------- real time polymerase chain reaction shRNA ------------------------ short hairpin RNA StAR protein ---------------- steroidogenic acute regulatory protein Vmax ---------------------------- maximum reaction rate vol ----------------------------- volume WT ----------------------------- wildtype mouse wt ------------------------------ weight X-gal -------------------------- 5-bromo-4-chloro-3-hydroxyindole vii Acknowledgements I extend immense gratitude to all my teachers who brought the best out of me, especially my thesis advisor, Professor Joe Beavo, who instilled in me much knowledge in matters both professional and personal. Joe shaped me into the scientist that I am today. Without the support and freedom that I received in the Beavo laboratory, this thesis would not have been possible. Additionally, Joe exposed me to a broad range of scientific topics and taught me to be critical when interpreting data. I also thank my committee members for their patience and insightful feedback on my research. Finally, I would like to thank the members of the Beavo laboratory for all of the helpful discussions and assistance throughout my graduate studies. On a more personal note, I thank my family and friends, especially my parents and my partner, Lincoln Ballard, for their emotional support and unconditional love. Without them, the process of pursuing this degree would be much more challenging and much less fun. viii