Elsevier

Neuropharmacology

Volume 60, Issue 6, May 2011, Pages 869-877
Neuropharmacology

Epac-mediated cAMP-signalling in the mouse model of Rett Syndrome

https://doi.org/10.1016/j.neuropharm.2011.01.002Get rights and content

Abstract

Rett Syndrome (RTT) is a neurodevelopmental disease thought to be caused by deficits in synaptogenesis and neuronal circuitry. cAMP is one of the key factors for neuronal outgrowth, plasticity and regeneration. We examined its homeostasis in RTT during early postnatal development of the essential part of the respiratory network, pre-Bötzinger complex. Using targeted expression of Epac1-camps sensor in neurons we quantified cAMP levels and their fluctuations in MeCP2−/y mice, an established model of RTT. Resting cAMP levels in the mutant were smaller than in the wild-type. cAMP transients elicited by depolarisation and stimulation of adenylate cyclase had also smaller amplitudes and faster time-courses. The anomalies in MeCP2 −/y mice were removed after inhibition of phosphodiesterase PDE4 with rolipram. Brief cAMP elevations triggered elongation of neuronal processes that was significantly bigger in the wild-type. The effects were observed after inhibition of protein kinase A and mimicked by activation of a guanine nucleotide exchange factor, Epac, with 8-(4-Chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (8-pCPT). The agonist reinforced bursting in preBötC neurons in the mutant and converted it to the wild-type. All actions of 8-pCPT were not reproduced by its non-active analogue and abolished by Epac signalling inhibitor Brefeldin A. We propose that disturbances in cAMP homeostasis in MeCP2 −/y mice can lead to inadequate Epac signalling. Concomitant defective development of respiratory circuits may be responsible for irregular breathing activity in RTT.

Highlights

► In this study we examine cAMP signalling in preBötC neurons in the mouse Rett model. ► We find lower cAMP levels due to enhanced PDE4 activity and Epac-mediated neurite outgrowth. ► We conclude that PDE4 and Epac are important in Rett Syndrome that can have therapeutic value.

Introduction

Diffusible second messenger cAMP is a multifaceted factor in cell signalling. Most of our knowledge about complex pathways of cAMP signalling has been derived from biochemical and pharmacological evidence. Recently developed genetically encoded fluorescent cAMP probes for single-cell imaging (Adams et al., 1991, Zaccolo et al., 2000, Ponsioen et al., 2004, Nikolaev et al., 2004) allow now to directly examine cAMP-related processes. The sensors have been successfully applied to various cell lines and primary cultures (Zaccolo et al., 2000, Ponsioen et al., 2004, Nikolaev et al., 2004) but not yet used to examine physiological processes in the living tissue. We previously explored genetically encoded probes to examine the topology of neuronal networks in the brain stem (Hartelt et al., 2008), modifications in morphology of neurons in relation to calcium homeostasis (Mironov et al., 2009b) and calcium-dependent changes in cAMP levels (Mironov et al., 2009a). Virus-mediated transduction delivers sensors in all neurons in the tissue and fills their interior. Visualisation of neuropil containing abundant synaptic connections is important to map the topology of living neuronal networks and examine their function. The approach is particularly suited to study functional plasticity of networks and its modifications during neuronal development and degeneration. The sensors can be also made to target specific types of neurons (Kasparov and Teschemacher, 2009).

In this study we examined cAMP homeostasis in neurons within pre-Bötzinger complex (preBötC) in the mouse model of Rett Syndrome (RTT). This disease represents a severe X-linked neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG binding protein 2, MeCP2 (Francke, 2006, Chahrour and Zoghbi, 2007). Affected females develop normally through first 6–18 months of life but then both neurological and psychiatric symptoms appear, including mental retardation with a loss of speech, development of autistic features, and disturbances in motor coordination and breathing. Respiratory activity is generated in the brainstem through coordinated activity of various CNS nuclei. The most important is pre-Bötzinger complex (preBötC), a compact kernel which generates rhythmic output closely related to the breathing pattern (Feldman and Del Negro, 2006). In RTT preBötC network shows functional disturbances which appear in early postnatal development and then aggravate (Stettner et al., 2007).

cAMP is a crucial factor in neurodevelopment and plasticity (Cohen and Greenberg, 2008). cAMP increases promote neurite elongation and decreases inhibit the outgrowth (Hutchins, 2010). We examined cAMP homeostasis using selective expression of Epac1-camps sensor in preBötC neurons and measured lower cAMP levels and faster kinetics in MeCP2 −/y mice. Both anomalies were corrected to the wild-type (WT) levels after inhibition of phosphodiesterase PDE4 with rolipram. cAMP increases induced elongation of neuronal processes that was more prominent in WT. The effects were independent from protein kinase A (PKA) and neurite outgrowth was stimulated by Epac, a guanine nucleotide exchange factor (Bos, 2003, Holz et al., 2006) that also stabilized the bursting activity in MeCP2 −/y neurons. The data thus indicate an important role of cAMP-dependent Epac-mediated signalling in the development and function of preBötC neurons.

Section snippets

Preparation

All animals were housed, cared for and euthanized in accordance with the recommendations of the European Commission (No. L358, ISSN 0378-6978), and protocols were approved by the Committee for Animal Research, Göttingen University. Experiments were performed using the mouse model for Rett Syndrome strain B6.129P2(C)-MeCP2tm1-1Bird (Guy et al., 2001). Mice were obtained from Jackson Laboratory (Bar Harbour, ME, USA) and maintained on a C57BL/6J background. Hemizygous mutant MeCP2−/y males were

Smaller cAMP levels in MeCP2 −/y neurons due to enhanced activity of PDE4

To determine absolute cAMP levels in the cytoplasm of neurons we calibrated Epac1-camps signals using 8-Bromo-2′-O-methyl-cAMP (BrOMecAMP, Kang et al., 2003), a compound with the same affinity to Epac1-camps as cAMP (Mironov et al., 2009a). For calibration we needed first to define a ‘zero’ cAMP level that was obtained by inhibiting AC with membrane-permeable inhibitor 2′5′-dideoxyadenosine (DDA, 100 μM). The drug produced smaller decreases of CFP/FRET ratio in MeCP2 −/y neurons (Fig. 1A) that

Discussion

In this study we tested the hypothesis that cAMP signalling can be distorted in MeCP2 −/y mice, lead to changes in the morphology of preBötC neurons and disturb their function. We show that in MeCP2 −/y neurons (i) cAMP levels are lower and change faster due to (ii) enhanced activity of PDE4; (iii) cAMP stimulates elongation of neuronal processes through activation of Epac signalling that is not dependent from PKA; (iv) 8-pCPT potentiates bursting and makes it regular and compatible to the

Acknowledgements

We greatly appreciate the gift of Epac1-camps construct from V. Nikolaev and M. Lohse (Univ. Würzburg, Germany). We are indebted to Nicole Hartelt for expert technical assistance and Cathy Ludwig for thorough reading of the manuscript. The work was supported by DFG grant MI 685/2-1 and funded by Deutsche Forschungsgemeinschaft through the DFG-Research Center for Molecular Physiology of the Brain.

References (39)

  • U. Francke

    Mechanisms of disease: neurogenetics of MeCP2 deficiency

    Nat. Clin. Pract. Neurol.

    (2006)
  • J. Guy et al.

    A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome

    Nat. Genet.

    (2001)
  • G.G. Holz et al.

    Cell physiology of cAMP sensor Epac

    J. Physiol.

    (2006)
  • B.I. Hutchins

    Competitive outgrowth of neural processes arising from long-distance cAMP signaling

    Sci. Signal

    (2010)
  • A.C. Jackson et al.

    Mechanism of spontaneous firing in dorsomedial suprachiasmatic nucleus neurons

    J. Neurosci.

    (2004)
  • S. Kasparov et al.

    The use of viral gene transfer in studies of brainstem noradrenergic and serotonergic neurons

    Philos. Trans. R. Soc. Lond. B Biol. Sci.

    (2009)
  • S. Kügler et al.

    Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area

    Gene Ther.

    (2003)
  • A. Lochner et al.

    The many faces of H89: a review

    Cardiovasc. Drug Rev.

    (2006)
  • A. Marcantoni et al.

    PDE type-4 inhibition increases L-type Ca2+ currents, action potential firing, and quantal size of exocytosis in mouse chromaffin cells

    Pflugers Arch.

    (2009)
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