University of Lisbon Institute of Pharmacology and Neuroscience, Faculty of Medicine Unit of Neuroscience, Institute of Molecular Medicine Setting GABA levels: GABA transporters modulation by adenosine receptors Ana Sofia Cristóvão Ferreira Tese orientada pela Professora Doutora Ana Maria Sebastião PhD in Biomedical Sciences Speciality in Neuroscience Todas as afirmações efectuadas no presente documento são da exclusiva responsabilidade do seu autor, não cabendo qualquer responsabilidade à Faculdade de Medicina de Lisboa pelos conteúdos nele apresentados. Lisbon 2012 A impressão desta dissertação foi aprovada pelo conselho científico da Faculdade de Medicina de Lisboa em reunião de 15 de Maio de 2012. ii The experimental work described in this thesis was performed at the Institute of Pharmacology and Neuroscience, Faculty of Medicine and Unit of Neuroscience, Institute of Molecular Medicine, under de supervision of Professor Ana Maria Sebastião. O trabalho experimental descrito nesta tese foi realizado no Instituto de Farmacologia e Neurociências, Faculdade de Medicina e Unidade de Neurociências, Instituto de Medicina Molecular, sob orientação da Professora Doutora Ana Maria Sebastião. iii iv Publications The scientific content of the present thesis has been included in the publication of the following original articles: Cristóvão-Ferreira S , Navarro G, Brugarolas M, Pérez-Capote K, Vaz SH, Fattorini G, Conti F, Lluis C, Ribeiro JA, McCormick PJ, Casadó V, Franco R, Sebastião AM (2011) Modulation of GABA transport by adenosine A1R-A2AR heteromers, which are coupled to both Gs- and G(i/o)-proteins . J Neurosci. 31, 15629-15639. Cristóvão-Ferreira S , Vaz SH, Ribeiro JA, Sebastião AM (2009) Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1. J Neurochem . 109, 336-347. Only the experiments performed by the author of this thesis were included in the corresponding results chapter. Experiments performed by other co-authors (the minority of the papers content) are referred as such and discussed . Other publications closely related to the content of this thesis: Sebastião AM, Cristóvão-Ferreira S, Ribeiro JA (2012) Downstream pathways of adenosine in Adenosine: a key Link between Metabolism and CNS Activity. Edited by Masino SA and Boison D. v Vaz SH, Jørgensen TN, Cristóvão-Ferreira S , Duflot S, Ribeiro JA, Gether U, Sebastião AM (2011) Brain-derived neurotrophic factor (BDNF) enhances GABA transport by modulating the trafficking of GABA transporter-1 (GAT-1) from the plasma membrane of rat cortical astrocytes . J Biol Chem. 286, 40464-40476. Vaz SH, Cristóvão-Ferreira S , Ribeiro JA, Sebastião AM (2008) Brain- derived neurotrophic factor inhibits GABA uptake by the rat hippocampal nerve terminals. Brain Res. 1219, 19-25. vi Para a minha mãe, vii viii “Tudo o que sabemos é uma impressão nossa, e tudo o que somos é uma impressão alheia” In “O Livro do Desassossego”, Bernardo Soares ix x Table of Contents 1 Introduction ..........................................................................1 1.1 GABA ................................................................................... 1 1.1.1 GABA Receptors.......................................................... 2 1.1.2 GABA metabolism ....................................................... 6 1.1.3 GABA transporters ...................................................... 8 1.2 The tripartite synapse and glial cells................................. 24 1.2.1 Astrocytes ................................................................. 24 1.3 Adenosine ......................................................................... 35 1.3.1 Adenosine synthesis.................................................. 37 1.3.2 Nucleoside transporters ........................................... 40 1.3.3 Adenosine degradation............................................. 43 1.3.4 Adenosine levels regulation at brain ........................ 47 1.3.5 Adenine nucleotides, adenosine and signaling ........ 48 1.3.6 Adenosine receptors and signaling pathways .......... 50 1.3.7 A1-A2A adenosine receptors interaction.................... 56 1.4 Heteromers of G protein coupled receptors .................... 57 2 Aims.................................................................................... 65 3 Techniques.......................................................................... 67 3.1 Isolated presynaptic terminals.......................................... 67 3.2 Primary cultures of astrocytes.......................................... 68 xi 3.3 Biotinylation assays........................................................... 69 3.4 BRET – bioluminescence resonance energy transfer ....... 70 35 3.5 GTP-γ-[ S]-assay............................................................... 73 4 Methods.............................................................................. 77 4.1 Reagents............................................................................ 77 4.2 Experimental protocols..................................................... 79 4.2.1 Synaptosomes isolation ............................................ 79 4.2.2 Cell lines and primary astrocytic cultures................. 80 3 4.2.3 [ H]GABA uptake assays............................................ 80 4.2.4 Biotinylation assays................................................... 82 4.2.5 Western Blot ............................................................. 84 4.2.6 Immunocytochemistry.............................................. 85 4.2.7 BRET .......................................................................... 85 35 4.2.8 [ S] GTP-γ- S assay.................................................... 86 4.3 Statistical analysis ............................................................. 87 5 Results ................................................................................ 89 5.1 Adenosine modulation of GAT-1-mediated GABA uptake by synaptosomes ............................................................................... 89 5.1.1 Rationale ................................................................... 89 5.1.2 Adenosine A 2A receptors tonically enhance GAT-1 - mediated GABA transport......................................................... 89 5.1.3 Adenosine A1 and A 2B receptors do not affect GAT-1 mediated GABA transport......................................................... 95 xii 5.1.4 Activation of adenylate cyclase mimics adenosine A 2A receptors activation and PKA blockade prevents the action of the A 2A receptors agonist.......................................................... 97 5.1.5 PKC constitutively inhibits GABA transport, and prevents A 2A receptors -mediated facilitation of GABA transport 99 5.1.6 PKA and PKC interaction ......................................... 103 5.1.7 Discussion................................................................ 107 5.2 Modulation of astrocytic GABA Transport by Adenosine A1–A2A Receptor Heteromers...................................................... 113 5.2.1 Rationale ................................................................. 113 5.2.2 Endogenous adenosine tonically modulates GABA uptake 114 5.2.3 Adenosine A1 receptors activation decreased and adenosine A2A receptors activation enhanced GABA uptake 117 5.2.4 Adenosine A 1 -A2A receptor heteromers in astrocytes................................................................................ 123 5.2.5 Adenosine A 1 or A 2A receptors activation, but not its blockade, leads to internalization of the A1-A2A receptor heteromers ............................................................................. 128 5.2.6 The adenosine A1-A2A receptor heteromer is coupled to G i/0 and G s proteins............................................................. 131 5.2.7 The A 1-A2A receptor heteromer signals through AC/PKA pathway ..................................................................... 138 5.2.8 Discussion................................................................ 141 xiii 6 General Conclusions .......................................................... 149 7 Future perspectives ........................................................... 153 8 Acknowledgments............................................................. 157 9 References ........................................................................ 161 10 Annex............................................................................ 213 xiv Figure Index Figure 1.1 – Schematic representation of the evidence of GABA as a neurotransmitter in mammalian cerebral cortex............................... 2 Figure 1.2 – Schematic representation of a GABAA receptor. ........... 3 Figure 1.3 – Schematic representation of GABAC receptor. .............. 4 Figure 1.4 – Schematic representation of GABAB receptor ............... 6 Figure 1.5 – Schematic representation of glutamate/GABA-glutamine cycle in GABAergic synapse. ............................................................... 8 Figure 1.6 – Schematic representation of a GABAergic synapse...... 10 Figure 1.7 – Schematic representation of GABA transport .............. 11 Figure 1.8 – Schematic representation of GAT-1............................. 13 Figure 1.9 – Chemical structure of adenosine.................................. 35 Figure 1.10 – Schematic representation of adenosine metabolism. 47 Figure 1.11 – Distribution of adenosine A1 and A2A receptors ....... 51 Figure 1.12 – Associated pathways to adenosine receptors ............ 55 Figure 3.1