Cyclic-Nucleotide Phosphodiesterases in the Central Nervous System E-Book
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- Herausgeber: John Wiley & Sons
- Kategorie: Wissenschaft und neue Technologien
- Serie: Wiley series in drug discovery and development
- Sprache: Englisch
This book reviews advances in understanding phosphodiesterases within the central nervous system and their therapeutic applications. A range of expert authors from both academia and industry describe these, then focus on the areas of greatest scientific and medical interest to provide more detailed coverage. Therapeutic and drug discovery applications are covered for diseases including Alzheimer's, Parkinson's, schizophrenia, erectile dysfunction, and spinal cord injuries. There is also a chapter on drug discovery tools such as in vitro assays and X-ray structures for medicinal chemistry studies.
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Veröffentlichungsjahr: 2014
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CONTENTS
Cover
Wiley Series in Drug Discovery and Development
Title Page
Copyright
Preface
Contributors
Chapter 1: Phosphodiesterases and Cyclic Nucleotide Signaling in the CNS
Introduction
The PDE Superfamily
The Properties of the Genes Encoding PDEs
Pattern of Gene and Protein Expression in the CNS
Mechanisms of Regulation of PDE Activity in the CNS
Mechanisms of Subcellular Localization of PDEs in the Cells of the CNS
PDEs and Compartmentalization of Signaling
Acknowledgments
References
Chapter 2: Putting Together the Pieces of Phosphodiesterase Distribution Patterns in the Brain: A Jigsaw Puzzle of Cyclic Nucleotide Regulation
Introduction
PDE1
PDE2
PDE3
PDE4
PDE5
PDE6
PDE7
PDE8
PDE9
PDE10
PDE11
Conclusions
References
Chapter 3: Compartmentalization and Regulation of Cyclic Nucleotide Signaling in the CNS
Introduction
New Tools to Study Cyclic Nucleotide Signaling: Fret-Based Biosensors
Specificity by Compartmentalization
Mechanisms Responsible for Cyclic Nucleotide Compartmentalization
Compartmentalization of Cyclic Nucleotide Effectors: The Role of Anchoring Proteins
Compartmentalization of the Signaling Machinery at the Plasma Membrane
Conclusions
Acknowledgments
References
Chapter 4: Pharmacological Manipulation of Cyclic Nucleotide Phosphodiesterase Signaling for the Treatment of Neurological and Psychiatric Disorders in the Brain
Introduction
PDE Localization Analysis in the Discovery Process
Molecular Pharmacology of PDEs
Current Status
Future Directions
Concluding Remarks
References
Chapter 5: Recent Results in Phosphodiesterase Inhibitor Development and CNS Applications
Introduction
Lead Discovery Approaches
Assay Methodology
PDE Chemotypes
Potential CNS Applications for PDE Inhibitors
Summary and Outlook
References
Chapter 6: Crystal Structures of Phosphodiesterases and Implication on Discovery of Inhibitors
Introduction
Overview of Structures of PDE Catalytic Domains
PDE4 Structures and Implication on the Design of Active Site Inhibitors
Conformation Variation of the PDE5 Catalytic Domain
Structures of GAF Domains
Structures of the Large PDE Fragments and Implication on Design of Allosteric Modulators
Concluding Remarks
References
Chapter 7: Inhibition of Cyclic Nucleotide Phosphodiesterases to Regulate Memory
Introduction
PDE1 and Memory
PDE2 and Memory
PDE4 and Memory
PDE5 and Memory
PDE9 and Memory
PDE10 and Memory
PDE11 and Memory
Future Directions
References
Chapter 8: Emerging Role For PDE4 in Neuropsychiatric Disorders: Translating Advances from Genetic Studies into Relevant Therapeutic Strategies
Introduction
PDE4 Signaling in Schizophrenia
PDE4 Signaling in Depression and Anxiety
PDE4 Signaling in Huntington's Disease
Future Perspectives
References
Chapter 9: Beyond Erectile Dysfunction: Understanding PDE5 Activity in the Central Nervous System
Introduction
PDE5 Inhibition as Possible Therapeutic CNS Target
Conclusions
References
Chapter 10: Molecular and Cellular Understanding of PDE10A: A Dual-Substrate Phosphodiesterase with Therapeutic Potential to Modulate Basal Ganglia Function
Introduction
Pde10A is a Member of the Superfamily of Cyclic Nucleotide Phosphodiesterases
PDE10A is Positioned to Play a Central Role in the Modulation of the Corticobasal Ganglia–Thalamocortical Loop
Current Pharmaceutical Landscape
Conclusions
References
Chapter 11: Role of Cyclic Nucleotide Signaling and Phosphodiesterase Activation in the Modulation of Electrophysiological Activity of Central Neurons
Introduction
Modulation of Neuronal Excitability and Synaptic Plasticity
Modulation of Cortical Neuronal Excitability by Cyclic Nucleotides and PDES
Modulation of Cortical Synaptic Plasticity by Cyclic Nucleotides and PDES
Modulation of Hippocampal Neuronal Excitability by Cyclic Nucleotides and PDES
Modulation of Hippocampal Synaptic Plasticity by Cyclic Nucleotides and PDES
Modulation of Striatal Neuronal Excitability by Cyclic Nucleotides and PDES
Modulation of Striatal Synaptic Plasticity by Cyclic Nucleotides and PDES
Modulation of Neuronal Excitability and Synaptic Plasticity by Cyclic Nucleotides and PDES: Midbrain and Brain Stem
Implications for the Treatment of Neurological Disorders
Conclusions
Acknowledgments
References
Chapter 12: The Role of Phosphodiesterases in Dopamine Systems Governing Motivated Behavior
Dopamine: A Central Regulator of Motivation and Volitional Behavior
Anatomical and Chemical Organization of Striatum
Phosphodiesterases and Dopamine Systems: Overlapping Tissue Distributions
Activity-Dependent Regulation of PDE Expression
DARPP-32 Regulates Cyclic Nucleotide-Dependent Dopamine Signaling and Behavior: A Monitor for PDE Activity
Specific PDE Isoforms Regulate Dopamine Signaling Behaviors
PDE10A
PDE4
Other PDE Isoforms with Emerging Roles in Volitional Behavior
PDE Isoforms, Dopamine Signaling, and Disease: Implications for Treatment
References
Chapter 13: Inhibition of Phosphodiesterases as a Strategy for Treatment of Spinal Cord Injury
Spinal Cord Injury: Obstacles to Regeneration
Rolipram in Spinal Cord Regeneration Research
Conclusions
Acknowledgments
References
Index
End User License Agreement
List of Tables
Table 1.1
Table 2.1
Table 5.1
Table 6.1
Table 7.1
Table 8.1
Table 12.1
List of Illustrations
Figure 1.1
Figure 1.2
Figure 1.3
Figure 1.4
Figure 2.1
Figure 2.2
Figure 2.3
Figure 3.1
Figure 4.1
Figure 4.2
Figure 4.3
Figure 4.4
Figure 4.5
Figure 4.6
Figure 4.7
Figure 4.8
Figure 4.9
Figure 4.10
Figure 4.11
Figure 4.12
Figure 5.1
Figure 5.2
Figure 5.3
Figure 5.4
Figure 5.5
Figure 5.6
Figure 5.7
Figure 5.8
Figure 5.9
Figure 5.10
Figure 5.11
Figure 5.12
Figure 5.13
Figure 5.14
Figure 5.15
Figure 5.16
Figure 5.17
Figure 5.18
Figure 5.19
Figure 5.20
Figure 5.21
Figure 6.1
Figure 6.2
Figure 6.3
Figure 6.4
Figure 6.5
Figure 6.6
Figure 6.7
Figure 6.8
Figure 6.9
Figure 7.1
Figure 7.2
Figure 7.3
Figure 8.1
Figure 9.1
Figure 9.2
Figure 9.3
Figure 9.4
Figure 10.1
Figure 10.2
Figure 10.3
Figure 11.1
Figure 11.2
Figure 11.3
Figure 11.4
Figure 11.5
Figure 12.1
Figure 12.2
Figure 12.3
Figure 13.1
Guide
Cover
Table of Contents
Preface
Chapter 1
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1721
Wiley Series in Drug Discovery and Development
Binghe Wang, Series Editor
A complete list of the titles in this series appears at the end of this volume.
Cyclic-Nucleotide Phosphodiesterases in the Central Nervous System
From Biology to Drug Discovery
Edited by
Nicholas J. Brandon
AstraZeneca Neuroscience
Cambridge, Massachusetts, USA
Anthony R. West
Rosalind Franklin University of Medicine and Science
North Chicago, Illinois, USA
Copyright © 2014 by John Wiley & Sons, Inc. All rights reserved
Published by John Wiley & Sons, Inc., Hoboken, New Jersey
Published simultaneously in Canada
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Library of Congress Cataloging-in-Publication Data:
Cyclic-nucleotide phosphodiesterases in the central nervous system : from biology to drug discovery / edited by Nicholas J. Brandon, Anthony R. West.
p. ; cm.
Includes bibliographical references.
ISBN 978-0-470-56668-8 (cloth)
I. Brandon, Nicholas J., editor of compilation. II. West, Anthony R., 1970- editor of compilation [DNLM:1. 3’,5’-Cyclic-AMP Phosphodiesterases–metabolism. 2. 3’,5’-Cyclic-AMP Phosphodiesterases–therapeutic use. 3. Central Nervous System–physiology. 4. Central Nervous System Diseases–drug therapy. 5. Drug Discovery. QU 136]
QP370
612.8–2–dc23
2013042742
Preface
Cyclic-nucleotide phosphodiesterases (PDEs) are critically involved in the regulation of cellular processes at work from cell birth to death. PDEs are produced by and operate within all cells of the body, and their key role in dampening or redirecting cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) signaling cascades makes them essential for cell health. In both the brain and spinal cord, PDEs show intricate patterns of cellular localization, both regionally and at the subcellular level. Such an infrastructure undoubtedly contributes to the tremendous computational power needed for the effective execution of sensorimotor, cognitive, and affective functions. On the flipside, we are entering a period of time when diseases of the central nervous system (CNS), which disrupt these essential functions, will affect more and more of us. For reasons described in this book, it is now clear that PDEs have enormous potential as targets for new medicines. In this book we have brought together the expertise of leading researchers from both basic and applied sciences to highlight the beautiful biology of the diverse superfamily of PDEs, as well as the medical potential of targeting PDEs for the treatment of disorders of the CNS. Indeed, numerous applications for small-molecule inhibitors selective for specific PDE isoforms are being investigated for the treatment of CNS diseases, including schizophrenia, depression, Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal cord injury, and others. Drug discovery for disorders of the CNS is exceptionally difficult, but undoubtedly, our understanding of PDE biology and PDE-based therapeutics will continue to evolve and hopefully lead to the development of novel medicines of value for patients suffering from these devastating disorders.
We thank all of our wonderful colleagues who have contributed chapters to this book, as well as the numerous reviewers who have provided constructive criticism of its content. We hope that this work will render important insights into PDE biology and therapeutics that will inspire a new generation of researchers interested in this field.
Nicholas J. Brandona
Anthony R. West
Contributors
Eva P.P. Bollen, Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands
Nicholas J. Brandon, AstraZeneca Neuroscience iMED, Cambridge, MA, USA
Erik I. Charych, Lundbeck Research USA, Paramus, NJ, USA
Marco Conti, Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of California, San Francisco, San Francisco, CA, USA
Marie T. Filbin, Department of Biological Sciences, Hunter College, City University of New York, New York, NY, USA
Joseph P. Hendrick, Intra-Cellular Therapies Inc., New York, NY, USA
Yingchun Huang, Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill, NC, USA; Biomedical and Pharmaceutical Department, Biochemical Engineering College, Beijing Union University, Beijing, China
Hengming Ke, Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill, NC, USA
Michy P. Kelly, Department of Pharmacology, Physiology & Neuroscience, University of South Carolina School of Medicine, Columbia, SC, USA
Frank S. Menniti, Mnemosyne Pharmaceuticals, Inc., Providence, RI, USA
Elena Nikulina, The Feinstein Institute for Medical Research, Manhasset, NY, USA
Akinori Nishi, Department of Pharmacology, Kurume University School of Medicine, Fukuoka, Japan
James O'Donnell, School of Pharmacy & Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY, USA
Niels Plath, H. Lundbeck A/S, Valby, Denmark
Jos Prickaerts, Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands
Olga A.H. Reneerkens, Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands
Wito Richter, Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine, University of California, San Francisco, San Francisco, CA, USA
David P. Rotella, Department of Chemistry and Biochemistry, Montclair State University, Montclair, NJ, USA
Kris Rutten, Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands
Akira Sawa, Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Christopher J. Schmidt, Pfizer Global Research and Development, Neuroscience Research Unit, Cambridge, MA, USA
Gretchen L. Snyder, Intra-Cellular Therapies Inc., New York, NY, USA
Alessandra Stangherlin, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK
Harry W.M. Steinbusch, Department of Psychiatry and Neuropsychology, School for Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands
Niels Svenstrup, H. Lundbeck A/S, Valby, Denmark
Sarah Threlfell, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
Huanchen Wang, Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill, NC, USA
Anthony R. West, Department of Neuroscience, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
Ying Xu, School of Pharmacy & Pharmaceutical Sciences, The State University of New York at Buffalo, Buffalo, NY, USA
Mengchun Ye, Department of Biochemistry and Biophysics and Lineberger Comprehensive Cancer Center, The University of North Carolina, Chapel Hill, NC, USA
Manuela Zaccolo, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
Han-Ting Zhang, Departments of Behavioral Medicine & Psychiatry and Physiology & Pharmacology, West Virginia University Health Sciences Center, Morgantown, WV, USA
Sandra P. Zoubovsky, Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD, USA
Chapter 1Phosphodiesterases and Cyclic Nucleotide Signaling in the CNS
Marco Conti and Wito Richter
Center for Reproductive Sciences, Department of Obstetrics, Gynecology and Reproductive Sciences, School of Medicine University of California, San Francisco San Francisco CA USA
Introduction
Discovery of PDEs, Historical Perspectives, and Progress in Understanding the Complexity of PDE Functions
Soon after the discovery of the second messenger cAMP by Sutherland and Rall [1], it was observed that cyclic nucleotides are unstable in tissue extracts. This observation paved the way for the identification of the enzymatic activities responsible for their destruction [1]. Sutherland and coworkers correctly attributed this activity to a Mg2+-dependent, methylxanthine-inhibited enzyme that cleaves the cyclic nucleotide phosphodiester bond at the 3′-position, hence the name phosphodiesterase (PDE) (Figure 1.1). With the discovery of cGMP and the improvement of protein separation protocols [2], it also became apparent that multiple PDE isoforms with different affinities for cAMP and cGMP and sensitivity to inhibitors coexist in a cell (Figure 1.1). Only with the application of protein sequencing and molecular cloning techniques has it been realized that 21 genes code for PDEs in humans and that close to 100 proteins are derived from these genes, forming a highly heterogeneous superfamily of enzymes (Figures 1.1 and 1.2) [3].
Figure 1.1 Timeline of the major discoveries related to the field of phosphodiesterases.
Figure 1.2 The domain organization of the different families of phosphodiesterases. Domains are depicted as “barrels” connected by “wires” indicating linker regions. Phosphorylation sites are shown as red circles with the respective kinase phosphorylating this site listed above. PDEs are composed of a C-terminal catalytic domain (shown in red) and distinct regulatory domains at the N-terminus. These include Ca2+/calmodulin (CaM)-binding sites (PDE1), GAF domains that function as cAMP or cGMP sensors (PDE2, PDE5, PDE6, PDE10, and PDE11), the UCRs that include a phosphatidic acid (PA)-binding site in PDE4, and the PAS domain (PDE8). The inhibitory gamma subunit of PDE6 is indicated as a yellow ellipse. Domains functioning as targeting sequences by mediating membrane–protein or protein–protein interactions are indicated as red striated barrels and the transmembrane (TM) domains of PDE3 are indicated in blue. The number of PDE genes belonging to each PDE family is indicated in parentheses beside the PDE family name.
Although PDEs were implicated early on in the control of intracellular levels of cAMP and cGMP and the termination of the neurotransmitter or hormonal signal, 30 additional years of research have been necessary to understand that PDEs are not simply housekeeping enzymes. The activity of PDEs is finely regulated by a myriad of regulatory loops and integrated in a complex fashion with the cyclic nucleotide signaling machinery and other signaling pathways. Blockade of PDE activity does not exclusively lead to an increase in cyclic nucleotides and a gain of function, as one would predict from the removal of cyclic nucleotide degradation. On the contrary, complex changes in cellular responses are associated with PDE inhibition, often causing loss of function, as documented by the phenotypes of natural mutations or engineered inactivation of the PDE genes [4–7]. These findings imply that PDEs and their regulation are indispensable to faithfully translate extracellular cues into appropriate biological responses. Indeed, in neurons as in other cells, the biological outcome of activation of a receptor is defined by the multiple dimensions of the cyclic nucleotide signal. This specificity of the response depends on the changes in concentration of the cyclic nucleotide, the time frame in which these changes occur, and the subcellular locale in which the nucleotides accumulate. Because cyclic nucleotide accumulation is dependent on the steady state of cAMP/cGMP production as well as hydrolysis, degradation by PDEs is a major determining factor of all three dimensions of the cyclic nucleotide signal.
In spite of seemingly comparable enzymatic functions, each of the several PDEs expressed within a cell appears to serve unique roles. This view is paradoxical because it implies, as fittingly summarized by L.L. Brunton, that “Not all cAMP has access to all cellular PDEs” [8]. As an extension of this concept, a PDE may play critical functions in a cell even if it represents a minor fraction of the overall hydrolytic activity, a view with considerable impact on pharmacological strategies targeting PDEs. The discovery of macromolecular complexes involving PDEs has confirmed this concept and added a new dimension to the function of these enzymes in signaling. In those complexes in which they are associated with cyclic nucleotide targets, it is likely that PDEs play an essential role in controlling or limiting the access of cyclic nucleotides to their effectors. Since protein kinase A (PKA), protein kinase G (PKG), GTP exchange protein activated by cAMP (EPAC), and cyclic nucleotide-gated (CNG) channels are tethered to specific subcellular compartments, PDEs likely contribute to the compartmentalization of cyclic nucleotide signaling and to the spatial dimension of the signal. PDEs may also have scaffolding properties within these complexes, opening the possibility that PDEs serve functions beyond their catalytic activity and that a dynamic formation and dissolution of these complexes may contribute to the allosteric regulation of PDE activities.
The PDE Superfamily
After several more PDE genes were discovered through homology screening of nucleotide sequence databases between 1996 and 2000 (PDE8, PDE9, PDE10, and PDE11), the completion of the Human Genome Project in 2001 eventually established that there are 21 PDE genes in humans [9]. Orthologs of all 21 genes are encoded in the genomes of rats and mice and might be present in the same number in other mammals. Metazoan model organisms such as Caenorhabditis elegans or Drosophila melanogaster express orthologs of some, but usually not all of the mammalian PDEs [3]. Based upon their substrate specificities, kinetic properties, inhibitor sensitivities, and, ultimately, their sequence homology, the 21 mammalian PDE genes are subdivided into 11 PDE families, each consisting of 1 to a maximum of 4 genes (Table 1.1). Most PDE genes are expressed as a number of variants through the use of multiple promoters and alternative splicing. The PDE6 genes, with only 1 transcript per gene reported, and PDE9A, for which more than 20 putative variants have been proposed, represent the extremes in the number of variants generated from individual genes. Together, close to 100 PDE proteins are generated in mammals, each likely serving unique cellular functions.
Table 1.1 The Properties of the Mammalian PDE Genes
Chromosome Region
a
Gene Symbol
Name/Aliases
Predominant CNS Distribution
b
Disease Association
c
References
2q32.1
PDE1A
Phosphodiesterase 1A, Ca
2+
/calmodulin-dependent
Cortex
Schizophrenia
[10]
12q13
PDE1B
Phosphodiesterase 1B, Ca
2+
/calmodulin-dependent
Striatum, hippocampus
7p14
PDE1C
Phosphodiesterase 1C, Ca
2+
/calmodulin-dependent
Cerebellum
11q13.4
PDE2A
Phosphodiesterase 2A, cGMP-stimulated
Striatum, hippocampus, cerebellum
12p12
PDE3A
Phosphodiesterase 3B, cGMP-inhibited
Striatum, hippocampus
RR and QT interval
[11]
11p15.1
PDE3B
Phosphodiesterase 3B, cGMP-inhibited
Hippocampus
19p13.2
PDE4A
Phosphodiesterase 4A, cAMP-specific (
Drosophila dunce
homolog, DPDE2, PDE2)
Cortex, hippocampus
1p31
PDE4B
Phosphodiesterase 4B, cAMP-specific (
Drosophila dunce
homolog, DPD4, PDE4)
Cortex, striatum, hippocampus
Schizophrenia, bipolar disorder, depression, alcohol responses, multiple sclerosis
[12–15]
19p13.1
PDE4C
Phosphodiesterase 4C, cAMP-specific (
Drosophila dunce
homolog, DPD1, PDE1)
Low level in CNS
5q12
PDE4D
Phosphodiesterase 4D, cAMP-specific (
Drosophila dunce
homolog, DPD3, PDE3, STRK1)
Cortex, hippocampus, brain stem
Stroke susceptibility, asthma, sleep disorders, neuroticism, schizophrenia
[10,16–21]
4q25–q27
PDE5A
Phosphodiesterase 5A, cGMP-specific
Cerebellum, hippocampus
5q31.2–q34
PDE6A
Phosphodiesterase 6A, cGMP-specific, rod, alpha
Retina
Retinitis pigmentosa
[5]
4p16.3
PDE6B
Phosphodiesterase 6B, cGMP-specific, rod, beta
Retina
Night blindness, congenital stationary type 3
[22]
10q24
PDE6C
Phosphodiesterase 6C, cGMP-specific, cone, alpha prime
Retina
Cone dystrophy
[7]
8q13–q22
PDE7A
Phosphodiesterase 7A, HCP1
Hippocampus
6q23–q24
PDE7B
Phosphodiesterase 7B
Striatum, hippocampus
15q25.3
PDE8A
Phosphodiesterase 8A
Cortex
Major depressive disorder
5q13.3
PDE8B
Phosphodiesterase 8B
Striatum, hippocampus
Pigmented nodular adrenocortical disease
21q22.3
PDE9A
Phosphodiesterase 9A
Cortex, cerebellum
Bipolar disorder
[23,24]
1q11
PDE10A
Phosphodiesterase 10A
Striatum
Autism
[25]
2q31.2
PDE11A
Phosphodiesterase 11A
Hippocampus CA1
Pigmented nodular adrenocortical disease
[26]
a
Shown is the chromosomal localization of the PDE genes in humans.
b
Expression data in human based on
in situ
hybridization; where not available, data from rat and mouse brain were used.
c
The association with inherited diseases is based on published data, GWAS (
http://gwas.nih.gov/
) analysis, and OMIM (
http://www.ncbi.nlm.nih.gov/omim
) data.
Nomenclature
Due to the large number of PDE variants present in mammals, an initial classification based on regulatory properties and inhibitor sensitivities of newly discovered enzymes as well as their order of discovery soon became inadequate. It was subsequently replaced with a consensus nomenclature in which the first two letters indicate the species followed by the three letters “PDE”, an Arabic numeral indicating the PDE family, a letter indicating the gene within the PDE family, and finally another Arabic numeral indicating the precise PDE variant. For example, HsPDE4D3 identifies the species as Homo sapiens, the PDE family as 4, the gene as D, and the variant as 3. This nomenclature was widely adopted in 1994 [27]. For a complete list of PDE genes and variants as well as information regarding the nomenclature used before 1994, please see http://depts.washington.edu/pde/pde.html or Ref. [28].
Overall Protein Domain Arrangement
Despite their multitude and diversity, all PDEs share several structural and functional properties. One of the most obvious is their modular structure consisting of a relatively conserved catalytic domain located in the C-terminal half of the protein and N-terminal domains that are structurally diverse, but all function to regulate enzyme activity (Figure 1.2). The C-terminal catalytic domain contains all residues required for catalysis and determines the enzyme kinetics unique to each PDE subtype. The characteristic features of the N-terminal regulatory domains are highly conserved modules such as Ca2+/calmodulin-binding domains (PDE1), GAF domains (cGMP-activated PDEs, adenylyl cyclase, and Fh1A; PDE2, PDE5, PDE6, PDE10, and PDE11), UCR domains (upstream conserved regions; PDE4), REC domains (receiver; PDE8), and PAS domains (period, aryl hydrocarbon receptor nuclear translocator (ARNT), and single minded; PDE8), as well as phosphorylation sites, which mediate the regulation of enzyme activity by posttranslational modifications and/or ligand binding. As a result of this modular structure, truncated PDEs encoding only the catalytic domain not only retain enzyme activity, but also exhibit kinetic properties and substrate specificity similar to those of the holoenzyme, whereas regulation of enzyme activity is lost or is altered compared to full-length proteins [29–31]. In most cases, the inhibitor sensitivity of the full-length proteins is also retained in the catalytic domain constructs. There are some exceptions, however. The catalytic domain of PDE4, for example, exhibits an affinity for the prototypical PDE4 inhibitor rolipram that is about 100-fold lower compared to full-length proteins, whereas the sensitivity toward structurally unrelated compounds is not affected by the truncation [30,31]. Thus, catalytic domain constructs may have limited value for the development of PDE4 inhibitors.
The regulatory domains, in turn, may function in a similarly independent manner. The GAF domains, for example, are found in PDEs, adenylyl cyclases, and the Escherichia coli Fh1A protein. Chimeras between the GAF domains of different PDEs (PDE5, PDE10, and PDE11) and the catalytic domain of the cyanobacterial cyaB1 adenylyl cyclase were found to be fully functional in that the PDE-GAF domains sense cyclic nucleotide levels and mediate activation of the catalytic domain of the cyclase [32,33]. This finding, together with the similar domain organization of all PDEs, led to the proposal that despite their structurally distinct N-termini, the mechanism by which the N-terminal domains regulate enzyme activity is perhaps conserved among all PDE subtypes. The functional properties of the different N-terminal regulatory domains are discussed in greater detail later in this chapter.
The unique combination of catalytic domain kinetics and regulatory domain properties defines a PDE family and is conserved among its members. The variants generated from a single PDE gene, in turn, contain with few exceptions the identical catalytic domain, but often possess variant-specific N-termini. These variants are divided into those that encode the entire regulatory module characteristic of a given PDE family, such as GAF domains or UCR domains, and those that encode only a portion of the regulatory module or lack it altogether. The latter include so-called short PDE4 variants, which contain only a portion of the UCR module, and PDE11 variants that lack GAF domains. Underlining the critical role of the regulatory domains, variants that encode the entire regulatory module exhibit similar mechanisms of regulation, whereas variants that lack part or all of the regulatory domains are either insensitive to ligand binding or posttranslational modification per se or respond differently (Figure 1.2) [3,34]. The extreme N-termini unique to individual PDE variants are encoded by variant-specific first exons (Figure 1.3) and often mediate subcellular targeting through protein–protein or protein–lipid interactions, thus allowing the cell to specifically target PDE variants to subcellular compartments [35].
Figure 1.3 Structure of the PDE4D locus. Schematic representation of the structure of the mammalian PDE4D locus (top) and the encoded mRNAs. Exons are presented as filled bars, introns are drawn as solid lines, and a noncoding exon is depicted as a striated box. Indicated are variant-specific first exons, long form (LF) exons shared by all so-called long PDE4 isoforms, and common exons shared by most PDE4D variants. The scheme is not drawn to scale.
Although the mechanisms of regulation of PDE activity have been described biochemically in great detail, structural aspects of enzyme regulation had remained elusive (see Chapter 6 for a detailed discussion). Pandit et al. [36] recently provided a major breakthrough on the question of how modification of the N-terminal domains by posttranslational modifications and/or ligand binding exerts its effect on the conformation of the distal catalytic domain, thereby controlling PDE activity. The authors crystallized a PDE2A that, although lacking some N- and C-terminal sequence of the holoenzyme, contains the critical components of the PDE2 structure: a tandem set of GAF domains linked to the C-terminal catalytic domain. PDE2A crystallized as a linear structure that extends along a GAF-A/GAF-B/catalytic domain axis. The enzyme forms head-to-head dimers with the dimer interface spanning the entire length of the molecule with interactions between the GAF-A and GAF-A, GAF-B and GAF-B, and between the two catalytic domains of the individual monomers. When the GAF domains are unoccupied, the substrate-binding pockets of the two catalytic domains are packed against each other, essentially closing off access to the substrate. As a mechanism for the allosteric activation of PDE2, the authors propose that cGMP binding to the GAF-B domain results in a reorientation of the linker regions connecting GAF-B and the catalytic domain, which in turn leads to a disruption of the dimer interface between the two catalytic domains, thus promoting an “open” conformation of the enzyme that allows substrate access. Both the general organization of the PDE2 structure and the mechanism of PDE activation proposed by Pandit et al. [36] are in agreement with many structure–function relationships observed in other PDEs. Most PDEs have been reported to form homo- or heterodimers [29,34,37–41], and critical dimerization domains were identified in the N-terminal domains. The catalytic domains also retain some affinity as evidenced by the fact that several PDE catalytic domains form dimers in purified protein preparations as well as in crystal structures [42–44]. In addition, most PDEs also possess the elongated structure described for PDE2 as indicated by their high frictional ratios [39,45,46]. Electron microscopic images of PDE5 and PDE6 show an elongated structure highly similar to the atomic structure of PDE2, with points of contact between the GAF-As, the GAF-Bs, and the catalytic domains of the individual monomers [47,48]. Dimerization mediated by the N-terminal domains of PDE2 plays a critical role in stacking the substrate-binding sites at the catalytic domain against each other, thus preventing substrate access. This is in agreement with the observation that N-terminal domains in various PDEs exert an inhibitory constraint on the active site, which can be uncovered through deletion mutagenesis or proteolytic digest of full-length enzyme [49,50]. Taken together, these similarities suggest that the atomic structure of PDE2 might represent a model for a general organization of PDEs and a mechanism of enzyme activation.
However, Burgin et al. [51] recently suggested an alternative mechanism of how inhibitory constraint and regulation of enzyme activity is achieved in PDE4. In crystal structures of a truncated PDE4, substrate access to the catalytic site is prevented not by stacking of the catalytic domains against one another, as proposed for PDE2 [36], but by direct binding of a helix in the regulatory UCR2 domains to the substrate-binding pocket in the catalytic domain. PDE4 variants are divided into so-called long forms that contain the complete UCR1/2 module and short forms that lack UCR1 but still contain all or a portion of UCR2. The constructs crystallized by Burgin et al. lack UCR1 and encode only a portion of UCR2, thus encoding a PDE4 that resembles short forms. There are significant structural and functional differences between long and short forms including oligomerization, enzyme activation, and inhibitor sensitivity [40]. Thus, it remains to be determined whether the mechanism of enzyme inhibition/activation proposed by Burgin et al. [51] reflects properties of all PDE4 isoforms or whether this model describes properties inherent only to short PDE4 isoforms, whereas long isoforms are regulated differently. If the former is the case, PDE4 regulation of enzyme activity would be different from models described for PDE2, PDE5, and PDE6. This in turn would suggest that distinct modes of regulating PDE activity evolved for the different PDE families.
Catalytic Site Properties and Interaction with the Substrates
PDEs are divalent metal ion-dependent enzymes and share with other metal-dependent phosphohydrolases an HD(X2)H(X4)N motif, which defines residues forming the metal ion-binding site. Much progress has been made in understanding the structure and properties of the catalytic domains since crystal structures have become available in 2000 [43]. The catalytic domain consists of 16 α-helices folded into a compact structure. Whereas the sequence homology of the catalytic domains can be as low as 25% for members of different PDE families, the three-dimensional structure of the catalytic domain aligns all residues that are invariant or semiconserved among all PDEs to form the substrate-binding pocket. These residues include an invariant glutamine that forms hydrogen bonds with the 1- and/or 6-positions of the cyclic nucleotides, several residues that form a “hydrophobic clamp” that anchors the purine ring, and residues that form two metal ion-binding sites, termed M1 and M2, which are positioned at the bottom of the substrate-binding pocket. Based on biochemical data and X-ray diffraction, M1 is likely occupied by Zn2+, whereas M2 is occupied by Mg2+ or Mn2+ in the native enzyme. The two metal ions function to activate the substrate phosphate and to coordinate a water molecule that acts as a nucleophile in the PDE reaction. Recent studies suggest that the water molecule coordinated by the metal ions may partially dissociate into a hydroxide ion [52–55], and that this hydroxide acts as the nucleophile on the phosphorus, eventually becoming part of the outgoing phosphate, whereas a nearby strictly conserved histidine assists with the protonation of the O3′ group.
Defining the first atomic structure of any PDE, Xu et al. [43] reported the crystal structure of the catalytic domain of PDE4B in 2000. Since then, crystal structures for the catalytic domain of most PDE families have been reported, providing further insight into the distinct mechanisms of inhibitor binding and substrate specificity [42,43,56–64].
On the basis of their substrate specificity, the 11 PDE families can be divided into three groups. PDE4, PDE7, and PDE8 selectively hydrolyze cAMP, whereas PDE5, PDE6, and PDE9 are selective for cGMP hydrolysis. The remaining PDEs (PDE1, PDE2, PDE3, PDE10, and PDE11) bind and hydrolyze both cyclic nucleotides with varying efficiency. An invariant glutamine residue that is conserved among all PDEs and that was shown to form hydrogen bonds with either AMP or GMP in crystal structures has been proposed as the major determinant of PDE substrate specificity. As both cyclic nucleotides were thought to bind to the substrate-binding pocket in the same conformation and the hydrogen-bonding character of the 1- and 6-positions of adenine and guanine is essentially reversed, a so-called glutamine switch mechanism was previously proposed to determine PDE substrate specificity [43,63]. According to this model, the amide group of the invariant glutamine can rotate by 180° to accommodate binding of either cAMP or cGMP. It was assumed that PDEs in which additional residues limit the mobility of the invariant glutamine are selective for one of the cyclic nucleotides, whereas PDEs that allow a free rotation of the invariant glutamine could bind both. However, several recent findings suggest that this cannot be the only mechanism that determines substrate specificity among PDEs. Mutation of this invariant glutamine in PDE5A, for example, did reduce affinity of the enzyme for its physiological substrate cGMP but did not enhance binding of cAMP [65]. In addition, mutation of an aspartic acid residue conserved among the cAMP-PDEs ablates the substrate specificity of PDE4 isoenzymes, suggesting that this residue represents an additional evolutionary conserved component of substrate specificity [66,67]. Most important are recent studies by Wang et al. [61,68] that demonstrate that PDE10 binds the substrates cAMP and cGMP in syn-conformation, whereas the PDE reaction products AMP and GMP dock in anti-conformation in crystal structures. This suggests that previous reports, which relied on the analysis of cocrystals of various PDEs with AMP or GMP, might not reflect the binding of substrates in the native enzyme. In addition, Wang et al. show that rotation of the invariant glutamine is unlikely the cause for the dual-substrate specificity of PDE10, as the position of this residue is locked through additional hydrogen bonds, and that PDE4 forms only one hydrogen bond with cAMP rather than two as suggested by the “glutamine switch model” [61,68]. Thus, in addition to the conserved glutamine residue, there are likely additional, perhaps PDE family-selective determinants of substrate specificity.
Properties of the Regulatory Domains
Three critical functions have been identified for the PDE sequences N-terminal to the catalytic domain. These are the regulation of enzyme catalytic activity, oligomerization, and subcellular targeting. These properties are examined in detail in the subsequent sections of this chapter.
The Properties of the Genes Encoding PDEs
Organization of the PDE Genes
A common feature of the PDE genes already present in the ancestral dunce PDE locus of the D. melanogaster is the complex arrangement of transcription start sites and exons. The dunce locus, coding for a PDE involved in learning, memory, and development, spans over more than 148 kb (1 kb = 103 bases or base pairs) of genomic DNA and includes at least 16 exons with exceedingly long introns that often contain exons of unrelated genes. This complex arrangement is further augmented in the mammalian PDE4 genes. For instance, the human PDE4D locus (5q12) spans more than a million base pairs with at least 25 exons and 10 different start sites (see Figure 1.3). The exons encoding the catalytic domain are clustered together, whereas the exons coding for the amino termini and the regulatory/dimerization domains (UCRs) are distributed over a large stretch of genomic DNA. Other PDE loci have similar characteristics. For instance, the PDE10A gene spans more than 200 kb and contains 24 exons, whereas the PDE11A gene covers approximately 300 kb of genomic DNA and contains 23 exons [69]. The four PDE11A variants have different amino termini due to separate promoters and transcriptional start sites [70–72]. The PDE1A gene spans over 120 kb and contains 17 exons, again with multiple promoters and start sites [73]. Comparison of PDE1B1 genomic sequence with other PDE genes has indicated that two splice junctions within the region encoding the catalytic domain are conserved in rat PDE4B and PDE4D, as well as in the Drosophila duncePDE [74,75]. This commonality in the splicing junction strongly suggests that the catalytic domains of PDEs are derived from a common ancestral gene, a view further supported by the high conservation of the amino acid sequence of the catalytic domain.
PDE loci have been associated with several neurological disorders (see Table 1.1). These associations are derived from genome-wide association studies (GWAS), meta-analyses, or candidate gene approach studies. It should be noted that numerous single-nucleotide polymorphisms (SNPs) have been identified in PDE loci and that SNPs are in linkage disequilibrium in several studies of patient populations (Table 1.1). However, nonsynonymous SNPs in the coding regions are very rare. For example, the PDE4D gene has 1919 SNPs in introns, but none in exons.
Multiple Transcripts
The presence of multiple transcripts derived from each PDE gene was initially revealed by Northern blot analysis. For instance, multiple RNAs ranging in size from 4.2 to 9.6 kb were detected for the Drosophila dunce PDE [76,77]. PDE4D cDNA probes hybridize to at least three or four different transcripts ranging between 2.3 and 5 kb depending on the tissue used [78]. PDE11A is expressed as at least three major transcripts of ∼10.5, ∼8.5, and ∼6.0 kb, thus again suggesting the existence of multiple subtypes [72]. Further studies using PCR and exon-specific primers have provided a better understanding of the exon composition of each transcript.
Most of the heterogeneity in transcript size is due to the presence of multiple transcription start sites. In general, these start sites are under the control of promoters that often function in a tissue- and cell-specific manner as described for PDE1 [79–81], PDE4 [82–84], PDE7 [85,86], and PDE9 [87]. This property provides one of several explanations for the complexity of the PDE genes, whereby different promoters have evolved to allow developmental and cell-specific expression of a given PDE gene. However, it is also clear that PDEs encoded in different transcripts exclude or include regions with regulatory function, and show differences in subcellular localization and interaction with other proteins [35,88]. Thus, multiple transcriptional units generate PDEs with distinct functions.
Alternate Splicing
Splicing and alternate exon usage has been reported for many PDEs and most of this splicing occurs at the amino terminus. PDE4 splicing variants have been extensively characterized and are generated mostly by alternate promoters and first exon usage. Most of the splicing variants predicted by transcript analysis have been confirmed with antibodies specific for the N-termini of PDE4s [84]. Different variants show differences in interaction with other proteins and in subcellular localization. The largest number of splicing variants has been reported for PDE9. Through PCR amplification and alignment of EST sequences, 20 N-terminal mRNA variants have been identified [87,89]. However, only the proteins encoded by PDE9A1 and PDE9A5 have been expressed and characterized [90]. Unlike PDE4, these variants use the same transcriptional start site but are alternatively spliced to produce unique mRNAs that are distinct at the 5′-end. The functional consequence(s) of these amino acid sequence changes is (are) unclear as the affected regions are outside the catalytic domain or any recognized regulatory domain. PDE1C is another example of alternative splicing where proteins with identical catalytic domain and divergent amino termini are generated. Although uncommon, splicing at the carboxyl terminus is also present, as shown for PDE1A, PDE1C, PDE7A, and PDE10A [91]. Several splicing variants have been detected for PDE10. Omori and coworkers have suggested that this splicing controls the PKA phosphorylation of the enzyme and possibly subcellular localization [92]. This possibility has recently been experimentally verified by Charych et al. [93] as detailed in Chapter 10.
The importance of transcript splicing is underscored by analysis of RNA processing of the PDE6B gene in patients with autosomal recessive retinitis pigmentosa [94]. An acceptor splice site mutation in intron 2 of the PDE6B gene leads to the accumulation of a pre-mRNA with intermediate lariats, generating a PDE6B transcript that is 12 nucleotides shorter than wild type. In the normal PDE6B mRNA, these 12 nucleotides code for four amino acids highly conserved in the putative noncatalytic cGMP-binding domain GAF-A of PDE6B and are probably important for the correct folding and function of the protein.
Promoter Regulation
Regulation of PDE protein synthesis was observed in the 1970s when it was shown that treatment of cultured cells with cAMP analogs produced a large increase in PDE activity, which was blocked by protein synthesis inhibition [95,96]. With the cloning of different PDE4 variants, it was demonstrated that transcriptional activation of specific PDE4s accounts for some of the increased PDE activities described. Using an endocrine model and hormones that regulate cAMP synthesis, Swinnen et al. were the first to show that the accumulation of mRNA for PDE4D1 and PDE4B2 is dependent on cAMP-mediated transcriptional activation of these open reading frames (ORFs) [78]. Subsequently, an intronic promoter controlling the transcription of PDE4D1 mRNA was identified and functionally characterized [97]. In cortical neurons, D'sa et al. demonstrated that dibutyryl-cAMP (db-cAMP) induces expression of PDE4B2 and PDE4D1/PDE4D2 [98], whereas the splice variants PDE4A1, PDE4A5, PDE4A10, PDE4B3, PDE4B1, PDE4D3, and PDE4D4, although present in these cells, were not regulated at the transcriptional level by db-cAMP. Dominant negative mutants of the cAMP response element-binding (CREB) protein suppress PDE4B2 promoter activity and a constitutively active form of CREB stimulates it, confirming CRE- and CREB-dependent cAMP-mediated transcription of the short PDE4 forms. Thus, cAMP-dependent regulation of PDE4 transcription is a negative feedback loop contributing to long-term adaptation of cAMP signaling. It should be pointed out that robust PDE4B2 synthesis is also induced by activation of Toll-like receptor 4 (TLR4) and signaling through NF-κB and related transcription factors, suggesting that additional signaling pathways control the expression of PDE4 short forms [6].
During the circadian rhythm and in synchrony with cAMP synthesis, an increase in PDE activity that peaks in the middle of the night has been reported in the pineal gland. This nocturnal increase in PDE activity results from a fivefold increase in abundance of PDE4B2 mRNA [99]. The increase in PDE4B2 mRNA is followed by increases in PDE4B2 protein and PDE4 enzyme activity. These changes are dependent on the activation of adrenergic receptors and require PKA activation. The findings in this pineal gland model are an additional demonstration that PDE4B2 expression is regulated by cAMP. They also document the involvement of this regulatory loop in the circadian rhythm, providing evidence that PDE induction is a physiologically relevant mechanism of feedback regulation.
Using promoter/reporter assays, CRE elements were also identified in the promoter region of PDE4D5 [100]. This PDE4D variant accumulates in response to increased cellular cAMP, although at much lower levels than that reported for the short PDE4D1/2 and PDE4B2 forms. Site-directed mutational analysis revealed that the CRE at position −210 from the transcription start site is the principal element underlying cAMP responsiveness. The authors further determined that cAMP induced PDE4D5 expression in primary cultured human airway smooth muscle cells, leading to upregulation of phosphodiesterase activity.
CREB-mediated and cAMP-dependent regulation of PDE expression is not restricted to PDE4. For instance, PDE7B, a cAMP-specific PDE that is predominantly expressed in the striatum, is also regulated at the transcriptional level by cAMP [101]. Transcriptional activation of rat PDE7B following activation of the dopaminergic system has been demonstrated in primary striatal cultures. RT-PCR analysis revealed that dopamine D1 agonists, forskolin, or 8-Br-cAMP stimulated PDE7B transcription in striatal neurons, whereas D2 agonists did not. The cAMP-dependent regulation of PDE7B transcription, like that of PDE4D and PDE4B genes, was also variant-specific, because only PDE7B1 transcription was activated by a D1 agonist. Also in this case, functional CRE elements were identified in the promoter region.
With the above exceptions, little information is available about the mechanisms of transcriptional regulation of other PDE genes in spite of the fact that numerous reports show altered PDE expression in pathological conditions or after pharmacological treatments. For instance, alterations in PDE7 and PDE8 isozyme mRNA expression were observed in Alzheimer's disease brains examined by in situ hybridization [102]. McLachlan et al. [103] found that PDE4D isoforms 1–9 were expressed in the hippocampus of healthy human adults as well as a patient with advanced Alzheimer's disease. However, the expression for the majority of the PDE4D isoforms was reduced in the Alzheimer's disease patient compared to normal controls, whereas PDE4D1, a short isoform under the transcriptional control of cAMP, was increased twofold. PDE4D is also regulated by treatment with antidepressants [104] and chronic haloperidol and clozapine treatment causes altered expression of PDE1B, PDE4B, and PDE10A in rat striatum [105]. The above are mostly correlative studies but imply that PDE promoters are finely regulated by a variety of extracellular signals and that pathological conditions affecting cyclic nucleotide signaling also affect PDE expression.
Pattern of Gene and Protein Expression in the CNS
General Concepts
Many of the PDE variants encoded in the human genome are expressed in a tissue- and cell-specific manner. Among the various tissues examined, the CNS is remarkable in the fact that it expresses one of the highest amounts of PDE protein and activity; essentially all PDE genes are expressed in the CNS and most cells express a multitude of variants. This complexity documents the tight control of cyclic nucleotide levels in cells whose primary function is processing and integration of information. Chapter 2 shows this clearly with some original data sets, which document PDE expression in the adult mouse brain.
The majority of studies examining PDE expression patterns in the brain have focused on PDE genes rather than individual PDE variants. This information is sufficient when exploring the therapeutic potential of PDE inhibitors, which are currently designed to inactivate all members of a PDE family or, at least, all variants generated from a single PDE gene. PDE inhibitors were often developed to inactivate the major PDE subtype expressed in the target cells or tissues as a strategy that has been successfully applied in the development of PDE5 inhibitors for erectile dysfunction, PDE4 inhibitors for inflammatory airway diseases, and PDE3 inhibitors for heart disease [91]. However, with the idea of compartmentalized cAMP signaling gaining momentum (see Chapter 3), it should be realized that the amount of a PDE subtype expressed in a cell is not proportional to its functional significance or therapeutic potential. While the predominantly expressed PDEs are certainly important, one cannot discount that PDE variants that contribute only a minor fraction of the total PDE activity in a cell may yet play critical physiological roles and may well be targeted for therapeutic intervention. In this context, the focus on where a PDE is expressed most abundantly does not necessarily predict specific functions. Furthermore, the localization of PDE variants to specific subcellular compartments is thought to be necessary to stabilize cAMP microdomains of signaling (see below). As a result, displacement of a unique PDE variant from signaling complexes, rather than inactivation of catalytic activity, has been proposed for future drug development [106]. As subcellular localization is often unique to individual variants generated, understanding the nature of these complexes becomes a critical issue to develop new therapeutic strategies.
Individual PDE Gene Pattern of Expression
PDE1
The three PDE1 genes, PDE1A, PDE1B, and PDE1C, are all expressed in the brain. PDE1A is widely distributed with high levels in cortex, hippocampus, cerebellum, olfactory bulb, and striatum. PDE1B message is also distributed widely, but is particularly enriched in striatum with PDE1B1 activity in mouse striatum being 3–17-fold higher than that in any other brain region. PDE1C is more selectively expressed in olfactory epithelium, cerebellum, and striatum [79–81,107–111]. PDE1B has received particular attention, as PDE1B knockout mice exhibit increased locomotor activity after D-amphetamine administration as well as spatial learning deficits. PDE1B knockout mice show increased levels of Thr34-DARPP-32 phosphorylation in response to dopamine D1 receptor stimulation suggesting that elevated cyclic nucleotide levels resulting from PDE1B inactivation act synergistically to dopaminergic agonism [112]. Inactivation of PDE1 has been proposed as a possible therapeutic approach in Parkinson's disease because antiparkinson agents, such as amantadine or deprenyl, were shown to inhibit PDE1 in vitro [113–116]. In addition, SNPs in the PDE1A gene are associated with remission of antidepressant treatment response [117]. Chapter 7 explores the effect of PDE1B inhibition in memory disorders.
PDE2
Although the cGMP-stimulated PDE2 is expressed in many tissues, the highest levels are found in the brain, where it is expressed in olfactory bulb and tubercle, cortex, amygdala, striatum, and hippocampus [108,118–122]. PDE2 inhibitors increase cyclic nucleotides in cortical and hippocampal neurons [123,124], promote enhanced long-term potentiation [123] and object recognition [125], and attenuate the learning impairment induced by acute tryptophan depletion [126]; all together suggesting that inactivation of PDE2 could boost memory functions. The potential of PDE2 inactivation in memory and cognition is presented in detail in Chapter 7.
PDE3
The two PDE3 genes, PDE3A and PDE3B, are widely expressed throughout the brain, with PDE3A being enriched in striatal and hippocampal regions [127–129]. PDE3B has been suggested to play a role in hypothalamic leptin signaling [130].
PDE4
These enzymes are widely expressed throughout the body, but highest levels of activity and protein of any tissue are found in the brain, suggesting a critical role of PDE4 in cyclic nucleotide signaling in the CNS. Indeed, the mammalian PDE4 genes are orthologs of the D. melanogaster dunce gene, whose ablation produces a phenotype of learning and memory deficits [131,132]. In addition, rolipram, the prototypical inhibitor defining the PDE4 family, was shown more than 25 years ago to produce antidepressant-like effects in rats and humans [133–135]. These studies have since been greatly expanded and PDE4 inhibitors are now investigated for potential therapeutic benefits for a plethora of CNS diseases, including Huntington's disease, Parkinson's disease, Alzheimer's disease, schizophrenia, and stroke [136].
While PDE4A, PDE4B, and PDE4D are all highly expressed in the brain, a detailed analysis reveals that their respective expression patterns are highly segregated, an observation that is further augmented if the expression patterns of individual variants, rather than the combined messages expressed from a PDE4 gene, are considered [82,98,104,137–148]. PDE4A is expressed at high levels in cortex, hippocampus, olfactory bulb, and brain stem. PDE4B displays a distinct pattern of accumulation with highest expression levels in striatum, amygdala, hypothalamus, and thalamus [12,83,149]. There is only a limited expression of PDE4C in the CNS, with some PDE4C found in cortex, some in thalamic nuclei and in the cerebellum in humans, but only in olfactory bulb in rat [148]. PDE4D is widely expressed with high levels in the cortex, olfactory bulb, and hippocampus [82].
The unique PDE4 expression patterns suggest distinct functions of PDE4 genes and this assumption has been confirmed by the distinct behavioral phenotypes of mice deficient in individual PDE4 genes. PDE4D is the primary target of the antidepressant effects of rolipram treatment [150]. However, the therapeutic use of PDE4 inhibitors has thus far been prevented by the significant side effects produced by this class of drugs, particularly emesis and nausea. PDE4D is highly expressed in the area postrema and nucleus of solitary tract, regions that are thought to mediate the emetic response, and PDE4DKO mice display a phenotype of shortened α2-adrenoceptor-mediated anesthesia, a correlate of emesis in mice [138,143,148,151]. This has led to the assumption that PDE4D inactivation is the cause and is inevitably tied to the occurrence of emesis and nausea. This finding provided the rationale for an effort to design drugs that either do not penetrate the brain or are more selective for the other PDE4 subtypes, in particular PDE4B. In both cases, these efforts have forsaken the potential therapeutic use of PDE4D inactivation in CNS disorders. However, a recent report by Burgin et al. [51] suggests an alternative strategy of drug design that would overcome the obstacle of unwanted side effects. The authors describe the development of allosteric PDE4 inhibitors that target specific conformations of PDE4 rather than behaving as purely competitive inhibitors at the active site and do not completely inactivate enzyme activity even at highest concentrations. Even though these compounds are highly selective for PDE4D, they exhibit a reduced emetic activity in various animal models while retaining full cognition-enhancing properties. The authors propose that the partial inhibition of enzyme activity is the key to dissociating therapeutic benefits from side effects because spatial and temporal properties of cAMP signaling are maintained to some extent, which in turn lowers target-based toxicity. Given the absence of emetic side effects, this class of compounds can be used to target PDE4D for CNS diseases.
PDE4B has been linked to several other CNS disorders, in particular schizophrenia. DISC1, a well-established genetic marker associated with schizophrenia, has been shown to interact with PDE4B [13]. In addition, several studies have shown altered, generally reduced expression of PDE4B in schizophrenia patients versus healthy controls [12,152]. The changed expression levels likely result from SNPs in the PDE4B gene that are associated with schizophrenia in a Scottish family as well as in Japanese, Finnish, Caucasian, and African American populations [12–14,152,153]. There might be population differences or other genetic factors involved because two other studies did not detect associations of PDE4B SNPs with schizophrenia [154,155]. PDE4B has multiple additional CNS effects. Mice deficient in PDE4B exhibit an anxiogenic-like behavior [156] as well as decreased prepulse inhibition, decreased baseline motor activity, and an exaggerated locomotor response to amphetamine [157]. Changes in expression levels of PDE4B variants have also been associated with long-term potentiation [158–160] and levels of PDE4B and PDE4A expression were associated with autism [161].
PDE4 as a therapeutic target for CNS disorders is discussed in greater detail in several subsequent chapters.
