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Role of Regulator of G-Protein Signaling Proteins in Serotonin and Opioid Mediated Behaviors

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Role of Regulator of G-Protein Signaling Proteins in Serotonin and Opioid Mediated Behaviors Role of Regulator of G-Protein Signaling Proteins in Serotonin and Opioid Mediated Behaviors by Nicolas B. Senese A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Pharmacology) in The University of Michigan 2017 Doctoral Committee: Professor John R. Traynor, Chair Assistant Professor Asim Beg Professor Margaret E. Gnegy Assistant Professor Emily M. Jutkiewicz Professor Henry I. Mosberg Nicolas B. Senese [email protected] ORCID Identifier: orcid.org/0000-0002-9615-8587 ACKNOWLEDGMENTS I would like to acknowledge our collaborators who have contributed their time and data to these projects, my thesis committee for their guidance over the years, and everyone in the Traynor lab past and present for their advice and expertise. I would also like to thank my friends and family who supported me throughout this entire process, and the Department of Pharmacology staff, administrators and faculty whose hard work makes this research possible. Finally, I would like to thank Dr. John Traynor for his mentorship, and unwavering support both personal and professional. ii TABLE OF CONTENTS ACKNOWLEDGMENTS ii LIST OF FIGURES vi LIST OF TABLES viii LIST OF ABBREVIATIONS ix ABSTRACT xii INTRODUCTION 1 CHAPTER 1. The Role of RGS and G-Proteins in the Regulation of Depressive Disorders 5 GPCRs in Depression 5 G-protein Subunits Involved in Depression and Antidepressant Action 7 RGS Protein Regulation of G-Protein Activity 12 RGS Proteins Involved in Depression and Antidepressant Action 13 RGS Insensitive G-Proteins 17 Conclusions 19 References 20 2. Loss of RGS Control at Gαi2 Produces Antidepressant-Like Behavior by an Action at 5-HT1A Receptors in the Hippocampus. 32 iii Abstract 32 Introduction 33 Materials and Methods 35 Results 39 Discussion 48 References 51 3. RGS Regulation of Opioid Receptor Signaling and Antinociception 58 RGS Regulation of Opioid Receptor Signaling and Antinociception 58 Use of RGS Insensitive G-Protein Variants to Study Opioid Signaling 70 Conclusions 72 References 73 4. Loss of RGS Control at Gαo Produces a Nociception/Orphanin FQ Receptor Dependent Hyperalgesia: Implications for a Balance Between Nociceptin Receptor and Mu-Opioid Receptor Systems 85 Abstract 85 Introduction 86 Materials and Methods 88 Results 91 Discussion 98 References 101 5. Overall Discussion 111 5-HT1AR-Mediated Antidepressant-Like Behaviors 111 Opioid-Receptor Mediated Hyperalgesic Behaviors 117 iv Conclusions 126 References 127 v LIST OF FIGURES FIGURE 0.1 Ribbon Diagram of the Mu-Opioid Receptor Crystal Structure Bound to a Morphinan Antagonist 1 0.2 Ribbon Diagram of RGS4 (Top) in Contact with Gαi1 (Bottom) Generated from a AlF4- Stabilized Crystal Structure 1 0.3 Depiction of GPCR Cycle with Regulation by RGS Proteins 2 1.1 Ratio of Non-Lipid Raft vs. Lipid-Raft Associated Gαs Protein Expression in Post-Mortem Tissue from Suicide Victims Vs. Controls 9 1.2 Antidepressant-Like Phenotype of Heterozygous (GS/+) and Homozygous (GS/GS) RGS Insensitive Gαi2 Knock-in Mice Compared to Wild Type (+/+) Littermates 17 2.1 Effects of the 5-HT1A Antagonist WAY100635 on Spontaneous Antidepressant- Like Behavior in Homozygous RGSi Gαi2 Expressing Mice (GS/GS) 39 2.2 Effects of Intrahippocampal 8-OH-DPAT Administration in Wild Type Mice 40 2.3 Recording from Hippocampal Slices in the Presence of 8-OH-DPAT from Wild Type and RGSi Gαi2 Knock-in Mice 43 2.4 Intra-Hippocampal CCG-203769 Administration Produces Antidepressant-Like Effects in Female Mice 45 2.5 Hippocampal [3H]8-OH-DPAT Binding and G-Protein Expression is Unaltered in Heterozygous RGSi Gαi2 Knock-in Mice 46 2.6 Spontaneous Antidepressant-Like Behavior in Mice Expressing RGSi Gαo is Not Reversed by the 5-HT1A Antagonist WAY100635 47 3.1 Heterozygous RGSi Gαo Knock-in Mice (Gαo +/GS) Display a Naltrexone Reversible Baseline Antinociceptive Phenotype on the Hot Plate Test 71 4.1 Baseline Mechanical Nociceptive Threshold in Heterozygous RGSi Gαo Knock- in Mice (+/GS) Compared to Wild Type (WT) Littermates 92 vi 4.2 Effect of NOPR Agonist Ro64-6198 on Mechanical Nociceptive Threshold in Wild Type Mice 93 4.3 Effect of NOPR Antagonist J-113397 Delivered Intracerebroventricularly (i.c.v.) on Mechanical Nociceptive Threshold in Heterozygous RGSi Gαo Knock-in Mice (+/GS) and Wild Type (WT) Littermates 94 4.4 Effects of NOPR Antagonist J-113397 Pretreatment on Mechanical Hyperalgesia Produced by Intraplantar Administration of a 2.5% λ-Carrageenan Solution Given 72 Hours Before Testing in Wild Type Mice 95 4.5 Stimulation of [35S]GTPγS Binding by N/OFQ in Whole Brain Membrane Homogenates from Heterozygous RGSi Gαo Knock-in Mice (+/GS) and Wild Type (WT) Littermates 97 4.6 Effect of NOPR Agonist Ro64-6198 on Mechanical Nociceptive Threshold in Heterozygous Gαo Knock-out (+/-) Mice and Wild Type (+/+) Littermates 98 5.1 Diagram of Brain 5-HT Network 112 5.2 Pilot 50C Warm Water Tail Withdrawal Test Results in Wild Type (WT) and Heterozygous RGSi Gαo Knock-in Mice (+/GS) Treated with NOPR Antagonist J- 113397 118 5.3 Pilot von Frey Test Results in Wild Type (WT) and Heterozygous RGSi Gαi2 Knock-in Mice (+/GS) Treated with NOPR Agonist Ro64-6198 120 5.4 MAPK Activity (Quantified as Ratio of pERK/Total ERK) in Whole Brain Homogenates from Heterozygous RGSi Gαo Knock-in Mice (+/GS) and Wild Type (WT) Littermates Administered Ro64-6198 or Vehicle i.p. 121 5.5 Effects of 50 μg Enkephalinase Inhibitor RB-101 Administered i.c.v. 122 5.6 Effects of a 15-Minute Swim Stress in Room Temperature Water on 52C Hot Plate Test Responding Before and After 10 mg/kg i.p. Naloxone 123 5.7 Pilot Study of Tolerance to Morphine Challenge One Week After Morphine Pellet Implantation in the Flank of a Wild Type Mouse on the 52C Hot Plate Test 124 5.8 15-Minute Elevated Plus Maze Results with Wild Type (WT) and Heterozygous RGSi Gαo Knock-in Mice (+/GS) Following i.p. Saline Administration 125 vii LIST OF TABLES TABLE 0.1 RGS Protein Families, with Conserved Domains and G-Protein Specificity Highlighted 3 2.1 Bmax and Kd of Hippocampal [3H]8-OH-DPAT Binding 47 3.1 General Overview of Findings Described in this Chapter Showing RGS Protein Regulation of MOPR, DOPR, KOPR and NOPR-Mediated Effects 60 4.1 [3H]N/OFQ Saturation Binding and Potency of [35S]GTPγS Uptake Stimulation by N/OFQ. 97 viii LIST OF ABBREVIATIONS Analysis of variance (ANOVA) artificial cerebrospinal fluid (aCSF) Bicinchoninic acid (BCA) cAMP response element-bindinG-protein (CREB) Central nervous system (CNS) Complete Freund's adjuvant (CFA) Cornu Ammonis 1 (CA1) Cyclic adenosine monophosphate (cAMP) Delta opioid receptor (DOPR) Dorsal raphe nucleus (DRN) Dorsal root ganglion (DRG) Fibroblast growth factor 2 (FGF2) G-protein-coupled receptor (GPCR) G-protein-coupled receptor kinase (GRK) G-protein-coupled inwardly-rectifying potassium channel (GIRK) G-protein gamma-like (GGL) Gamma-Aminobutyric acid (GABA) Glycogen synthase kinase 3 beta (GSK3β) GTPase activatinG-protein (GAP) Guanosine diphosphate (GDP) Guanosine triphosphate (GTP) Input resistance (IR) Intracerebroventricular (i.c.v.) ix Intraperitoneal (i.p.) Intraplanter (i.pl.) Kappa opioid receptor (KOPR) Major depressive disorder (MDD) Messenger ribonucleic acid (mRNA) Mitogen-activated protein kinase (MAPK) Monoamine oxidase inhibitor (MAOI) Mu opioid receptor (MOPR) N-methyl-D-aspartate (NMDA) Nicotinamide adenine dinucleotide phosphate (NADPH) Nociceptin receptor (NOPR) Nociceptin/Orphanin FQ (N/OFQ) Periaqueductal gray (PAG) Regulator of G-protein signaling (RGS) Resting membrane potential (RMP) Rho guanine nucleotide exchange factor (RhoGEF) RGS homology (RH) RGS insensitive (RGSi) Selective serotonin reuptake inhibitors (SSRI) Serotonin (5-HT) Serotonin 1A receptor (5-HT1AR) Serotonin norepinephrine reuptake inhibitor (SNRI) Single nucleotide polymorphism (SNP) Sodium dodecyl sulfate (SDS) Standard error of measurement (SEM) Subcutaneous (s.c.) Tail suspension test (TST) Triton-X 100 (TTX100) x Triton-X 114 (TTX114) Wild type (WT) xi ABSTRACT Multiple classes of drugs, including antidepressants as well as opioid analgesics, exert their therapeutic effects at least in part by direct or indirect actions at G-protein-coupled receptors (GPCR’s). This class of receptor propagates a signal inside the cell by activating heterotrimeric G-proteins (comprised of Gα and Gβγ subunits). Following receptor activation GDP bound to the Gα subunit of the heterotrimer is exchanged for GTP, followed by separation of the Gα and Gβγ subunits. Both Gα and Gβγ then activate downstream signaling pathways inside the cell. The regulator of G-protein signaling (RGS) proteins are a class of intracellular regulatory proteins that serve as negative modulators of GPCR signaling. They exert their actions by binding to active Gα-GTP subunits and accelerating the hydrolysis of GTP to GDP with subsequent reformation of the Gα/βγ heterotrimer, thus terminating signaling. In this work, I describe the use of mice expressing Gα subunits of one of two types (either Gαi2 or Gαo) that do not bind to RGS proteins and so are RGS insensitive (RGSi) to gain a better understanding of how RGS proteins regulate both antidepressant-like and antinociceptive behaviors. Site-specific microinjections of a serotonin 1A receptor (5HT1AR) antagonist and agonist are used to show that mice expressing RGSi Gαi2 have a robust antidepressant-like phenotype dependent on hippocampal 5-HT1AR activity. Ex vivo recording from hippocampal tissue confirms that a 5-HT1AR agonist inhibits cellular activity more effectively in mice expressing RGSi Gαi2. Furthermore, I demonstrate that hippocampal administration of an RGS4/19 inhibitor (CCG203769) produces antidepressant-like effects. I show that mice expressing RGSi Gαo have a complex phenotype including increased and decreased sensitivity to noxious stimuli consistent with alterations in both nociceptin receptor (NOPR) and mu opioid receptor (MOPR) activity. The balance between the antinociceptive MOPR and pronociceptive NOPR systems is disturbed in the RGSi Gαo mice and in wild type mice during inflammatory pain.
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