DIPLOMARBEIT Effects of the Atypical Antipsychotic Clozapine On
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DIPLOMARBEIT Effects of the atypical antipsychotic clozapine on recombinantly expressed GABAA receptor subtypes zur Erlangung des akademischen Grades Dr. med. univ. ausgefuehrt am Zentrum fuer Hirnforschung unter der Anleitung von Prof. Dr. Margot Ernst, PhD Luca Leandro Silva Pita 05/2018 Contents 0.1 Acknowledgements . .i 0.2 Abbreviations . ii 0.3 Abstract . iv 0.4 Abstract [german] . .v 1 Introduction 1 1.1 GABA Metabolism . .2 1.2 GABA Transport . .3 1.3 GABAA Receptors . .5 1.3.1 Structure and Distribution . .5 1.3.2 Drug-receptor Interactions . 12 1.3.3 Endogenous GABAA Receptor Ligands . 13 1.3.3.1 GABA . 13 1.3.3.2 Other Endogenous Ligands . 14 1.3.4 Exogenous GABAA Receptor Ligands . 15 1.3.4.1 Benzodiazepines . 15 1.3.4.2 Anesthetics . 17 1.3.4.3 Other GABAA Receptor Ligands . 20 1.4 GABAB Receptors . 22 1.5 Schizophrenia . 25 1.5.1 Epidemiology . 26 1.5.2 Pathomechanism . 26 1.5.3 Pharmacological Management . 27 1.6 Clozapine . 29 2 Materials and Methods 32 2.1 Two-Electrode Voltage Clamp . 32 2.1.1 Xaenopus laevis oocytes . 32 2.1.2 Preparation of oocytes . 32 2.1.3 Electrophysiological recordings . 34 3 Results 36 3.1 α1β2 ................................ 37 3.2 α2β3 ................................ 38 3.3 α1β2γ2 ............................... 39 3.4 α2β3γ1 ............................... 40 3.5 α2β3γ2 ............................... 41 3.6 Summary . 42 4 Discussion 44 4.1 Exploring a possible intra-subunit binding pocket . 44 4.2 Agranulocytosis . 49 5 Summary and Outlook 50 List of tables 51 List of figures 53 Bibliography 55 0.1 Acknowledgements I wish to thank, first and foremost, Margot Ernst for the generous integration into her research group. Her encouraging way to allow for sovereign pursuit of emerging interests while providing a reliable framework with continuous, knowledgeable and heartfelt support promotes the formation of new ideas. I feel privileged having worked with someone who radiates true curiousity and dedication about her field. Moreover I want to share the credit of this work with everyone I encoun- tered during my time at the CBR - thank you for interesting conversations, fun times and emerging friendships. i 0.2 Abbreviations 2AG ................. 2-arachidonylglycerol THDOC .............3 α5α-tetrahydrodeoxycorticosterone DHP .................5 α-dihydroprogesterone AMPA .............. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid GABA .............. γ-aminobutyric acid BBB ................. blood brain barrier BZD ................. benzodiazepines CNS ................. central nervous system CLZ ................. clozapine D2 ................... dopamine D2 receptor DZP ................. diazepam DMSO .............. dimethylsulfoxide EC ................... effective concentration ECD ................. extracellular domain FMZ ................. flumazenil GPCR ............... G protein-coupled receptor GAT ................. GABA transporter GAD ................ glutamate decarboxylase GlyR ................ glycine receptor ii ICD ................. intracellular domain MDD ................ major depressive disorder mRNA .............. messenger ribonucleic acid NMDAR ............ N-methyl-D-aspartate receptor PKA ................. protein kinase A RT-PCR ............ real-time polymerase chain reaction SLC6 ................ solute carrier family 6 TMD ................ transmembrane domain TEV ................. two-electrode voltage clamp VFTD ............... venus fly-trap domain VGAT ............... vesicular GABA transporter VGCC ............... voltage-gated calcium channel WHO ................ World Health Organization XR .................. xaenopus ringer iii 0.3 Abstract Clozapine (CLZ) is a tetracyclic compound with outstanding clinical ef- fectiveness in the treatment of psychotic symptoms even in patients resis- tant to high doses of typical neuroleptics [Naber and Lambert, 2009,Attard and Taylor, 2012]. While CLZ has been shown to interact with multiple targets [Ashby and Wang, 1996, Michel and Trudeau, 2000, Korpi et al., 1995, Khokhar et al., 2018], its action at subtypes of the GABAA receptor family presents a unique feature when compared to the typical antipsychotic haloperidol [Korpi et al., 1995]. Treatment with CLZ is limited by its serious and potentially fatal side effects, which include increased seizure suscepti- bility and agranulocytosis [Pacia and Devinsky, 1994, Alvir et al., 1993]. As GABAergic interneurons form an undisputed core component of cor- ticolimbic circuity, defects in GABA transmission have become a central element in a number of models for psychiatric disease such as schizophrenia and bipolar disorder [Benes and Berretta, 2001]. In this work we studied CLZ's effects on five different recombinantly expressed GABAA receptor subtypes in electrophysiological experiments on Xenopus laevis oocytes. We show extensive negative allosteric modulation of GABA elicted currents at micromolar CLZ concentrations in all studied subtypes. Our results confirm that the existence of a γ subunit is no premise for CLZ sensitivity. The maximum mean inhibition at 100umol CLZ is significantly higher in α2 containing assemblies (α2β3γ2, α2β3γ1 and α2β3) compared to α1 containing assemblies (α1β2 and α1β2γ2). In light of hints gained from structural models of analog proteins, we explore a tentative intra-subunit binding site candidate for CLZ and related molecules. Our findings prompt for a deeper examination of a possible link between some of CLZs clinical effects and its negative modulatory action on GABAA receptors. iv 0.4 Abstract [german] Clozapin (CLZ) ist ein tetracyclischer Wirkstoff mit herausragender klinis- cher Wirksamkeit in der Behandlung psychotischer Symptome in Patienten, die nicht auf typische Neuroleptika ansprechen [Attard and Taylor, 2012]. Waehrend der Wirkmechanismus von CLZ unzureichend geklaert ist, kon- nten Interaktionen mit verschiedenen Zielmolekuelen experimentell gezeigt werden [Ashby and Wang, 1996, Michel and Trudeau, 2000, Korpi et al., 1995]. Die Beeinflussung von Stroemen in Subtypen der GABAA Rezeptor- familie stellt eine einzigartige Eigenschaft im Vergleich zum typischen Neu- roleptikum Haloperidol dar [Korpi et al., 1995]. Die Behandlung mit CLZ ist begrenzt durch ihre schweren und potentiell toelichen Nebenwirkungen [Pa- cia and Devinsky, 1994, Alvir et al., 1993]. In dieser Arbeit wurde die Wirkung von CLZ auf fuenf verschiedene rekombinant exprimierte GABAA Rezeptor Subtypen in elektrophysiologis- chen Experimenten an Xaenopus laevis Oozyten untersucht. In allen un- tersuchten Subtypen zeigen wir eine ausgepraegte Reduktion von GABA induzierten Stroemen durch mikromolare CLZ Konzentrationen. Die Ergeb- nisse zeigen, dass die γ Untereinheit keine Vorraussetzung fuer CLZ Sensitiv- itaet ist. Die maximale mittlere Inhibition bei 100umol CLZ ist signifikant hoeher in α2 beinhaltenden Subtypen (α2β3γ2, α2β3γ1 und α2β3) verglichen mit α1 beinhaltenden Subtypen (α1β2 und α1β2γ2). Im Licht der Erkenntnisse aus Vergleichen mit Strukturmodellen analoger Proteine schlagen wir eine intra-subunit Bindestelle als Kandidat fuer die Vermittlung der negativ modulatorischen Effekte von CLZ vor. Die Ergeb- nisse regen zu einer tieferen Auseinandersetzung mit dem potentiellen Zusam- menhang zwischen negativ modulatorischen Effekten auf GABAA Rezep- toren und einigen von CLZs klinischen Effekten an. v 1. Introduction The human CNS is a network comprised of approximately 100 billion neu- ral cells connected through a large number of synaptic interfaces. These synapses allow for phasic signal transmission between cells through neuro- transmitter release. Neurotransmitters trigger excitatory (depolarizing) or inhibitory (hyperpolarizing) changes in the postsynaptic cell as a result of interactions with target molecules (receptors). Neurotransmitter release and action also occurs at extrasynaptic locations, leading to an overall change in the system's excitability; a principle referred to as tonic signaling. Once the net depolarization of a neural cell reaches a certain threshold, an action potential is triggered, ultimately enabling subsequent signal transmission. In healthy brains, excitation and inhibition are finely attuned, con- tributing to an environment promotive for information flow and cellular health [Wolfe et al., 2010,Schonfeld-Dado and Segal, 2009]. A disruption of this equilibrium can be observed in a multitude of neuropsychiatric disor- ders, including schizophrenia and epilepsy [Schnitzler and Gross, 2005]. While glutamic acid constitutes the principal excitatory transmitter, γ- aminobutyric acid (GABA) and glycine carry out inhibitory neurotrans- mission in mammals. GABA transmission occurs at an estimated 30% of mammalian synapses, making it a principle signalling mechanism. Its ac- tion goes beyond simple inhibition, as GABA can also exhibit excitatory, shunting and modulatory effects [Ernst et al., 2018]. Exemplary, GABAer- gic interneurons are involved in the integration of synaptic inputs through 1 oscillatory modulation of membrane potentials [Schnitzler and Gross, 2005]. The early hypothesized purely inhibitory nature of GABA was first chal- lenged by observation of depolarizing effects in immature neurons [Ben-Ari et al., 1989]. Later these effects were found to be present and important even in the adult CNS [Spitzer, 2010]. Besides the GABA systems fundamental physiological roles, GABA re- ceptors constitute molecular targets of essential and widely employed drugs such as benzodiazepines and anesthetics. Research into the GABA sys- tem has become