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Regulation of GABAAR Signaling and Neuroadaptations in Response to Diazepam by Joshua M. Lorenz-Guertin B.S. Grand Valley State

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Regulation of GABAAR Signaling and Neuroadaptations in Response to Diazepam by Joshua M. Lorenz-Guertin B.S. Grand Valley State University, 2014 Submitted to the Graduate Faculty of School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2019 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Joshua M. Lorenz-Guertin It was defended on July 26, 2019 and approved by Michael Palladino, Professor, Pharmacology & Chemical Biology Alexander Sorkin, Professor, Cell Biology Simon Watkins, Professor, Cell Biology Gregg Homanics, Professor, Anesthesiology & Perioperative Medicine Dissertation Director: Tija Jacob, Assistant Professor, Pharmacology & Chemical Biology ii Copyright © by Joshua M. Lorenz-Guertin 2019 iii Regulation of GABAAR Signaling and Neuroadaptations in Response to Diazepam Joshua M. Lorenz-Guertin, PhD University of Pittsburgh, 2019 Despite 50+ years of use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of GABA, the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABAARs). Sustained treatment with BZD drugs is intimately associated with the development of tolerance, dependence, withdrawal and addiction. BZD efficacy diminishes after prolonged or high dose acute exposure, with tolerance to the sedative/hypnotic effects forming most quickly. We investigated the adaptive mechanisms occurring during initial exposure to the classical BZD, Diazepam (DZP), and the molecular signature of the mouse brain during established sedative tolerance. We found cultured neurons treated 24 h with DZP presented no change in surface or synaptic levels of γ2-GABAARs. In contrast, both γ2 and the key inhibitory synaptic scaffolding protein gephyrin levels were decreased after a single DZP treatment in vitro and in vivo. Live-imaging and label-free quantitative proteomics further revealed alterations in γ2 subunit surface trafficking, internalization and lysosomal targeting. In comparison, mice treated seven days with DZP had altered GABAAR subunit composition, reduced responsiveness to DZP, and tonic inhibition was diminished. Furthermore, DZP increased excitatory NMDA receptor subunit levels and function. State of the art mass spectrometry experiments revealed increased CaMKII subunits, which are positive regulators of NMDA receptors and involved in tolerance to other drugs. Downstream bioinformatics analysis confirmed robust synaptic plasticity after DZP. Together, we describe a time-dependent downregulation of synaptic GABAAR function after initial DZP exposure iv followed by an adaptive increase in excitatory neurotransmission, neuronal remodeling and altered synaptic GABAAR composition. v Table of Contents Acknowledgements .................................................................................................................... xiv 1.0 Introduction ............................................................................................................................. 1 1.1 The GABA Type A Receptor ......................................................................................... 1 1.2 GABAAR Pharmacology ................................................................................................ 3 GABAAR Clinical Agents ................................................................................... 3 BZD Clinical Use and Tolerance ....................................................................... 4 1.3 GABAAR Regulation and Surface Localization .......................................................... 6 Key GABAergic Synapse Components ............................................................. 6 Extrasynaptic GABAARs .................................................................................. 10 1.4 GABAAR Intracellular Trafficking ............................................................................ 13 Assembly and Forward Trafficking ................................................................ 13 Internalization, Recycling and Lysosomal Degradation................................ 15 1.5 Activity-Dependent Plasticity of GABAAR Synapses ................................................ 20 1.6 Advancing our Understanding of Dynamic GABAAR Trafficking and Regulation .............................................................................................................................................. 23 2.0 Designing an Optical Tool to Measure Multistage GABAAR Trafficking ....................... 25 2.1 Introduction .................................................................................................................. 25 2.2 Methods and Materials ................................................................................................ 28 Cell culture and Transfection .......................................................................... 28 DNA Constructs and Antibodies ..................................................................... 28 MG Dyes ............................................................................................................. 29 vi Immunocytochemistry and Confocal Microscopy ......................................... 29 Live-Cell Imaging .............................................................................................. 30 Western Blot ...................................................................................................... 32 Electrophysiology .............................................................................................. 32 Statistics ............................................................................................................. 33 2.3 Results ............................................................................................................................ 34 Expression and Function of γ2pHFAP in HEK293 Cells ................................ 34 γ2pHFAP is Clustered at Synapses in Neurons and Can be Used to Monitor Multistage Receptor Trafficking .............................................................................. 38 pH γ2 FAP Reveals Enhanced GABAAR Synaptic Turnover in an In Vitro Bicuculline Seizure Paradigm ................................................................................... 48 2.4 Discussion ...................................................................................................................... 51 3.0 Early Diazepam Exposure Alters GABAAR Intracellular Trafficking and Accelerates Synaptic Exchange ................................................................................................. 54 3.1 Introduction .................................................................................................................. 54 3.2 Methods and Materials ................................................................................................ 57 Cell Culture, Transfection, Expression Constructs and Mice ...................... 57 Reagents, Antibodies, and MG Dye ................................................................. 57 Fixed and Live-Imaging .................................................................................... 58 Lysosomal Targeting Assay .............................................................................. 59 NH4Cl Intracellular Imaging ........................................................................... 59 Intermolecular FRET Imaging, Characterization and Analysis .................. 60 Synaptic Exchange Rate FRAP Imaging ........................................................ 61 vii Quantitative PCR .............................................................................................. 62 Western Blot and Immunoprecipitation ......................................................... 63 Membrane and Subcellular Fractionation ................................................... 64 Co-immunoprecipitation ................................................................................ 64 Mass Spectrometry and Data Processing ...................................................... 65 Bioinformatics Analysis .................................................................................. 66 Statistics ........................................................................................................... 67 3.3 Results ............................................................................................................................ 67 12-24h Diazepam Decreases the GABAAR γ2 Subunit and Gephyrin through Post-Translational Mechanisms ................................................................................ 67 GABAAR Composition and Intracellular and Surface Trafficking are Changed by DZP In Vitro .......................................................................................... 73 Label-Free Quantitative Proteomics of γ2 GABAAR Following DZP Injection In Vivo ......................................................................................................................... 85 3.4 Discussion ...................................................................................................................... 94 4.0 Inhibitory and Excitatory Synaptic Neuroadaptations in the Diazepam Tolerant Brain ..........................................................................................................................................
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