University of Groningen Novel cyclic AMP signalling avenues in learning and memory Ostroveanu, A IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2009 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Ostroveanu, A. (2009). Novel cyclic AMP signalling avenues in learning and memory. s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. 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Financial support from the graduate school of Behavioral and Cognitive Neuroscience and the University of Groningen for the publication of this thesis is gratefully acknowledged. school of behavioral and cognitive nenuroscience Cover photos: A. Ostroveanu Cover design: A. Ostroveanu Printed by: Ipskamp Drukkers RIJKSUNIVERSITEIT GRONINGEN Novel cyclic AMP signalling avenues in learning and memory Proefschrift ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op vrijdag 1 mei 2009 om 16.15 uur door Anghelus Ostroveanu geboren op 25 januari 1976 te Ciocanesti, Roemenie Promotores: Prof. dr. P.G.M. Luiten Prof. dr. E.A. van der Zee Prof. dr. U.L.M. Eisel Copromotor: Dr. I.M. Nijholt Beoordelingscommissie: Prof. dr. M. Schmidt Prof. dr. M. Verhage Prof. dr. B. Roozendaal ISBN: 978-90-367-3750-0 A man's real possession is his memory. In nothing else he is rich, in nothing else he is poor. Dreamthorp: Essays written in the Country Alexander Smith-Scottish essayist & poet (1830 - 1867) to my dear Oana and the loved ones left home Contents Chapter 1 Introduction and aim of the thesis 9 Chapter 2 A-kinase anchoring protein 150 in the mouse brain is concentrated 39 in areas involved in learning and memory Chapter 3 Both exposure to a novel context and associative learning induce an 63 upregulation of AKAP150 protein in mouse hippocampus Chapter 4 Inhibition of PKA anchoring to A-kinase anchoring proteins impairs 75 consolidation and facilitates extinction of contextual fear memories Chapter 5 Detailed analysis of mAKAP expression in the brain of young and 95 old mice Chapter 6 Exchange protein activated by cyclic AMP 2 (Epac2) plays a specific 119 and time-limited role in memory retrieval Chapter 7 General discussion 141 Summary of the thesis 171 Nederlandse samenvatting 175 Acknowledgements 179 List of publications 181 Curriculum vitae 183 Chapter 1 Introduction and aim of this thesis Chapter 1 Contents: 1. cAMP signaling in learning and memory 2. Cyclic AMP dependent protein kinase 3. PKA in learning and memory 4. Compartmentalization of PKA signaling through AKAPs 5. A-kinase anchoring protein 150 6. Muscle AKAP 7. Role of AKAPs in learning and memory 8. Tools for AKAP research 9. Exchange protein directly activated by cAMP 10. Aim and outline of the thesis 11. References 10 Introduction and aim of this thesis 1. cAMP signaling in learning and memory In the molecular mechanisms that underlie the formation of all memories from simple adaptive responses to complex declarative memories, nerve cells and their interacting functional networks are subject to a large variety of signaling molecules including neurotransmitters, hormones, cytokines and matrix molecules. All these messengers can exert their influence to the same nerve cell. At the same time each nerve cell according to the model of Hebb can participate in different memory traces and respond to a variety of temporal and spatial dynamics. An obvious question then coming up is how a single individual neuron can handle the myriads of stimuli necessary to build memories during life, or in other words: how do nerve cells organize the where and when in their extensive cellular domain? So far, a large number of signalling proteins have been shown to be involved in learning and memory processes. One of the most important molecules involved is cyclic adenosine monophosphate (cAMP, cyclic AMP or 3'-5'-cyclic adenosine monophosphate), a second messenger ubiquitously expressed and shown to be involved in many other biological processes besides learning and memory. This is e.g. evidenced by the fact that there are more than 90.000 scientific publications concerning cAMP since its discovery as a diffusible intracellular second messenger produced in response to hormone action (Sutherland and Rall, 1958). In the brain, activation of cAMP signaling occurs after stimulation of adenylyl cyclases by stimulatory G-proteins after binding of an extracellular ligand (e.g. adrenaline) to a GPCR and by Ca2+ through the Ca2+-binding protein calmodulin (Wang and Storm, 2003). Initially, the importance of cAMP signaling in learning and memory was demonstrated in genetic and pharmacological studies in invertebrate models for associative learning such as Aplysia (Byrne and Kandel, 1996), Drosophila (Waddell et al., 2000), and in honeybees (Apis mellifera) (Hammer and Menzel, 1995). Soon after, evidence for the role of cAMP in learning and memory in mammals was published. Genetic manipulations of several components of the cAMP pathway, such as augmentation of adenylyl cyclase activity (Pineda et al., 2004) or altering the expression of G-proteins (Bourtchouladze et al., 2006), 11 Chapter 1 led to severe memory impairments in mice. Although cAMP was acknowledged as crucial molecule in learning and memory, the precise role of cAMP in these processes remained unclear due to contradictory results from other pharmacological and genetic studies. While the elevation of cAMP levels in the hippocampus by forskolin, an activator of adenylyl cyclase, improved memory for passive avoidance tasks in rats (Bernabeu et al., 1997), pharmacological activation of the cAMP pathway in the prefrontal cortex was shown to impair working memory performance (Taylor et al., 1999). Very recently, it was shown that transgenic mice in which cAMP levels are spatially and temporally elevated in forebrain regions, exhibit enhanced memory consolidation and retrieval for contextual fear conditioning (Isiegas et al., 2008). Altogether, these results suggest that cAMP signals are mediated by different intracellular mechanisms and their actions dependent on the brain region involved. Thus cAMP appears to play a complex role in distinct cognitive processes in different brain regions. In this thesis we will investigate the role of two, recently discovered, cAMP signaling avenues, A-kinase anchoring proteins (AKAPs) and exchange protein activated by cAMP (Epac), in memory processes. 2. Cyclic AMP dependent protein kinase One of the main targets of cAMP is cAMP-dependent protein kinase (PKA). Mammalian PKA includes four regulatory (RIα, RIβ, RIIα, RIIβ) and three catalytic (Cα, Cβ, Cγ) subunits, each encoded by a separate gene (McKnight et al., 1988; Doskeland et al., 1993). Two major types of mammalian PKA, type I (PKA I, with RIα and RIβ dimers) and type II (PKA II, with RIIα and RIIβ dimers), were initially described by their pattern of elution from diethylaminoethylcellulose (DEAE) cellulose columns (Tasken et al., 1993, Francis and Corbin, 1999). Both type I and type II PKA are expressed in the mammalian brain (Cadd and McKnight, 1989). In the absence of cAMP, PKA consists of an inactive heterotetramer of two catalytic subunits bound to two regulatory subunits (Taylor et al, 1990). Each regulatory subunit contains two tandem cAMP binding sites, a high affinity 12 Introduction and aim of this thesis site and a low affinity site (Taylor et al., 1990). Activation of PKA occurs upon increasing cAMP levels. Accordingly, cAMP binding to these two sites located on the R subunits, results in the dissociation of the holoenzyme and the release of the monomeric C subunits which in turn can phosphorylate serine and threonine residues on targeted proteins (Taylor et al., 1990, Wang et al., 1991, Gibbs et al., 1992). The C subunits are broad-spectrum serine/threonine kinases that could potentially target numerous proteins, therefore signalling specificity for PKA is required. A basic specificity of PKA action is supported by the fact that there is a specific subcellular localization of PKA isoforms. For example, RI is found throughout the cytoplasm, whereas RII is localized to nuclei, nucleoli, the microtubule organizing center, the Golgi apparatus, and the plasma membrane (Griffiths et al., 1990, Li et al., 1988). Interestingly, even within the same subcellular compartment the PKA isoforms can have a differential localisation. For example RIIα and RIIβ have been demonstrated to localize differently in the Golgi-centrosomal area (Keryer et al., 1999). The specificity of PKA action can also be achieved by its affinity to cAMP. The PKA isoforms become activated at different concentrations of cAMP: type Iα is activated when the cAMP concentration reaches 10 nM, type Iβ at 100 nM, type IIα at 200 nM and type IIβ at 600 nM (Taylor et al, 1990).