Recent Advances in Parkinsons Disease, Volume 183: Part I: Basic Research (Progress in Brain Research)

PROGRESS IN BRAIN RESEARCH

VOLUME 183

RECENT ADVANCES IN PARKINSON’S DISEASE: BASIC RESEARCH EDITED BY

ANDERS BJO¨RKLUND Wallenberg Neuroscience Centre Division of Neurobiology Lund University Lund, Sweden

M. ANGELA CENCI Basal Ganglia Pathophysiology Unit Department of Experimental Medical Science Lund University Lund, Sweden

AMSTERDAM – BOSTON – HEIDELBERG – LONDON – NEW YORK – OXFORD PARIS – SAN DIEGO – SAN FRANCISCO – SINGAPORE – SYDNEY – TOKYO

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK 360 Park Avenue South, New York, NY 10010-1710 First edition 2010 Copyright Ó 2010 Elsevier B.V. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-444-53614-3 ISSN: 0079-6123 For information on all Elsevier publications visit our website at elsevierdirect.com

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List of Contributors D.M. Alessi, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA C. Baunez, Laboratoire de Neurobiologie de la Cognition (LNC), UMR6155 CNRS/Aix-Marseille Université, Marseille, France H. Bergman, The Interdisciplinary Center for Neural Computation; Institute for Medical Research IsraelCanada (IMRIC), Department of Medical Neurobiology (Physiology), The Hebrew University, Jerusalem, Israel R.E. Burke, Departments of Neurology, Pathology and Cell Biology, Columbia University, New York, NY, USA P. Calabresi, Fondazione Santa Lucia IRCCS, Rome, Italy; Clinica Neurologica, Università degli Studi di Perugia, Ospedale S. Maria della Misericordia, Perugia, Italy A.R. Carta, Department of Toxicology, University of Cagliari, Cagliari, Italy M.A. Cenci, Basal Ganglia Pathophysiology Unit, Department of Experimental Medical Science, Lund University, Lund, Sweden S. Chan, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA M.R. Cookson, Cell Biology and Gene Expression Unit, Laboratory of Neurogenetics, National Institute on Aging, Bethesda, MD, USA T.M. Dawson, NeuroRegeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA V.L. Dawson, NeuroRegeneration and Stem Cell Programs, Institute for Cell Engineering; Department of Neurology; Department of Physiology; Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA M. Day, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA R.L.A. deVries, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA M. di Luca, Department of Pharmacological Sciences, University of Milano, Milano, Italy S. Elias, Institute for Medical Research Israel-Canada (IMRIC), Department of Medical Neurobiology (Physiology), The Hebrew University, Jerusalem, Israel M. Fournier, Laboratory of Molecular Neurobiology and Neuroproteomics, Brain Mind Institute, The Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland M.J. Frank, Department of Cognitive, Linguistic, and Psychological Sciences, Department of Psychiatry and Human Behavior, and Brown Institute for Brain Science, Brown University, Providence, RI, USA v

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F. Gardoni, Department of Pharmacological Sciences, University of Milano, Milano, Italy T. Gasser, Hertie Institute for Clinical Brain Research, Department of Neurodegenerative Diseases, Tübingen, Germany; DZNE, German Center for Neurodegenerative Diseases, Tübingen T. Gertler, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA V. Ghiglieri, Fondazione Santa Lucia IRCCS, Rome, Italy J.A. Goldberg, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA P. Gubellini, Institut de Biologie du Développement de Marseille-Luminy (IBDML), UMR6216 CNRS/ Aix-Marseille Université, Marseille, France J.N. Guzman, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA G. Heimer, Institute for Medical Research Israel-Canada (IMRIC), Department of Medical Neurobiology (Physiology), The Hebrew University, Jerusalem, Israel V. Jackson-Lewis, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA A. Kachroo, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Boston, MA, USA C. Konradi, Center for Molecular Neuroscience and Kennedy Center for Research on Human Development, Departments of Pharmacology and Psychiatry, Vanderbilt University, Nashville, TN, USA H.A. Lashuel, Laboratory of Molecular Neurobiology and Neuroproteomics, Brain Mind Institute, The Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland I. Martin, NeuroRegeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA; Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA M. Morelli, Department of Toxicology, University of Cagliari, Cagliari, Italy A. Oueslati, Laboratory of Molecular Neurobiology and Neuroproteomics, Brain Mind Institute, The Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland J.L. Plotkin, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA S. Przedborski, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA M. Rivlin-Etzion, The Interdisciplinary Center for Neural Computation; Institute for Medical Research Israel-Canada (IMRIC), Department of Medical Neurobiology (Physiology), The Hebrew University, Jerusalem, Israel J. Sanchez-Padilla, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA M.A. Schwarzschild, MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Boston, MA, USA W. Shen, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA D.J. Surmeier, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA X. Tian, Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

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M. Tocilescu, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA C. Vives-Bauza, Departments of Neurology, Pathology and Cell Biology, and the Center for Motor Neuron Biology and Disease, Columbia University, New York, NY, USA T.V. Wiecki, Department of Cognitive Linguistic, and Psychological Sciences, Department of Psychiatry and Human Behavior, and Brown Institute for Brain Science, Brown University, Providence, RI, USA

Preface Research on Parkinson´s disease (PD) is one of the most dynamic fields of modern neuroscience. It is an excellent example of how clinical and basic research can fruitfully interact and inspire each other in a truly translational way. During the decades after the discovery of dopamine in the late 1950s the field was dominated by pharmacological and neurochemical approaches. Since the discovery of the role of alphasynuclein and its role in PD pathogenesis in the late 1990s, PD research has entered a new exciting phase of development, and its scope has broadened to include dynamic molecular and genetic approaches. This had led to the discovery of further genetic mutations accounting for familial forms of the disease (Parkin, DJ-1, Pink-1, and leucine-rich repeat kinase 2), and spurred intense molecular biological investigations on the mechanisms of neurodegeneration in PD. In addition to molecular genetics, other fields of PD research have undergone a dramatic development during the past 20 years. Significant progress has been made modelling PD in animals both on a symptomatic level and on a mechanistic perspective. The current availability of a diversified range of models in different species provides neuroscientists with articulate tools to study molecular mechanisms, test pathophysiological hypotheses and identify new treatment principles. The discovery that PD motor symptoms and treatment-induced dyskinesias are dramatically ameliorated by high-frequency stimulation of some deep basal ganglia nuclei has prompted efforts on the part of both neurophysiologists and computational neuroscientists to decipher the basic neural operations of the basal ganglia in health and disease. Finally, technological developments in the area of brain imaging have provided exciting new opportunities for pathophysiological investigations, differential diagnosis and treatment monitoring in PD patients. These two companion volumes of Progress in Brain Research were composed to capture all the richness and complexity of PD as a topic for basic, translational and clinical investigation. A year ago, when we approached leading researchers in the different subfields to contribute, the vast majority of the invited authors enthusiastically accepted the invitation and delivered contributions that turned out to represent the utmost state-of-the art in each given field. It is with great pleasure and pride that we now present this collection of review chapters to a broad audience of readers. The chapters have been grouped into two volumes and five sections. The first volume covers basic and molecular investigations of the mechanisms of neurodegeneration in PD (Section I: Genetic and molecular mechanisms of neurodegeneration in PD) and the secondary adaptations that affect the basal ganglia at both the single cell level and the system level (Section II: Cellular and system-level pathophysiology of the basal ganglia in PD). The second volume focuses on translational and clinical aspects of PD research reviewing animal models of PD from drosophila to non-human primate species (Section I: Animal models of PD) the very dynamic area of functional neuroimaging (Section II: Exploring PD with brain imaging) and the most challenging therapeutic developments (Section III: Frontiers in PD treatment). Like no other neurological disease, PD is inspiring enormously diversified research themes and approaches in a way that would have been impossible to foresee some 10 years ago. An increasing number ix

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of investigators, also from areas outside neuroscience, have joined the PD research community and are now contributing to the richness and diversity of this field. Over the last 15 years, several international PD patient-initiated, non-profit organizations have dramatically improved the funding possibilities for this area of research which is now advancing at an extremely rapid pace. It is our hope that this formidable development of knowledge and technologies will deliver novel options for treatment – and eventually a cure – for all who suffer from this disease. In closing we would like to express our warmest thanks to all the authors for their outstanding contributions and to Gayathri Venkatasamy, our Developmental Editor at Elsevier, for her expert and patient assistance. Lund, June 11th 2010 Anders Bjorklund ¨ M. Angela Cenci

SECTION I

Genetic and molecular mechanisms of neurodegeneration in PD

A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 1

Identifying PD-causing genes and genetic susceptibility factors: current approaches and future prospects Thomas Gasser
Hertie Institute for Clinical Brain Research, Department of Neurodegenerative Diseases, T€ ubingen, Germany, and DZNE, German Center for Neurodegenerative Diseases, T€ ubingen

Abstract: Over the last years, a plethora of genetic findings have completely changed our views on the aetiology of Parkinson’s disease (PD). Linkage studies and positional cloning strategies have identified mutations in a growing number of genes which cause monogenic autosomal-dominant or autosomalrecessive forms of the disorder. While these Mendelian forms of PD are relatively rare, high-throughput genotyping and sequencing technologies have more recently provided evidence that low-penetrance variants in at least some of these genes also play a direct role in the aetiology of the common sporadic disease. In addition, rare variants in other genes, such as the Gaucher’s disease-associated glucocerebrosidase A, have also been found to be important risk factors at least in subgroups of patients. Thus, an increasingly complex network of genes contributing in different ways to disease risk and progression is emerging. These findings provide the ‘genetic entry points’ to identify molecular targets and readouts necessary to design rational disease-modifying treatments. Keywords: Parkinson’s disease; Genetics; Genetic risk factors; DNA polymorphisms

innovations of the 1980s, such as polymerase chain reaction amplification of DNA fragments and the discovery of polymorphic micro-satellite repeat elements in the genome (usually consisting of repetitive di-, tri- or tetranucleotide sequences which proved to be extremely useful as landmarks (‘DNA markers’) to map the genome) hand in hand with the development of appropriate statis­ tical tools and computer programmes, led to the mapping and cloning of a large number of genes

Introduction The progress of molecular genetic technology over the last 25 years has revolutionized our understanding of Parkinson’s disease (PD) and many other common complex disorders. The technological


Corresponding author. Tel.: 07071/2986529; Fax: 07071/294839;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83001-8

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which cause – when mutated – monogenic dis­ eases, that is inherited disorders following a Men­ delian mode of transmission (Gasser, 2009a). Most of these classic neurogenetic disorders such as Huntington’s disease, myotonic dystrophy or spinal muscular atrophy, to name just a few, are relatively rare. Nevertheless, with the identifica­ tion of these rare disease genes ‘neurogenetics’ has become part of the mainstream of neurology (Harbo et al., 2009). During the 1990s it became apparent that in some rare cases the much more common and typically sporadic neurologic disorders such as Parkinson’s disease (PD) (Denson and Wszolek, 1995), Alzheimer’s disease (AD) (Goate et al., 1991) or amyotrophic lateral sclerosis (Rosen et al., 1993) could also run in families following a Mendelian pattern of inheritance. In fact, it turned out that many of these monogenic variants of common dis­ eases resemble the typical sporadic forms to a large degree both clinically (with the exception that age of onset is often younger in patients with inherited forms of these disorders) and pathologically, sug­ gesting that the molecular pathways discovered through the relevant genes in hereditary forms may also be of importance in sporadic cases. The same gene identification strategies used in classical neuro­ genetic diseases were successfully used to show that the Mendelian forms of the respective common dis­ orders were also caused by mutations in single genes. The identification of those disease genes, such as SNCA, the gene encoding alpha-synuclein (aSYN) for PD, or APP, the gene coding for the amyloid precursor protein in AD, was a crucial step in the elucidation of the chain of molecular events which lead to neurodegeneration in these disorders. It was then only in recent years that accumulating evidence suggested that the close resemblance between familial and sporadic forms on the clinical and pathologic level also has its correspondence on the genetic level: common genetic variants in genes identified in monogenic forms or in genes belonging to the identified pathways have been found to modify the risk to develop a sporadic disease.

The advent of micro-array technology which provided the opportunity to study hundreds of thousands or even millions of genetic variants (usually single-nucleotide polymorphisms, ‘SNPs’) in a large cohort of patients and controls has provided the basis for a new generation of genetic studies, genome-wide association studies (GWAS) in order to go beyond the analysis of single candi­ date genes and to systematically analyse, in an unbiased way, the genetic risk profile of complex diseases. While most GWAS evaluate the role of common genetic variability as risk factors for a disease, this approach has already been success­ fully applied also to quantitative traits such as age of onset in PD (Latourelle et al., 2009) or to laboratory values such as serum levels of uric acid (Dehghan et al., 2008). Another extension of this technology is the combination with gen­ ome-wide transcriptome analysis (Elstner et al., 2009), promising a deeper insight into relevant gene regulation networks. The next technological revolution is already under way. Massive-parallel sequencing will make large-scale whole exome or even whole gen­ ome sequencing feasible. This will be necessary to identify the suspected multitude of rare genetic variants in different genes which are thought to explain another substantial fraction of the genetic risk for common diseases. Methods are being developed to deal with the increasingly complex and vast amount of informa­ tion generated by current and future sequencing technology.

Identification of monogenic forms of PD by positional cloning strategies Autosomal-dominant forms of inherited PD The classic approaches of linkage analysis and positional cloning have been the uniquely success­ ful strategies to identify genes causing the autoso­ mal-dominantly inherited diseases including the major forms of familial PD.

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This strategy relies on the availability of large and clinically well-characterized families, usually with at least 8–10 affected family members. By studying the co-segregation of genetic (DNA)mar­ kers (most often highly polymorphic dinucleotide repeat elements, above-mentioned micro-satellite polymorphisms, were used), the genetic locus of the disease-causing gene in a given family can be narrowed down to a region of several million base pairs (megabases, Mb) of DNA. The statistical method to estimate the likelihood that a particular set of neighbouring DNA markers (a so-called haplotype) are co-inherited with a disease gene as a result of its physical proximity on the chromo­ some (i.e. that DNA markers and disease gene are ‘linked’) is called linkage analysis. The most important prerequisite for this type of study, in addition to the availability of sufficiently large families, is the unequivocal classification of affected and unaffected family members. Erroneous classi­ fication, which in many age-related complex dis­ eases is a real possibility, will lead to false linkage results (Gasser, 2008). When a disease locus is identified with sufficient confidence (a so-called lod score of >3 is equivalent to a genome-wide pvalue of 0.05 and is considered to be significant evidence), all the genes in the identified region have to be sequenced and analysed for potentially disease-causing mutations. Of course, not all of the identified sequence variants in a linked region are pathogenic. This means that either the demonstra­ tion of mutations in several independent families co-segregating with a disease is necessary (amount­ ing in effect to a replication of the initial finding) or the careful functional studies in model systems are required to prove pathogenicity.

PARK1 (alpha-synuclein) It was by classic linkage analysis that Polymero­ poulos et al. mapped the disease locus in a large Italian family with autosomal-dominant PD to the long arm of chromosome 4 (Polymeropoulos et al., 1996). In this family, more than 40

individuals were identified with a relatively earlyonset form of PD (average age of onset was 46 years), a high rate of dementia and an unusually severe and rapid course (average disease duration less than 10 years), segregating as an autosomaldominant trait (Golbe et al., 1990). Only a year later, the same group identified a putative diseasecausing mutation in a known gene of the region, alpha-synuclein (the gene is abbreviated as SNCA, the protein as aSYN). It was a single base-pair change (a G to A transition) at position 209 of the coding sequence, leading to the change from alanine to threonine at position 53 of the aSYN protein (A53T) (Polymeropoulos et al., 1997). As expected, the mutation co-segregated with the disease in the affected family, but this in itself is no proof of pathogenicity. Depending on the size of a linked genetic region, the affected members in a family will share a large number of candidate genes and consequently all genetic var­ iants that are located in this region. In fact, the causative role of the SNCA mutation initially was doubted by some researchers based on the surpris­ ing fact that the highly homologous mouse SNCA gene contains the threonine thought to be patho­ genic in humans at position 53. It was therefore of great importance to find additional independent sequence variants segregating with PD in other families. Eventually, those variants were found, although they are extremely rare: only two further pathogenic point mutations in SNCA have been recognized, leading to the exchange A30P (Krüger et al., 1998) and E46K (Zarranz et al., 2004), respectively, each in a single, large, domi­ nant family, reflecting the high penetrance of these mutations. SNCA point mutations are very rare and have not been found in large cohorts of patients with sporadic PD (Berg et al., 2005). Further important insight into the link between SNCA and PD came from the discovery of gene multiplication mutations. A family with autoso­ mal-dominant parkinsonism, also frequently accompanied by dementia, had been mapped to the short arm of chromosome 4 (4p15) and had therefore been thought to be genetically distinct

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from the families with SNCA mutations (Farrer et al., 1999). The affected members resembled those with SNCA mutations both clinically and pathologically to a remarkable degree (see below). It was therefore not too surprising when it became apparent that the assignment to chro­ mosome 4p was in fact due to a genotyping error and that the disease also co-segregated with the SNCA locus in this family. However, direct sequencing of the SNCA gene did, however, not reveal any putative pathogenic mutations. Instead, Singleton and co-workers found a triplication of the entire locus containing the SNCA gene in the affected members of this pedigree (Singleton et al., 2003). This finding is of particular relevance with respect to the molecular pathogenesis of PD, as it suggests that not only structurally altered aSYN can cause PD but that also the wild-type aSYN protein is pathogenic, if it is overexpressed. In fact, the triplication is associated with a roughly twofold over-expression of the aSYN protein in the brain of affected individuals, as shown by Western blotting on autopsy material (Singleton et al., 2003). Subsequently several additional families with SNCA triplications, and also with SNCA duplica­ tions, have been found (Ibanez et al., 2009). In those gene locus multiplication families, a clear dose dependence of the pathogenic effect was observed: SNCA duplications, that is a 50% increase of the gene dosage (three instead of the usual two gene copies) leads to relatively lateonset dopa-responsive parkinsonism resembling typical PD, while triplications are associated with an earlier disease onset, a high prevalence of dementia and a rapid disease course. This could be even shown in a single family in whom different branches segregated a duplication and a triplica­ tion of a 1.5 Mb genomic fragment containing the SNCA gene (Fuchs et al., 2007). The identification of the first SNCA mutations by Polymeropoulos and co-workers soon leads to the discovery that the encoded protein (aSYN) is the major fibrillar component of the Lewy body and Lewy neurites (Spillantini et al., 1997), the

protein aggregates which had long been recog­ nized as the pathologic hallmark in familial as well as sporadic cases of the disease (Fig. 1). The currently favoured hypothesis states that the amino acid changes in aSYN lead to an increased tendency of the protein to form oligomers and fibrillar aggregates (Goedert et al., 1998; Karpinar et al., 2009), eventually resulting in neuronal dys­ function and cell death, although the precise rela­ tionship between mutations, aggregate formation and their deleterious effects on neurons is still unknown. Many studies favour the hypothesis that the mature aggregates (i.e. Lewy bodies and Lewy neuritis detected on the light microscopy level) are not themselves the toxic moiety, but rather an attempt of the cell to clear much smaller toxic oligomers (Cookson and van der Brug, 2007), while the elusive oligomers are the true toxic moiety (Conway et al., 2000). Mutation carriers from the first family described with an SNCA mutation (the ‘Contursi’ kindred)

Fig. 1. aSYN immunopositive neuronal inclusions in the dorsal motor vagal nucleus of a patient with an A30P SNCA mutation. Marked aSYN pathology is obvious with numerous Lewy bodies (arrowheads) and Lewy neurites (arrows). Figure kindly provided by Prof. R. Krüger, Hertie Institute for Clinical Brain Research, Tübingen.

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clinically had relatively early onset of a mostly akinetic/rigid form of PD with rapid progression and commonly also suffered from dementia. The clinical spectrum was later confirmed and extended in additional families carrying the same mutation, in whom prominent autonomic distur­ bances were also noted (Spira et al., 2001). A very similar clinical picture with early dementia and autonomic disturbances, thereby more resem­ bling a variant of PD called ‘dementia with Lewy bodies’ (DLB) rather than typical PD, was described in the family with the E46K mutation (Zarranz et al., 2004). On the other hand, a more typical late onset of parkinsonian symptoms with only late development of relatively mild dementia was described in a German family with the A30P mutation (Krüger et al., 1998). Although this family is relatively small and therefore this conclu­ sion rests only on very few cases, there appear to be mutation specific differences in disease presentation. Pathologically, fibrillar aSYN aggregates (i.e. Lewy pathology) were found in all patients with SNCA mutations, both point mutations and multi­ plications, not only in the substantia nigra but also in other brain stem nuclei and widespread in mesocortical and neocortical neurons, again com­ patible with a diagnosis of DLB (Spira et al., 2001). Interestingly, in mutation-positive cases, aSYN pathology was also found in oligodendro­ glial cells, a feature thought to be typical for multi­ ple system atrophy (MSA) (Dickson et al., 1999). This large overlap of pathologic features of PD, DLB and MSA in cases with SNCA mutations strongly supports the close aetiologic link between these disease entities.

PARK8 (LRRK2) Another locus for a dominant form of PD was first mapped in a large Japanese family to the pericen­ tromeric region of chromosome 12 and named PARK8, again by a classic linkage analysis approach. Affected members in this family

showed typical L-dopa-responsive parkinsonism with onset in their fifties (Funayama et al., 2002). By positional cloning, missense mutations in the gene for leucine-rich repeat kinase 2 (LRRK2) (Paisan-Ruiz et al., 2004; Zimprich et al., 2004) were found to be disease causing. The gene spans a genomic region of 144 kb, with 51 exons encoding 2527 amino acids, and to date, at least six verified disease-causing mutations have been identified. Mutations in the LRRK2 gene are clearly the most common cause of dominantly inherited PD discovered so far. In a number of studies across several different populations between 5 and 15% of dominant families carry mutations in LRRK2 (Berg et al., 2005; Di Fonzo et al., 2005). The single most common mutation, G2019S, is respon­ sible for familial PD in up to 7% of familial cases in different Caucasian populations (Di Fonzo et al., 2005; Kachergus et al., 2005; Nichols et al., 2005). This mutation has also been found in about 1–2% of sporadic patients of European descent (Gilks et al., 2005), indicating that the mutation has a reduced penetrance, which was estimated between 35 and 70% (Goldwurm et al., 2005; Ozelius et al., 2006) and which must be taken into account in genetic counselling. Even higher G2019S prevalence rates of up to 40% were found in genetically more isolated populations, such as the Ashkenazi Jewish or the North African Arab populations, both in sporadic and in familial cases (Lesage et al., 2006; Ozelius et al., 2006) due to genetic founder effects. Despite its reduced penetrance, the G2019S mutation is usually thought of as a true ‘disease­ causing mutation’, because it is very rare in all control populations studied so far. Other more common variants in the LRRK2 gene, on the other hand, appear to act more like risk factors of modest effect sizes. The G2385R (Farrer et al., 2007) variant, for example, was found not only in approximately 9% of Chinese patients with PD but also in about 3% of controls. The same role of a risk allele has been suggested for the R1628P exchange (Lu et al., 2008; Ross et al., 2008).

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Clinical signs and symptoms of LRRK2-related disease closely resemble typical sporadic PD. This is also true for age of onset, which is on average in the late fifties and only slightly below that in non-mutation-carrying PD patients (Healy et al., 2008). However, age at onset as well as severity of the disease may be highly variable, even within families. Pathological changes in patients with LRRK2 mutations are consistent with typical Lewy body PD in most cases reported so far and also include diffuse Lewy body disease, nigral degeneration with­ out distinctive histopathology and rarely even pro­ gressive supranuclear palsy-like tau aggregation. LRRK2 mutations may therefore be an upstream event in the cascade leading to neurodegeneration with different pathologies. Although the natural sub­ strate and disease-relevant function of LRRK2 is unknown, cell culture studies suggest that pathogenic mutations seem to be associated with increase, rather than a loss, of kinase activity (Gloeckner et al., 2006), raising the interesting possibility that kinase inhibition may be a potential therapeutic strategy.

Autosomal-recessive parkinsonism The strategies of linkage mapping and positional cloning can also be used to identify loci in genes responsible for autosomal-recessive monogenic diseases. This mode of inheritance is characterized typically by the occurrence of the disease in sib­ lings while the parents are obligatory heterozygous mutation carriers and usually remain healthy. Autosomal-recessive PD has clinically been first recognized and characterized in Japan (Ishikawa and Tsuji, 1996). Sibling pairs with PD often have much earlier age of onset compared with patients with the sporadic disease, which is why the term ‘autosomal-recessive juvenile parkinsonism’, has been coined. Since families with a recessive disease are usually much smaller than multigenerationaldominant pedigrees, linkage analysis is only successful if several families mapping to the same locus are included into a study.

PARK2 (Parkin) It was in autosomal-recessive families with very early-onset parkinsonism that the first recessive PD gene was mapped to the long arm of chromo­ some 6 in the vicinity of the gene for superoxide dismutase 2 (SOD2) (Matsumine et al., 1997). Because of the suspected role of toxic oxygen radicals in the pathogenesis of PD, SOD2 was a plausible candidate gene. However, no sequence variants in this gene could be identified. Rather, a number of different mutations were found in a very large neighbouring gene which was then called Parkin (PRKN) (Kitada et al., 1998). PRKN mutations turned out to be a common cause of parkinsonism with early onset, particularly in individuals with evidence of recessive inheri­ tance. Nearly 50% of sibling pairs with PD were found to have PRKN mutations (Lücking et al., 2000), if at least one of them had an age of onset below 45 years. As recessive diseases often appear to be sporadic, particularly in societies with rela­ tively small families, because statistically only 25% of the offspring of two heterozygous mutation car­ riers will be homozygous for the disease allele, PRKN mutations are also responsible for the major­ ity of sporadic cases with very early onset (before age 20) and are still common (25–40%) when onset is between 20 and 35 years. PRKN mutations are rare in sporadic cases with onset later than 45 years.

Other recessive forms of parkinsonism Mutations in the PINK1 gene (PARK6) have been identified as another cause for autosomalrecessive early-onset parkinsonism (Valente et al., 2004), again following a linkage mapping approach in several multiplex recessive families (Valente et al., 2001). This gene is particularly interesting within the context of the findings link­ ing PD to mitochondrial dysfunction and oxidative stress, as PINK1 encodes a mitochondrially located protein. Mutations in the PINK1 gene are less common than PRKN mutations in most

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populations studied and probably account for only 1–2% of early-onset cases (Hatano et al., 2004; Rogaeva et al., 2004; Rohe et al., 2004; Valente et al., 2004). From work with animal models it has become quite clear that PINK1 acts in a common pathway with PRKN (Dodson and Guo, 2007). However, the natural substrate of the kinase activity of PINK1 is still unknown. Mutations in the DJ-1 gene (PARK7) are yet another rare cause of autosomal-recessive parkin­ sonism (Bonifati et al., 2002; Healy et al., 2004; Hedrich et al., 2004). The clinical picture with early onset and slow progression is similar to the other recessive parkinsonian syndromes. Follow­ ing the initial discovery of two mutations in an Italian and a Dutch family (Bonifati et al., 2002), only a few additional bona fide pathogenic muta­ tions [one homozygous (Hering et al., 2004) and one compound heterozygous (Abou-Sleiman et al., 2003)] have been identified. While mutations in the genes named above all cause a ‘pure’ form of early-onset parkinsonism, an increasing number of genes have been found to cause more complex phenotypes which includes, in addition to parkinsonism, often dystonia, spas­ ticity, and dementia (Klein et al., 2009). The common denominator for all those cases is that they represent Mendelian forms of the dis­ ease, which are relatively rare, can be tackled by classic approaches of linkage analysis and posi­ tional cloning, and can be modelled in different cellular and animal model systems, which is invaluable for a better understanding of the mole­ cular pathways leading to dopaminergic neurode­ generation (Gasser, 2009b).

Rare genetic variants causing or pre-disposing to PD While the genes described above are generally thought of as high-penetrance disease-causing genes, the genetic epidemiology of LRRK2­ associated PD has already made clear that the boundary between disease genes and risk factors is more a matter of semantics than of biology.

While some mutations such as R1441C or Y1699C have so far only been found in families with clear autosomal-dominant inheritance and thus are con­ sidered to be high-penetrance disease genes, the G2019S variant is clearly a disease gene with mark­ edly reduced penetrance, which is still very rare in unaffected individuals, while the variants G2385R or R1628P are found in >1% of the healthy Chinese population and thus must be considered relatively ‘common’ genetic variants, which are associated with an approximately threefold increased risk to develop PD. In addition to the concept that rare mutations cause rare genetic diseases and – on the other hand – common variants are associated with a mod­ erate increase of relative risk for common disorders, another concept has emerged over the last several years: the rare variant-common disease hypothesis. It states that multiple rare variants in a potentially large number of genes may each be significant (par­ tial) causes in a relatively small proportion of patients with a common disease such as PD. This hypothesis is exemplified in the role of mutations in the gene for glucocerebrosidase (GBA) in PD. About 10 years ago, astute clinical observation suggested that patients with Gaucher’s disease, an autosomal-recessive, usually childhood-onset lysoso­ mal storage disease associated with a wide variety of organ manifestations, had a conspicuous tendency to develop PD in later life (Machaczka et al., 1999; Tayebi et al., 2001). Gaucher’s disease is caused by mutations in the gene for glucocerebrosidase, GBA, which is located in chromosome 1q21. GBA is an enzyme of the ceramide pathway, and its deficiency leads to the excessive storage of its substrate, glucosylceramide, within lysosomes of many differ­ ent cell types, including neurons and macrophages (Grabowski, 2008). Following the initial reports of a clinical association of Gaucher’s disease with PD it was observed that heterozygous, otherwise healthy, carriers of GBA mutations, that is the relatives of Gaucher patients, also have an increased risk to develop PD. This association, which was initially observed in Ashkenazi Jewish families in whom the carrier frequency for GBA mutations is particularly

10

high, was later confirmed in a number of larger patient series from different Jewish and non-Jewish populations (Aharon-Peretz et al., 2004; De Marco et al., 2008; Mata et al., 2008), in a study comprising autopsy-proven cases (Goker-Alpan et al., 2006) and, most recently, in a large meta-analysis (Sidransky et al., 2009). This association is now firmly established. For example, a recent study showed that the prevalence of GBA mutations in British patients with sporadic PD is 3.7%, while the frequency of these variants is less than 1% in the general population. Mutations in the GBA gene therefore are the most common risk factor for development of PD in this population detected so far (Neumann et al., 2009). The relative risk conferred by heterozygous carrier status for the development of PD varies, for different mutations and in different studies, from about 4 to 20, with the large meta-analysis suggesting a relative risk of about 4 for the N370S mutation, the most common variant allele in Ashkenazi Jews, and about 6 for the most common mutation in non-Jews, L444P (Sidransky et al., 2009). This estimation of modest but significant rela­ tive risks to develop the disease explains why so far no large ‘GBA-families’ have been identified. As far as it is known today there is no way to distinguish PD patients with GBA mutations from those without, both clinically and pathologically. As a group, GBA-positive patients have a slightly earlier age of onset (55 vs. 59 years) and a some­ what higher prevalence of dementia (Sidransky et al., 2009). Neuropathologic examination of 17 GBA mutation carriers showed typical PD changes, with widespread and abundant SNCA pathology, and most also had neocortical Lewy body pathology (Neumann et al., 2009).

Common risk variants for PD: candidate gene and whole genome association studies Association studies have been widely used in an attempt to identify common genetic variations that carry a mild to moderately increased risk to

develop a disease. Over the years, literally hun­ dreds of studies have been published, but unfortu­ nately, only very few of them have produced robust and reproducible results. The reasons for the failure of this approach are manifold: 1. Most studies were greatly underpowered in relation to the small increases in the relative risk that today are known to be conferred by common genetic variants (usually the odds ratios are in the range of 1.2–2, with some exceptions, for example apolipoprotein E for AD). 2. The choice of candidate genes was often based on rather arbitrary rationales with very weak experimental or epidemiologic evidence. As the gene-mapping studies in monogenic diseases have shown, newly identified genes most often could not have been predicted based on the current knowledge of pathogenesis. 3. In most studies only arbitrarily chosen individual genetic variants were interrogated; thus it was a priori unlikely that the causative variant or a variant in high linkage disequilibrium, tagging the risk-conferring variant, would be among those studied. Finally, it is not easy to match patient and con­ trol cohorts with respect to their genetic back­ ground. Often, due to different recruiting strategies, these cohorts differ in their genetic composition (a problem called undetected popula­ tion stratification, which today can be easily resolved in GWAS, see below). Due to different allele frequencies in different populations, spur­ ious associations can be detected. Due to these shortcomings in study design, so far not a single association study result concerning PD truly withstood the test of time and replication, with the notable exceptions of candidate gene associa­ tion studies of the SNCA and MAPT genes, two genes that initially were identified by mapping disease genes in monogenic families (see below). Technical advances in sequencing technologies including a vast increase in speed and accuracy of genotyping individual sequence changes (SNPs)

11

together with a rapid increase in knowledge about the haplotype structure of the human genome paved the way for a more systematic study of genetic variability and its relationship to common diseases such as PD. This is nicely exemplified in the way that variability in SNCA and in the gene for the microtubule-associated protein tau (MAPT) is now recognized as the major genetic risk factors in sporadic PD. Since aSYN aggregates are widely accepted to be the hallmark neuropathologic change in PD and the role of SCNA point mutations and gene multiplications is clearly established in familial PD, it was only obvious to search for a possible association of genetic polymorphisms in the SNCA gene with sporadic PD. SNCA variability, putatively influencing the level of SNCA gene expression, became a particularly plausible candi­ date after the discovery that multiplications of the SNCA locus and thus a gene dosage effect of the wt aSYN were the cause for dominant PD. Early studies had produced somewhat conflict­ ing results, because again, in hindsight, those stu­ dies were underpowered given the relatively small odds ratios that are known today. Nevertheless, the majority of association studies, including a large meta-analysis of studies interrogating the ‘NACP-REP1’ polymorphism, a complex repeat element located about 10 kb upstream of the SNCA coding region (Maraganore et al., 2006), supported the assumption of a role of SNCA var­ iants in sporadic PD. Müller et al. performed the first systematic study of SNPs in the SNCA gene, analyzing more than 50 variants distributed over the entire gene in more than 600 patients and a similar number of controls, thereby capturing nearly all of the genetic variability of the gene. They found that the SNCA gene consists of two major haplotype blocks: one comprising the pro­ motor and exon 1–4 and another one spanning exons 5 and 6 and the 30 -untranslated region of the gene. The strongest association signal was detected with SNPs in 30 -haplotype block, although a second, somewhat weaker signal in the promotor region was also detected. Due to a

certain degree of linkage disequilibrium between those regions, it was not possible to definitively separate the signals. On the other hand, as a num­ ber of different regulatory mechanisms are likely to determine SNCA expression, it would not be surprising if multiple regions of the gene were found to be involved. Since then a number of additional association studies were able to replicate these results in dif­ ferent populations (Mizuta et al., 2006; Winkler et al., 2007). Most of them found 30 -variants to be most significantly associated with the disease. Further evidence for a role of genetic variants in the SNCA gene came from GWAS (see below). The history of the association of MAPT, the gene encoding the microtubule-associated protein tau (MAPTau), is less straightforward. The gene has been studied as a candidate gene for neuro­ degenerative diseases for many years. Initially, MAPTau was identified as the main component of paired helical filaments, the intracellular hallmark neuropathology of AD (Goedert et al., 1988). Tau aggregates also form the major pathol­ ogy in several other diseases, the so-called tauo­ pathies, which include a subset of patients with a dementia syndrome called frontotemporal lobar degeneration (FTLD), and also atypical parkinso­ nian syndromes, such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) (Williams, 2006). Point mutations in MAPT, which either affect its amino acid sequence or the splicing of its isoforms, were then identified to cause a monogenic form of FTLD, called FTLD-17 or, today, FTLD-Tau (Hutton et al., 1998). Given the prominent tau-pathology in PSP and CBD, it was not surprising when it was reported that genetic variability in MAPT, initi­ ally a dinucleotide repeat, was found to be asso­ ciated with these disorders (Conrad et al., 1997). A closer study of this genetic region on chromo­ some 17 revealed that this variant was part of an extended haplotype, spanning about 1.5 Mb. Two major forms of this haplotype exist, called H1 and H2, with H1, which occurs on about 80% of Cau­ casian chromosomes, conferring the higher risk.

12

Despite their large size, H1 and H2 haplotypes do not recombine, because they are positioned in inverse orientation on the chromosome (Zody et al., 2008). As a consequence, the association cannot be pinned down more accurately by recombination mapping. However, Pittman and co-workers identified an H1 sub-haplotype (H1c), which showed a stronger association with PSP than the H1 parent haplotype, and suggested that the risk-conferring variant was located to a 22-kb intronic region of MAPT (Pittman et al., 2005). Although the major pathology of PD is com­ posed of aSYN and not MAPTau, the MAPT gene was studied as a candidate gene for typical PD, because the genomic region containing MAPT produced positive, although not genomewide significant, lod scores in a linkage study of 174 multiplex PD families (Scott et al., 2001). Somewhat surprisingly, a rather strong association signal was found in a subsequent study (Martin et al., 2001), a finding later confirmed and refined in other studies (Kwok et al., 2004; Skipper et al., 2004; Tobin et al., 2008; Zabetian et al., 2007). Again, this association was recently confirmed in genome-wide approaches.

Genome-wide association studies The advent of array technologies has taken the analysis of human genetic variability and its influ­ ence on the development of complex diseases to a new level. This technology allows to simulta­ neously genotype hundreds of thousands of SNPs, the most frequent form of genetic variants, in a large number of individuals at relatively low costs. In parallel, analytic tools have been devel­ oped to deal with the vast amount of data which are generated by these techniques. In recent years GWAS have been extremely fruitful in identifying common (a frequency of more than 5% of a genetic variant in a population is considered to be common) risk variants of rela­ tively low individual effect strength in many

important complex human diseases such as type 2 diabetes, atherosclerosis or AD, to name just a few (Harold et al., 2009; Kronenberg, 2008; Latourelle et al., 2009). Although there are more than 3 million single-nucleotide variants compared with the published reference sequence in any given individual genome, most of the genetic variability can be captured by genotyping approximately 500 000 carefully chosen SNP markers. This is due to the fact that the human genome consists of distinct segments, typically 10 000–50 000 base pairs in length, which are usually inherited en bloc. This so-called haplotype bloc structure of the human genome has been mapped and depos­ ited in publicly available data banks (the interna­ tional HapMap Project) and allows to predict, with high probability, the genetic variants at an adjacent locus within this bloc. GWAS have been able to reliably identify genetic variants which convey a relative risk of about 1.2–1.5 if populations on the order of 5 000–10 000 individuals are analysed. However, based solely on association studies it is usually not possible to determine which of the many variants in a haplotype bloc are functionally rele­ vant. Many of the risk-conferring variants are likely not to alter the amino acid sequence of one of the genes in the region; rather, biologically relevant variants will probably influence genetic regulatory networks, for example gene expression through alteration of transcription factor binding sites, alternative splicing or mRNA stability, for example by changing sequences detected by micro-RNAs. The elucidation of the biologic mechanisms underlying an association is therefore still a challenge for molecular biology. GWAS have overcome many of the problems of candidate gene association studies that have been described above. For example, the ‘genetic back­ ground’ is easily corrected for by taking into account the overall genetic ‘likeness’ of the SNP profile of the probands in the study. Individuals who differ with respect to their genetic back­ ground can be easily excluded from the analysis. A major challenge of GWAS is the fact that

13

genotyping of 500 000 variants per individual results in a huge number of statistical tests to be performed and thus a considerable problem of multiple testing. Although not all 500 000 tests can be considered to be truly independent, the mere number of tests leads to a large number of nominally significant associations. Typically sev­ eral thousand results with a nominal p-value of 10–3 to 10–5 are obtained, the vast majority simply by chance. In most of the recent studies, however, this has not been much of a problem. Sufficiently powered studies provided results with p-values of genome-wide significance applying stringent Bon­ ferroni corrections (the genome-wide threshold is usually at a p-value in the order of 10–7), and results of genome-wide significance have usually proven to be reproducible in subsequent studies. Therefore, the danger of false-positive results is low if adequate corrections are applied. However, it is unknown how many of the results with nom­ inally significant p-values below the genome-wide threshold are true associations that are presently being missed. In PD, the first GWAS was published in 2005. A total of 198 345 SNPs were genotyped in 443 sib­ ling pairs discordant for PD. In a second stage, the top 1793 PD-associated SNPs (p < 0.01) and 300 genomic control SNPs were typed in 332 matched case-unrelated control pairs (Maraganore et al., 2005). Given today’s knowledge of the effect strengths of common risk variants in PD, this study was significantly underpowered and none of the detected associations reached genomewide significance after Bonferroni correction. Nevertheless, it demonstrated the feasibility of this approach in a complex disorder such as PD and other studies soon followed. The next GWAS again used a relatively small sample, 267 PD patients and 270 neurologically normal controls (Fung et al., 2006). More than 408 000 unique SNPs were genotyped, without detecting an association signal. Increasing the study cohort to almost 900 patients and controls, Pankratz and co-workers, using Affimetrix 550K mapping chips, still did

not detect associations that survived stringent Bonferroni correction for multiple testing. How­ ever, SNPs in the SNCA and MAPT gene were among their top hits (Pankratz et al., 2009), with p-values of 5.5 × 10–5 and 2.0 × 10–5, and odds rations of 1.35 and 0.56, respectively. The close concordance with previous candidate gene studies of these loci (Martin et al., 2001; Mueller et al., 2005) supported these findings. Only recently in a still larger two-stage GWAS studying a total of more than 5000 PD subjects and 8000 controls, SNCA and MAPT again showed the strongest association signal and finally this study was powered sufficiently to identify those regions unequivocally with genome-wide signifi­ cance (p-values < 10–7) (Simon-Sanchez et al., 2009). As in the candidate gene study reported earlier (Mueller et al., 2005), the most strongly associated SNPs in SNCA were located in the 30 ­ region of the gene, in a haplotype block containing exons 5 and 6 as well as the 30 -untranslated region (30 -UTR) (Fig. 2). The relative risk conferred by genetic variability in SNCA was only about 1.3. However, the risk allele is common (about 40% of the population), and therefore this variant explains about 9% of the disease risk on the popu­ lation level. Based on this converging evidence it is now firmly established that genetic variability in SNCA influences the risk to develop typical spora­ dic PD. Very similar results with respect to SNCA have been obtained in a simultaneous study in Japan (Satake et al., 2009). However, it is still unclear how changes in the non-protein coding sequence of SNCA influence PD risk. Possible mechanisms are differential binding of enhancers or suppressors of transcrip­ tion (Fuchs et al., 2008), alterations in splicing, or, particularly with respect to variants in the 30 -UTR, differential binding of micro-RNAs. So far no direct in vivo evidence has been produced that clearly supports any one of those possibilities. Interestingly, the MAPT locus has also been confirmed with genome-wide significance as a risk factor in sporadic PD in the European, but not in the Asian, population (Satake et al., 2009;

14

Fig. 2. The genomic and haplotype structure of the SNCA locus (encoding SNCA in light grey) and p-values of SNPs investigated as part of a genome-wide association study (Simon-Sanchez et al., 2009). Each dot in the upper panel represents a single-nucleotide variant (SNP) tested for association in a region on chromosome 4, from base pair 90 500 000–91 500 000 (x-axis) with its respective pvalue (y-axis). In the lower panel, the haplotype structure of the region is symbolized. Figure kindly provided by Dr. A. Singleton.

Simon-Sanchez et al., 2009). In Asians, the taulocus is not polymorphic. Whether SNCA and MAPT act independently as risk factors as sug­ gested by two GWAS (Pankratz et al., 2009; Simon-Sanchez et al., 2009) or synergistically, as proposed in a candidate gene study by Goris et al. (2007), is still unclear. While evidence has been provided that the biologic effect of the MAPT haplotype is mediated by increased transcriptional activity of the risk haplotype (Simon-Sanchez et al., 2009), this does not seem to be the case for SNCA. An interaction on the protein level is possible, as co-staining of Lewy bodies with anti­ bodies to aSYN and MAPTau has been demon­ strated (Duda et al., 2002), and the concept of ‘cross-seeding’ of different proteins has been

discussed for a number of protein aggregation diseases such as the polyglutamine diseases and the prion disorders (Derkatch et al., 2004). Still larger study populations will probably reveal even more common risk alleles in future GWAS.

Future strategies Rapid further advances in sequencing technolo­ gies have already set the stage for next generation of analysis, which will take the form of whole exome or whole genome sequencing. While these technologies today are still rather expensive and the bioinformatics to deal with the enormous

15

amount of data generated are still a formidable challenge, the pace of technological progress is certain to increase in the next years and therefore a significant contribution to the understanding of complex diseases can be expected. A combination of technologies, such as whole genome haplotyp­ ing or sequencing combined with transcriptome analysis or RNA sequencing in tissues and cell types, will generate huge libraries mapping genetic expression networks with so far almost unimagin­ able complexity.

Whole exome sequencing Several technologies have been developed to sequence all expressed exons of the human nuclear genome. Basically, these technologies comprise multiple steps, including target enrich­ ment, actual sequencing, and data analysis. In a first step, hybridization techniques, using solidphase or emulsion-based short sequences repre­ senting all ~180 000 exons of the human genome, are used to enrich for the desired target sequences. Then, the enriched fragments are sequenced in a ‘massive-parallel’ fashion, that is each sequence is usually read multiple times (usually 10- to 30-fold coverage is required). Finally, the sequence data are assembled and aligned to produce a consensus sequence. So far, whole exome sequencing has revealed a large number (several thousands) of novel var­ iants in each sequenced individual. It will be a formidable challenge to devise strategies to eval­ uate the biological relevance of these variations. It is expected that whole exome sequencing will be particularly useful to identify rare variants of moderate to high effect strength. One potential application of whole exome sequencing that has been already successfully applied is the identification of rare autosomaldominant or autosomal-recessive disease genes following an approach that can be considered to be a ‘shortcut’ to the classic positional cloning approach described above (Ng et al., 2010). If

the entire exome of two affected members of a family (or better two or three families) with a rare monogenic disease, who are separated by at least four to six meiotic events (e.g. first- or seconddegree cousins) is sequenced, the number of potentially pathogenic shared novel variants is relatively limited, particularly if, as in a recessive disease, loss-of-function mutations are suspected. It is conceivable that a significant proportion of cases even in a late-onset neurodegenerative dis­ order such as PD might be due to rare recessive monogenic causes which could be identified using this approach. Another application may be the identification of relatively rare risk alleles of moderate effect, similar to the GBA mutations described above (Sidransky et al., 2009), which usually escape detection by whole genome SNP-genotyping approaches, because they are, individually, too rare, although in their entirety, they may still explain a significant proportion of cases. Although the distinction is of course somewhat arbitrary, by definition, risk alleles (as opposed to ‘disease-caus­ ing mutations’) also occur in the general popula­ tion. It will therefore be necessary to genotype a very large number of patients and controls to be sure of a significant disease association. The quan­ titative contribution of rare variants to a complex disease such as PD is, however, still completely unknown.

Whole genome sequencing When the first finished grade human reference genome [NCBI build 36 (International Human Genome Sequencing, 2004)] was published in 2004, it was almost inconceivable that only 3 years later the personal genome of one of the pioneers of genomic science J. Craig Venter would be published (Levy et al., 2007). Only a year later, the personal genomic sequence of James D. Watson was made public (Wheeler et al., 2008). Since then a rapidly growing number of individual genomes has been sequenced and

16

published, most recently an example of a medical application, the identification of a mutation in a patient with a rare form of Charcot–Marie–Tooth neuropathy (Lupski et al., 2010). In each of the individual genomes sequenced, more of 3 million single-nucleotide variants deviat­ ing from the published reference sequence and sev­ eral hundreds of thousands of larger structural variations usually copy number repeats were detected. In a recent study of the entire genome of five men from sub-Saharan Africa, 1.3 million novel DNA differences genome-wide including more than 13 000 coding changes were identified. Again, it will be a challenge for years to come to study the functional relevance of all this variability. Whole genome sequencing will certainly replace even whole exome sequencing within a foreseeable future. Today basically the same tech­ nology platforms are used as in whole exome sequencing, except for the initial target enrich­ ment step. Because of the much larger number of sequences that have to be read, a whole genome sequence is still estimated to cost more than 100 000 dollars (Metzker, 2009). Therefore only a few examples have been published. Never­ theless the ‘1000-dollar genome’ will probably be available within the next 3–5 years.

Conclusion The unravelling of the genetic underpinnings of complex diseases, such as PD, has just begun. Known Mendelian forms of PD and known genetic risk factors presently explain at most about 20% of all cases, on the general population level. It is often assumed that the common form of sporadic PD results from an ‘interaction of genetic and environmental factors’. While this is probably true if ‘environmental factors’ are defined broadly and would include, for example, the aging process, which can be seen as the major ‘environmental’ risk factor for PD, it is likely that the potential of genetics in explaining the multiple causes of PD is far from exhausted.

Abbreviations APP AD aSYN GWAS MAPT MAPTau

Low-coverage genome sequencing An international collaborative project called the ‘1000 genomes project’ aims to establish a catalo­ gue of human genetic variants that have a preva­ lence of at least 1% in the populations studied. This goal is achieved by sequencing a large num­ ber of individuals, but not with 30-fold coverage, as is necessary to obtain a complete and reliable sequence of an individual subject, but only with the coverage of about fourfold. The data of this project are already used to improve the informa­ tion gathered from whole genome SNP analyses, because many of the rarer variants which are not represented on the genotyping arrays can be deduced (‘imputed’) using this database.

MSA PD SNCA SNP

amyloid precursor protein Alzheimer’s disease alpha-Synuclein (protein) genome-wide association studies microtubule-associated protein tau (gene) microtubule-associated protein tau (protein) multiple system atrophy Parkinson’s disease alpha-synuclein (gene) single-nucleotide polymorphism

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A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 2

The impact of genetic research on our understanding of Parkinson’s disease Ian Martin†,‡, Valina L. Dawson†,‡,§,k and Ted M. Dawson†,‡,k, †

NeuroRegeneration and Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA ‡ Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA § Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA k Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

Abstract: Until recently, genetics was thought to play a minor role in the development of Parkinson’s disease (PD). Over the last decade, a number of genes that definitively cause PD have been identified, which has led to the generation of disease models based on pathogenic gene variants that recapitulate many features of the disease. These genetic studies have provided novel insight into potential mechanisms underlying the aetiology of PD. This chapter will provide a profile of the genes conclusively linked to PD and will outline the mechanisms of PD pathogenesis implicated by genetic studies. Mitochondrial dysfunction, oxidative stress and impaired ubiquitin–proteasome system function are disease mechanisms that are particularly well supported by genetic studies and are therefore the focus of this chapter. Keywords: alpha-synuclein; LRRK2; PINK1; Parkin; DJ-1; mitochondrial dysfunction; oxidative stress; ubiquitin-proteasome system

principally by symptoms of resting tremor, brady­ kinesia, rigidity and postural instability, although additional dysfunction of non-motor systems is fre­ quently present (Savitt et al., 2006). The primary symptoms, which together constitute parkinson­ ism, arise from a profound degeneration and loss of dopaminergic neurons in the substantia nigra pars compacta, which leads to a marked depletion of the neurotransmitter dopamine in the striatum, a key region of the basal ganglia that regulates movement (Dauer and Przedborski, 2003).

Introduction Parkinson’s disease (PD) is the most common neu­ rodegenerative movement disorder, affecting approximately 1% of the population at 65 years of age, increasing to 5% at 85 years (Van Den Eeden et al., 2003). Clinically, PD is characterized 

Corresponding author. Tel.: (410) 614 3359; Fax: (410) 614 9568; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83002-X

21

22

Identifying a role for genetics in PD The main discoveries that have shaped our under- standing of PD are outlined in Fig. 1. Until the end of the twentieth century, PD was predominantly

considered a non-genetic disease caused by envir­ onmental factors and aging (Farrer, 2006). This notion was propagated early-on by the emergence of parkinsonism in survivors of the encephalitis epidemic that occurred from 1917 to 1928

Timeline of key discoveries on PD pathogenesis 1817

James Parkinson publishes the first formal description of PD entitled “An Essay on the Shaking Palsy,” establishing Parkinsonism as a recognized medical condition.

1912

Friedrich Lewy first describes concentric inclusion bodies in nigral cells of patients presenting with “paralysis agitans” (Obsolete name for Parkinsonism). These inclusions were later named Lewy bodies and Lewy neurites.

1919

Konstantin Tretiakoff observes a reduction in pigmented cells of the substantia nigra in brains of PD patients.

1920s The post-encephalitic parkinsonism (PEP) epidemic following a worldwide outbreak of Influenza in 1918 suggests possible link between PD development and viral exposure. 1950s Arvid Carrlson reports a high concentration of dopamine in the basal ganglia which, in rabbits, becomes depleted by treatment with reserpine. Reserpine-treated rabbits are incapable of voluntary movement, similar to patients with severe PD. These symptoms could be alleviated by administering L-DOPA indicating a role for dopamine deficiency in causing PD and leading to clinical use of L-DOPA. 1983

Exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) associated with acute-onset of PD. Chemical similarity of MPTP to the herbicide paraquat leads to studies linking pesticide exposure to PD.

1997

Identification of α-synuclein mutations as an underlying cause of familial PD. Subsequent studies suggest that aggregates of misfolded α-synuclein may be toxic to dopamine neurons and impair α-synuclein’s role in vesicular transport.

1997

Mutations in Parkin linked to AR-JP. Evidence suggests impaired ubiquitin–proteasome function and mitochondrial dysfunction associated with loss of Parkin E3 ligase function instrumental in PD pathogenesis.

2003

DJ-1 mutations linked to early-onset PD. DJ-1 is important in protecting against oxidative stress and cell death illustrating central importance of these factors to PD pathogenesis.

2004

Loss-of-function Pink1 mutations linked to PD and subsequently shown in model systems to result in mitochondrial dysfunction and cell death.

2004

Pathogenic LRRK2 mutations identified, many later shown to possess increased kinase activity to generic substrates. Translational inhibitor 4E-BP may be in vivo substrate, suggesting a role for general protein translation in PD pathogenesis.

Fig. 1. Timeline of key discoveries in PD pathogenesis. Parkinson’s disease was first formally described in 1817 by James Parkinson. From then until the late twentieth century, advances were made in describing the pathological features of PD and in understanding possible causes of the disease, such as exposure to pesticides and MPTP. The recent discovery of multiple genetic causes of PD has generated insight into novel mechanisms of PD aetiology. Studies using genetic models of PD will, no doubt, continue to advance our understanding of disease pathogenesis.

23

(Poskanzer and Schwab, 1963) and later fuelled by discoveries from epidemiological studies that par­ kinsonism is associated with exposure to certain pesticides and to 1-methyl-4-phenyl-1,2,3,6-tetrahy­ dropyridine (MPTP) via narcotic use (Elbaz and Moisan, 2008; Langston et al., 1983). A major con­ tribution of heredity to development of the disease was thought unlikely due to epidemiological studies which indicated no effect of heritability on lifetime risk of developing PD (Cookson et al., 2005). A role for genetics in PD was also not supported by crosssectional twin studies that suggested low concor­ dance rates in monozygotic and dizygotic twins (Marttila et al., 1988; Ward et al., 1983). This was despite the fact that since as early as the 1880s, clinicians had been noting familial aggregation of parkinsonism (Gowers, 1900; Leroux, 1880) and that numerous families inherited PD in a Mendelian fashion (Bell and Clark, 1926) suggesting a genetic contribution to disease. For example, studies on multiple populations in the mid-twentieth century indicated that the emergence of many cases of parkinsonism was consistent with an autosomal dominant mode of inheritance (Allen, 1937; Mjones, 1949). Finally, over the last decade, linkage mapping studies have resulted in the identification of distinct genetic loci that definitively cause familial PD (Farrer, 2006) (Table 1). These discoveries have resulted in a paradigm shift in perceptions towards the contribution of genetics to PD that extend beyond early-onset familial disease, since variants in a-synuclein and leucine-rich repeat kinase 2

(LRRK2) contribute to the lifetime risk of sporadic PD in the population (Cookson et al., 2005; Satake et al., 2009). Importantly, recent advances in the genetics of PD have led to cell and animal models of disease that promote our understanding of mole­ cular pathways underlying the pathogenesis of PD (Dawson et al., 2002; Moore and Dawson, 2008). Although monogenic and sporadic forms of PD are not clinically or pathologically identical, they exhibit common core features such as parkinsonism and loss of nigral dopamine neurons suggesting that they share common mechanisms of disease and that genetics should, therefore, provide clues to the aetiology of sporadic PD (Cookson et al., 2005). The following section describes the genes that have been definitively linked to PD, the normal function of their gene products and how pathogenic mutations are thought to impact cell systems. This outline will form the basis for a subsequent discus­ sion of how genetics has impacted our understand­ ing of PD pathogenic mechanisms.

Dominantly inherited mutations a-Synuclein Studies on a large family of Italian descent (the Contursi kindred), with apparent autosomal dominant PD, led to the discovery of a PD sus­ ceptibility locus on the long arm of chromosome 4 (Polymeropolous et al., 1996) that was later

Table 1. Genes underlying familial PD

Locus

Gene

Inheritance pattern

Typical age of onset

PARK1 and PARK4 PARK2 PARK6 PARK7

SNCA

Dominant

24–65

Parkin PINK1 DJ-1

Recessive Recessive Recessive

16–72 20–40 20–40

PARK8

LRRK2

Dominant

32–79

Phenotype characteristics Parkinsonism (progression related to gene dose) with common dementia and autonomic dysfunction Slow-progressing Parkinsonism Slow-progressing Parkinsonism Slow-progressing parkinsonism sometimes with behavioural disturbances Classic Parkinson’s disease

Gene variants that segregate with PD are listed. The inheritance pattern, typical age of disease onset and characteristic phenotypes observed are described.

24

identified as the SNCA gene that encodes a-synu­ clein (Polymeropolous et al., 1997). The 209G>A (Ala53Thr) mutation found in this family was followed by the discovery of two further PDassociated SNCA missense mutations: 88G>C (Ala30Pro) (Kruger et al., 1998) and 188G>A (Glu46Lys) (Zarranz et al., 2004). Patients with SNCA point mutations typically develop promi­ nent dementia and an earlier onset of parkinson­ ism than in sporadic PD (Farrer, 2006). In addition to point mutations, duplication or tripli­ cation of SNCA has been found in kindreds with classic PD or sometimes parkinsonism with auto­ nomic dysfunction and dementia (Chartier-Harlin et al., 2004; Singleton et al., 2003). The strong association between SNCA mutations or multi­ plications and PD suggests a central role for a-synuclein in PD pathogenesis (Moore et al., 2005). Furthermore, comparison of patients with duplications and triplications of SNCA reveals that age of onset is younger and disease progres­ sion faster with gene triplication (which results in an approximate doubling of plasma a-synuclein levels) (Farrer et al., 2004; Miller et al., 2004; Ross et al., 2008) suggesting that a-synuclein expres­ sion level and disease severity are related. This correlation between a-synuclein expression levels and PD susceptibility is further supported by stu­ dies in patients with sporadic PD in which allelic variability in regions of the SNCA promoter (especially the Rep1 region) is associated with risk of developing PD (Farrer et al., 2001; Pals et al., 2004; Tan et al., 2000), although this remains somewhat controversial (DeMarco et al., 2008; Spadafora et al., 2003; Tan et al., 2003). a-Synuclein exists mainly as a 140 amino acid protein whose precise function is unknown (Moore et al., 2005). a-Synuclein is expressed in neurons throughout the mammalian nervous system where it resides predominantly at pre-synaptic terminals associated with vesicles and membranes (Bonini and Giasson, 2005; Fortin et al., 2004; Kahle et al., 2000). Interest­ ingly, a-synuclein is natively unfolded in solu­ tion, although it adopts an alpha-helical-rich

conformation when associated with membranes (Ferreon et al., 2009). The precise function of a-synuclein is unclear, although its association with synaptic vesicles sug­ gests a possible role in neurotransmission. Indeed, studies in yeast and mammalian systems suggest that a-synuclein may regulate synaptic vesicle trafficking via binding to lipids (Jenco et al., 1998; Nemani et al., 2010; Outeiro and Lindquist, 2003). A recent report describes a possible role for a-synuclein in the assembly of soluble NSF attachment protein receptors (SNARE) complex between vesicle and pre-synaptic membranes, which is crucial for priming and recycling of synap­ tic vesicles (Bonini and Giasson, 2005; Chandra et al., 2005). Deletion of the co-chaperone cysteine-string protein-a in mice results in neuro­ degeneration with underlying impairment in SNARE complex assembly (Chandra et al., 2005). The authors further report that transgenic expression of a-synuclein attenuates inhibition of SNARE complex formation and prevents neuro­ degeneration. Despite this, genetic knock-out studies in mice have indicated that the absence of a-synuclein has no significant effect on the pool size of recycling synaptic vesicles, synaptic plasticity or dopamine uptake and release from nerve terminals (Chandra et al., 2004) suggesting that a-synuclein may not be required for regulat­ ing synaptic vesicle release or uptake under nor­ mal conditions and may, instead, be protective following exposure to certain cell stressors. Mutations and multiplications in the SNCA gene may cause PD through a gain-of-toxic-function mechanism as suggested by their dominant inheri­ tance pattern. When mutated or at elevated concen­ trations, a-synuclein has a propensity to develop a b-sheet-rich structure that readily polymerizes into oligomers (Sharon et al., 2003) and higher order aggregates such as fibrils (Conway et al., 1998) in cells, animal models and human brain (Lee et al., 2002; Miller et al., 2004; Outeiro et al., 2008; Sharon et al., 2003). Insoluble a-synuclein fibrils are a major component of hallmark PD inclusions called Lewy bodies and Lewy neurites, present in

25

perikarya and neurites, respectively. In PD, Lewy bodies and neurites can be found in both dopami­ nergic and non-dopaminergic neurons of the brain­ stem and in the cortex (Farrer, 2006). Importantly, Lewy bodies are found in a number of neurodegen­ erative diseases involving SNCA mutations includ­ ing PD, parkinsonism with dementia and dementia with Lewy bodies (Mart´ı et al., 2003). This estab­ lishes a link between these diseases with distinct clinical features but shared pathology. Controversy exists as to whether Lewy bodies and neurites are a cause or consequence of PD, and some evidence suggests that they might actually have a protective role by acting to sequester toxic a-synuclein oligo­ mers (Olanow et al., 2004; Tanaka et al., 2004). Fuelling this controversy are the findings that cer­ tain SNCA mutations (A53T and A30P) promote oligomerization but not fibrillization of a-synuclein, and moreover that Lewy bodies are frequently absent from the brains of PD patients with genetic mutations (Ahlskog, 2009; Conway et al., 2000; Gaig et al., 2007). Accordingly, emerging evidence suggests that pre-fibrillar oligomers and protofibrils are the toxic species responsible for PD pathology (Conway et al., 2000; Danzer et al., 2007; Goldberg and Lansbury, 2000; Kayed et al., 2003; Masliah et al., 2000). a-Synuclein oligomers, akin to other amyloidogenic oligomers cause elevated Ca2þ influx into cells in vitro, possibly by altering membrane stability or permeability by forming membrane pores (Danzer et al., 2007; Demuro et al., 2005). Elevated intracellular Ca2þ levels may promote cellular toxicity through increased generation of reactive oxygen species and resultant oxidative damage. Whether the pathogenic a-synuclein species is oligomeric, fibrillar or both, it is reasonably clear that aggregates of this protein are toxic in primary neuronal cultures (Petrucelli et al., 2002; Tanaka et al., 2001; Xu et al., 2002; Zhou and Freed, 2005), invertebrate animal models (Feany and Bender, 2000; Kuwahara et al., 2006; Lakso et al., 2003; Park and Lee, 2006; Periquet et al., 2007) and in rodent (Lo Bianco et al., 2002; St Martin et al., 2007) and non-human primate

models involving viral vectors to deliver a-synuclein to the substantia nigra (Kirik et al., 2003; Yasuda et al., 2007). Aggregation may be promoted by numerous factors including mitochondrial complex I inhibitors paraquat and rotenone (Manning-Bog et al., 2002; Sherer et al., 2002, 2003). Evidence linking exposure to these compounds with the occurrence of sporadic PD suggests a possible role for a-synuclein aggregates in sporadic disease. Additionally, oxidative and nitrative damage, which accumulate in the brains of many species including humans during aging, may promote aggre­ gation of a-synuclein to toxic species (Cole et al., 2005; Leong et al., 2009; Ostrerova-Golts et al., 2000; Qin et al., 2007). Tyrosine nitration of a-synu­ clein is found in the PD brain and has been shown to accelerate a-synuclein aggregation in vitro (Giasson et al., 2000) by a mechanism that may include reduced efficiency of a-synuclein degradation by calpain I and 20S proteasome (Hodara et al., 2004). Lastly, the interaction of a-synuclein with other amyloidogenic proteins such as tau (Giasson et al., 2003) or amyloid-b (Masliah et al., 2001) may synergistically drive fibrillization of these proteins. For example, amyloid-b peptides were shown to promote intraneuronal a-synuclein aggregation in cell cultures and transgenic mice expressing both a-synuclein and amyloid-b neuronally devel­ oped more a-synuclein-immunoreactive inclusions than singly a-synuclein transgenic mice. These dou­ ble transgenic mice also exhibited motor deficits and impaired learning and memory before mice expressing transgenic a-synuclein only (Masliah et al., 2001). A clear relevance of this stimulatory effect on a-synuclein aggregation applies to patients with clinical and pathological features of both PD and Alzheimer’s disease (e.g. those with the Lewy body variant of Alzheimer’s disease). While a-synuclein is seen to undergo aggregation and post-translational modifications, and these events may lead to its toxicity to neurons, the effects of a-synuclein responsible for causing cell death are not yet clear. Nevertheless, several theories have been put forth to explain a-synuclein toxicity and are briefly outlined here. As already mentioned,

26

a-synuclein oligomers can form pore-like structures and annular rings of a-synuclein were previously observed in brains of patients with multiple system atrophy, a synucleinopathy. Neural cells expressing mutant forms of a-synuclein (A53T and A30P) exhibited non-selective cation pores that increased both basal and depolarization-induced intracellular Ca2þ levels (Furukawa et al., 2006). Furthermore, cells expressing mutant a-synuclein were more sensitive to iron-generated reactive oxygen species, unless treated with Ca2þ-chelating agents, suggest­ ing that elevated intracellular Ca2þ levels were responsible for the increased vulnerability of these cells to toxic insults. Given that a portion of a-synuclein has been observed to localize to mitochondrial membranes of dopamine neurons (Li et al., 2007; Nakamura et al., 2008), and the central involvement of mitochondria in PD patho­ genesis, an obvious question is whether such pores form in mitochondrial membranes to promote mitochondrial dysfunction. Over-expression of a-synuclein in cells was reported to induce abnor­ mal morphology and dysfunction of mitochondria together with increased oxidative stress (Hsu et al., 2000). Since there was little change in cell viability, this suggests that mitochondrial deficits were not secondary to cell death, but a direct consequence of a-synuclein over-expression. Another potential mechanism of a-synuclein toxicity supported by the interaction of a-synu­ clein with synaptic vesicles is that increased or mutant a-synuclein expression interferes with synaptic neurotransmission. Wild-type or A30P a-synuclein was shown to impair catecholamine release from chromaffin and PC12 cells associated with an accumulation of ‘docked’ vesicles at the pre-synaptic membrane (Larsen et al., 2006). Furthermore, a-synuclein over-expression to levels predicted to result from gene multiplication impaired neurotransmitter release in mice through a mechanism involving reduced size of the recy­ cling vesicle pool (Nemani et al., 2010). Uptake of dopamine into synaptic vesicles may also be perturbed by a-synuclein. Mutant a-synuclein over-expression was reported to downregulate

the vesicular monoamine transporter 2 which may lead to increased cytosolic levels of dopamine (lotharius et al., 2002). Pathogenic species of a-synuclein are not good substrates for proteaso­ mal degradation and aggregates can directly bind to 20/26S proteasomal subunits inhibiting proteo­ lytic activity (Snyder et al., 2003). a-Synuclein may also affect protein degradation through inhi­ biting lysosomal function (Stefanis et al., 2001) and chaperone-mediated autophagy (Cuervo et al., 2004). Recent data suggest that chaperonemediated autophagy is important in the regulation of the neuronal survival factor MEF2D (myocyte enhancer factor 2D) and that a-synuclein expres­ sion can disrupt this leading to cell death (Yang et al., 2009). Hence, a-synuclein aggregates appear to exert toxic effects on numerous cell functions. The rela­ tive contributions of these effects to neuronal cell death are not well understood and may vary depending on cell type and other circumstances such as the type and amount of a-synuclein patho­ genic species present. Teasing apart primary effects of a-synuclein toxicity from secondary will be important for identifying therapeutic targets for preventing cell death (Cookson, 2009).

Leucine-rich repeat kinase 2 Since the first identification of pathogenic LRRK2 mutations in 2004 (Paisan-Ruiz et al., 2004; Zimprich et al., 2004a, 2004b), mutations in this gene are now the most common known cause of familial PD worldwide (Webber and West, 2009). Autosomal dominantly inherited LRRK2 mutations exist in families from diverse ethnic backgrounds and mostly give rise to PD pheno­ types that are highly similar to those of typical late-onset PD. This suggests that understanding the effects of mutant LRRK2 on disease patho­ genesis has the potential to generate substantial insight into sporadic PD mechanisms. Interest­ ingly, despite consistency of clinical phenotypes, LRRK2 mutant carriers can exhibit diverse

27

neuropathology occasionally lacking Lewy bodies, even between individuals with the same mutation (Gaig et al., 2007; Zimprich et al., 2004b). LRRK2 encodes a large, 280 KDa protein with initial studies indicating potential roles in cytoske­ letal dynamics, protein translation control, mito­ gen-activated protein kinase (MAPK) pathways and apoptotic pathways. LRRK2 contains numer­ ous domains, namely ankyrin-like repeats, leucine-rich repeats, COR (C-terminal of ROC) WD-40 domain and a catalytic GTPase/kinase region. Interestingly, LRRK2 kinase activity appears to require a functional GTPase domain (West et al., 2007) and possibly LRRK2 dimer formation (Sen et al., 2009). LRRK2 undergoes autophosphorylation and phosphorylates a num­ ber of protein substrates in vitro. Analysis of the human kinome indicates that the kinase domain of LRRK2 and its homologue LRRK1 are most simi­ lar in sequence to the receptor-interacting protein kinase and death-domain containing interleukin receptor-associated kinase families and to a lesser extent, MAPK kinase (MAPKK) kinases. Several of the most clear and common pathogenic muta­ tions (G2019S, I2020T and R1441C/G) are found in the central catalytic region and may result in increased kinase activity in vitro (West et al., 2007), although it is important to note that not all PD-associated LRRK2 mutations increase kinase activity and only the G2019S mutation has consistently been found to increase kinase activity to date (Greggio and Cookson, 2009). Much effort is currently focused on attempting to identify kinase substrates and pathogenic mechanisms linked to altered kinase activity. A role for LRRK2 in protein translation control has been put forth by the identification of the translational inhibitor eukaryotic initiation factor 4E-binding protein (4E-BP) as a LRRK2 sub­ strate both in vitro and in a Drosophila model (Imai et al., 2008; Tain et al., 2009). 4E-BP in its non-phosphorylated state interacts with eukaryo­ tic initiation factor 4E (eIF4E), preventing activity of eIF4E within the protein translation machinery thereby inhibiting protein translation (Khalegpour

et al., 1999). Phosphorylation of 4E-BP disrupts its interaction with eIF4E and stimuli that affect 4E-BP phosphorylation such as oxidative stress and activation of the mammalian target of rapa­ mycin pathway can impact protein translation indicating that 4E-BP phosphorylation is asso­ ciated with increased protein translation. Overexpression of human LRRK2 in mammalian cells or a Drosophila orthologue (dLRRK) in flies was shown to result in increased 4E-BP phosphoryla­ tion at two threonine sites (Thr37/Thr46) leading to secondary phosphorylation by additional kinases at other sites including Ser65/Thr70 (Imai et al., 2008). The authors also reported that RNAi-mediated silencing of LRRK2 in cells or loss-of-function dLRRK mutation in flies led to a decrease in 4E-BP phosphorylation at these sites supporting the possibility that 4E-BP is a kinase substrate of LRRK2. Finally, the authors showed that dopamine neuron pathology associated with dLRRK mutations was suppressed via overexpression of 4E-BP suggesting that increasing 4E-BP activity might attenuate PD pathology. Another recent study in Drosophila has strength­ ened a potential link between LRRK2, 4E-BP activity and PD pathology (Tain et al., 2009). Increased 4E-BP activity resulting from loss of dLRRK or administration of rapamycin was suffi­ cient to suppress pathology in PTEN-induced putative kinase 1 (PINK1) and Parkin mutants raising the possibility that an involvement of general protein translation in PD pathology might be relevant to other PD-associated genes. Another candidate substrate for LRRK2 kinase activity is moesin (Jaleel et al., 2007). Moesin is a member of the ezrin/radixin/meosin (ERM) pro­ tein family whose primary role is to anchor the cytoskeleton to the plasma membrane. Jaleel et al. found that moesin could be phosphorylated by LRRK2 at Thr558 and to a lesser extent at Thr526. One caveat is moesin phosphorylation could only be observed after denaturating moesin via heating and even then, phosphate was minimally incorporated suggesting that moesin may be a weak kinase substrate of LRRK2.

28

Despite this, a recent study on developing neurons supports the possibility that moesin is a LRRK2 substrate in vivo (Parisiadou et al., 2009). Phosphorylated ERM protein accumulated more in developing neurons from G2019S LRRK2 transgenic mice and less in LRRK2 knock-out mice than controls (Parisiadou et al., 2009). Furthermore, the extent of ERM phosphorylation correlated negatively with neurite outgrowth suggesting that LRRK2 mutations may perturb normal neuronal development. Based on sequence similarity between LRRK2 and MAPKK kinases, which are involved in the MAPK signalling pathway and important to cellular stress responses, Gloeckner et al. recently used in vitro studies to probe MAPK kinases as potential substrates for LRRK2 kinase activity (Gloeckner et al., 2009). These studies revealed phosphorylation of MKK3, MKK4, MKK6 and MKK7 by LRRK2 and moreover that PD-linked G2019S or I2020T mutations in LRRK2 exhibit increased phosphotransferase activity as well as enhanced autophosphorylation. Whether these changes are due to LRRK2 kinase activity is not clear since the authors did not use kinase-dead ver­ sions of LRRK2 as a control. Since phosphorylation of MAPK kinases within their activation loop is linked to increased downstream phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun, it might be expected that increased LRRK2 kinase activity would lead to higher phosphorylated JNK and/or c-Jun levels. However, this is inconsistent with prior studies in cell system (West et al., 2007) in which LRRK2 over-expression does not appear to increase levels of phosphorylated JNK or c-Jun. Hence, the simplest explanation here is that there exists disparity between kinase activity observed in vitro and that found in intact cells. For any putative LRRK2 kinase substrate iden­ tified in vitro, it will be imperative to determine its relevance as a substrate in vivo where conditions affecting protein localization, activity and struc­ ture are far more complicated. Moreover, since existing studies largely support an increase in kinase activity following certain mutations in

LRRK2 (e.g. G2019S), it will be important to assess whether the phosphorylation of any putative substrate is enhanced in the presence of mutant LRRK2 relative to wild-type LRRK2. Numerous studies show that expression of mutant LRRK2 causes cell death. Over-expression of mutant LRRK2 in primary neuronal cultures leads to rapid cell death possibly by apoptosis, while comparable expression of wild-type LRRK2 has only subtle effects on cell viability (Greggio et al., 2006; Ho et al., 2009; Iaccarino et al., 2007; MacLeod et al., 2006; Smith et al., 2005, 2006; West et al., 2007). A proposed link between increased LRRK2 kinase activity and neuronal death in PD requires further investiga­ tion in vivo, although a dominant mode of inheritance (consistent with a toxic-gain-of-func­ tion) and preliminary studies in cell culture are supportive of this. Multiple investigators have discovered that pathogenic LRRK2 mutants engineered to ablate kinase activity are substan­ tially less toxic in cultured cells than kinaseactive counterparts (Gregio et al., 2006; Smith et al., 2006; West et al., 2007) indicating that kinase activity contributes to cellular toxicity. One caveat is that not all pathogenic LRRK2 mutations appear to result in elevated kinase activity based on measurements of autopho­ sphorylation or generic kinase substrate phos­ phorylation. A possibility to consider here is that pathogenic LRRK2 mutations might alter kinase activity towards specific substrates that are key to LRRK2-mediated toxicity but not a universal increase in kinase activity to all sub­ strates. Perhaps identification of true in vivo LRRK2 substrates will permit definitive assess­ ment of the role of kinase activity in LRRK2­ mediated PD pathogenesis. Despite considerable recent progress, much remains to be understood about the contribution of mutant LRRK2 to PD pathogenesis. Given the pervasiveness of these mutations in PD, unlocking these mysteries will surely have broad implications in understanding fundamental mechanisms of PD development and for therapeutic strategies aimed at LRRK2.

29

Recessive mutations Compelling evidence implicates loss-of-function mutations in three genes, Parkin, PINK1 and DJ­ 1, in autosomal recessive PD and some sporadic cases (Dodson and Guo, 2007). Recent work has demonstrated key roles for all three gene products in preserving mitochondrial function and protecting against reactive oxygen species. This underscores the central role of mitochondrial dysfunction and oxidative stress in PD pathogen­ esis, reinforcing previous studies linking sporadic PD cases with mitochondrial poisons such as MPTP and paraquat. Recessively inherited mutations in a fourth gene, ATP13A2, are linked to Kufor-Rakeb syndrome, a pallidopyramidal syndrome featuring parkinsonism together with behavioural and cognitive disorders (Najim al-Din et al., 1994). Large-scale association studies showed that ATP13A2 genetic variants do not segregate with PD within families indicating that these mutations likely do not contribute to disease risk (Vilarino-Guell et al., 2009).

Parkin Mutations in Parkin were originally linked with autosomal recessive juvenile-onset parkinsonism in three unrelated Japanese families in 1997 (Kitada et al., 1998). Homozygous loss-of-function or compound heterozygous Parkin mutations account for approximately 50% of all familial early-onset cases of PD with point mutations being the most frequent genetic lesion and with deletions, duplications and exonic rearrangements also contributing to Parkin-linked PD (Mata et al., 2004). Although the majority of Parkin-associated PD is inherited in an autosomal recessive manner, there is some evidence to suggest that Parkin haploinsufficiency due to polymorphisms in the promoter or coding regions may associate with increased susceptibility to late-onset PD (Farrer, 2006). Clinically, patients with Parkin mutations are L-dopa responsive and exhibit slower disease

progression often accompanied by early-onset dystonia. Interestingly, Parkin-linked disease may be associated with an absence of Lewy body pathology, a finding that is inconsistent with these being causal in disease pathogenesis. However, Lewy body pathology is observed in some cases of Parkin-linked PD. Parkin encodes a protein of 465 amino acids consisting of an N-terminal ubiquitin-like domain, a central linker region and a C-terminal RING domain containing two RING-finger domains (Moore et al., 2005). Parkin demonstrates E3 ubi­ quitin protein ligase activity, tagging protein lysine residues with ubiquitin. Attachment of polyubi­ quitin chains to proteins via lysine K48 usually targets them for degradation via the 26S proteasome, whereas monoubiquitylation and polyubi­ quitination through K48 or K63 can influence other pathways such as intracellular signalling, DNA repair, endocytosis, transcriptional regula­ tion and protein trafficking (Mukhopadhyay and Riezman, 2007; Sandebring et al., 2009). While the majority of the Parkin pool is loca­ lized to the cytoplasm and vesicular structures (Kubo et al., 2001; Shimura et al., 2000), a portion is found associated with the outer mitochondrial membrane (Darios et al., 2003). Several recent studies on Drosophila and mice have revealed a key role for Parkin in regulating mitochondrial function and protection against oxidative stress (Deng et al., 2008; Greene et al., 2003; Palacino et al., 2004; Park et al., 2006; Yang et al., 2006), which is discussed further in the section on mito­ chondrial dysfunction and oxidative stress. Most mutations in Parkin appear to impair its E3 ligase activity or interactions with E2 enzymes such as UbcH7 and UbcH8 (Shimura et al., 2000; Zhang et al., 2000). Although it is not definitively known that loss of Parkin’s E3 ligase activity is sufficient for development of PD, a prominent hypothesis is that Parkin mutations lead to toxic accumulation of its substrates due to impaired ubiquitin–protea­ some function (UPS) (Dodson and Guo, 2007). Through extensive in vitro investigations, a num­ ber of putative Parkin substrates have been

30

identified including the aminoacyl-tRNA synthase cofactor p38 (Corti et al., 2003), far upstream ele­ ment-binding protein-1 (Ko et al., 2006), cyclin E (Staropoli et al., 2003), Parkin-associated endothelin receptor-like receptor (Imai et al., 2001), synphilin-1 (Chung et al., 2001a, Chung et al., 2001b), synaptotagmin XI (Huynh et al., 2003), CDCrel-1 (Zhang et al., 2000) and alpha/ beta-tubulin (Ren et al., 2003). From this set, only the p38 subunit of aminoacyl-tRNA synthase and far upstream element-binding protein-1 have been demonstrated to accumulate in the brains of patients with both Parkin mutations and Parkin null mice (Ko et al., 2005, 2006) highlighting the importance of determining the authenticity of all other putative substrates in vivo. Further studies will also be required to determine the roles of these substrates in PD pathogenesis.

PINK1 A second locus for autosomal recessive earlyonset parkinsonism was discovered initially in a large Sicilian family mapped to the short arm of chromosome 1p35–p36 (Valente et al., 2001) and later extended to eight additional families from four European countries (Valente et al., 2002). Subsequent work revealed that within this locus, mutations in PINK1 are linked to PD (Valente et al., 2004). Atypical clinical phenotypes have been reported in PINK1-linked PD, including dys­ tonia, psychiatric disturbances and sleep benefit (Hatano et al., 2004; Tan and Dawson, 2006; Valente et al., 2004). PINK1 is a cytosolic and mitochondrially localized protein kinase which contains an N-terminal mitochondrial targeting sequence followed by a predicted transmembrane domain, suggesting that PINK1 may be an integral transmembrane protein possibly in the mitochon­ drial inner membrane with which it closely associ­ ates (Silvestri et al., 2005). However, a recent study indicates that the kinase domain of PINK1 faces out into the cytosol (Zhou et al., 2008). Existing evidence from cell and animal models

suggests that PINK1 is important for protection against cell death related to mitochondrial dys­ function and oxidative stress (Clark et al., 2006; Deng et al., 2008; Exner et al., 2007; Hoepken et al., 2007; Wood-Kaczmar et al., 2008). Although several PD-associated mutations reduce PINK1 kinase activity, it is not clear whether loss of kinase activity is required for PD pathogenesis since disease-associated mutations are found both within and outside of the kinase domain. It seems, however, that kinase activity is required for the protective function of PINK1 against pro­ apoptotic agents since staurosporine-induced cell death was substantially reduced by wild-type PINK1 over-expression, whereas an equivalent increase in kinase-inactive PINK1 mutant had no protective effect (Petit et al., 2005). Additionally, recent in vivo data suggest that phosphorylation of the mitochondrial chaperone TNF-receptor-asso­ ciated protein 1(TRAP1) by PINK1 is important for the protective action of PINK1 against oxida­ tive stress-induced cell death (Pridgeon et al., 2007). The authors also reported that the ability of PINK1 to phosphorylate TRAP1 is impaired by PD-associated G309D, L347P and W437X PINK1 mutations suggesting a possible connection between these mutations, impaired PINK1 sub­ strate phosphorylation and cell death. PINK1 was recently demonstrated to regulate mitochon­ drial Ca2þ efflux in mammalian neurons, and loss of PINK1 was associated with mitochondrial Ca2þ overload and consequent respiration inhibition via increased reactive oxygen species generation and opening of the mitochondrial permeability transi­ tion pore (Ghandi et al., 2009).

DJ-1 Mutations in DJ-1 were originally associated with early-onset PD in 2003 (Bonifati et al., 2003) and are known to be very rare, accounting for less than 1% of early-onset cases. The DJ-1 protein is a member of the ThiJ/PfpI family of molecular chaperones that are induced by oxidative stress

31

(Dodson and Guo, 2007). Consistent with this, DJ-1 deficiency in Drosophila increases cell death caused by reactive oxygen species (ROS)-generat­ ing species linked to PD in humans (Muelener et al., 2005). Additional studies revealed that a conserved cysteine residue in human (CanetAviles et al., 2004), mice (Andres-Mateos et al., 2007) and Drosophila (Meulener et al., 2006) of DJ-1 is modified under conditions of oxidative stress, and this modification is necessary for the protective effects of DJ-1. Furthermore, evidence in mice indicates that DJ-1 acts as an atypical per­ oxiredoxin-like peroxidase to scavenge H2O2 pro­ duced by mitochondria (Andres-Mateos et al., 2007). Accordingly, DJ-1 knock-out mice exhibit elevated mitochondrial H2O2 and reduced activity of mitochondrial aconitase activity levels, although the pathological consequences of this are uncertain since there was an absence of dopaminergic neuron degeneration in these mice (Andres-Mateos et al., 2007). Hence, DJ-1 may act as a cellular redox sensor, which becomes activated under oxidative conditions to provide protection against ROSmediated damage. Numerous functions have been ascribed to DJ-1, including protease, transcrip­ tional co-activator and molecular chaperone func­ tions, although which of these, if any, contribute to its protective role in PD remains to be determined.

Impact of genetic research on understanding mechanisms of PD pathogenesis Genetic studies over the last decade have resulted in the identification of pathogenic gene variants that underlie familial PD and in some cases con­ tribute to the lifetime risk of developing sporadic PD. By linking these genes to PD and understand­ ing the biological roles of the products they encode, a wealth of insight has been generated into mechanisms of PD pathogenesis. These pathogenic genes also yield understanding of possible relationships between PD and disorders with overlapping clinical or neuropathological features that may share common mechanisms.

For example, neuronal a-synuclein accumulation often in Lewy bodies can be found in a number of neurodegenerative disease including PD, demen­ tia with Lewy bodies, multiple system atrophy and pure autonomic failure (Goldstein and Sewell, 2009; Ko¨ vari et al., 2009; Kramer and SchulzSchaeffer, 2007) suggesting that a-synuclein aggregation is a common mechanism in these diseases. Genetic studies have in some instances corroborated pathogenic mechanisms indicated by environmental factors such as a central involve­ ment of oxidative stress and mitochondrial dys­ function in PD aetiology. Additionally, genetic studies have implicated protein mishandling due to dysfunction of the UPS in development of PD. Since perturbations in the UPS or mitochondrial function both lead to the same pathological out­ come, that is loss of dopamine neurons and devel­ opment of PD, it is likely that an important relationship exists between these functions that converge on dopamine neuron viability. The model presented in Fig. 2 illustrates molecular pathways in PD pathogenesis implicated by genetic studies and how these pathways may be connected.

Mitochondrial dysfunction and oxidative stress A role for mitochondrial dysfunction in PD patho­ genesis is supported by studies on a number of gene products linked to PD. Knock-out studies in animals clearly indicate that two PD-linked genes, Parkin and PINK1, have key roles in preserving mitochondrial function and that loss-of-function mutations in these genes can lead to PD. Droso­ phila Parkin null mutants exhibit mitochondrial pathology marked by enlarged size and rarified cristae, as well as enhanced sensitivity to oxidative stress, apoptotic muscle degeneration, significant (albeit slight) degeneration of a subset of dopa­ mine neurons and reduced life span (Greene et al., 2003; Pesah et al., 2004). Parkin null mice, which exhibit nigrostriatal deficits without nigral degeneration, do not have gross changes in striatal

32

Parkin

Pink1

Ubiquitin­ proteasome system dysfunction

Mitochondrial dysfunction ? α-Synuclein aggregates (oligomers/fibrils)

Oxidative stress

DJ-1

↓ ATP

↑[Ca]2+

Impaired vesicular transport

SNCA

Accumulated parkin substrates

? Neuronal dysfunction & death ? Aberrant protein translation

LRRK2 Fig. 2. Pathogenic mechanisms implicated by genetic studies of PD. This model links genetic mutations (autosomal recessive mutations in blue boxes and autosomal dominant mutations in purple boxes) to neurodegeneration via pathways involving mitochondrial dysfunction, oxidative stress and impaired UPS. Loss-of-function mutations in PINK1 or Parkin cause PD possibly through a mechanism involving mitochondrial pathology and dysfunction. Deleterious effects of mitochondrial dysfunction include reduced ATP generation and oxidative stress due to elevated ROS generation. Loss of Parkin’s E3 ubiquitin ligase activity may also lead to dopamine neuron toxicity via impaired UPS and accumulation of Parkin’s substrates. Loss of DJ-1 antioxidant function may promote neuronal oxidative stress, as might reduced respiratory chain function and elevated intracellular calcium influx via pores created by a-synuclein oligomers. LRRK2 mutations might be linked to PD through altered protein translation further supporting a role for protein turnover in PD pathogenesis.

mitochondrial morphology but do experience mitochondrial dysfunction evidenced by reduced activity of multiple respiratory chain complexes along with decreased antioxidant capacity that results in increased oxidative damage (Palacino et al., 2004). Intriguingly, complete loss of PINK1 function in flies led to phenotypes that are highly similar to Parkin null flies (Clark et al., 2006). The subtle neuronal death observed in PINK1

or Parkin mutants can be prevented by overexpression of antioxidants (Wang et al., 2006; Whitworth et al., 2005) supporting the contention that oxidative stress is important to PD pathology. Indeed, oxidative damage to cell macromolecules is consistently observed in the substantia nigra of PD patients (Jenner, 2003), and elevated reactive oxygen species generation may occur as a result of impaired respiratory chain function.

33

Several lines of evidence suggest a genetic inter­ action between PINK1 and Parkin. For example, over-expression of Parkin rescued all phenotypes resulting from PINK1 deficiency, although a reci­ procal rescue effect of PINK1 over-expression in Parkin mutants was not found (Park et al., 2006). Similarly, loss of mitochondrial potential, abnor­ mal mitochondrial morphology and reduced cris­ tae seen in HeLa cells with PINK1 deficits can be rescued by increased expression of wild-type but not PD-associated mutant Parkin (Exner et al., 2007). These lines of evidence have led to the hypothesis that Parkin acts downstream of PINK1 to preserve mitochondrial function (Dod­ son and Guo, 2007). Recent studies in Drosophila have suggested a possible link between PINK1, Parkin and mitochondrial function by demonstrat­ ing that both appear to regulate mitochondrial dynamics by either promoting fission or inhibiting fusion of the organelle (Deng et al., 2008; Poole et al., 2008). Genetic manipulations of the fly mitofusin homologue (Mfa), Opa1 (optic atrophy 1) or drp1 in favour of mitochondrial fission are sufficient to rescue mitochondrial pathology, cell death and muscle degeneration in Parkin or PINK1 mutants (Deng et al., 2008). However, these data and their relevance to human disease should be interpreted cautiously due to discrepan­ cies in mitochondrial morphology abnormalities that exist between fly and mammalian model sys­ tems. Nonetheless, impaired mitochondrial respiration has been detected in peripheral tissues taken from human PD patients with PINK1 (Hoepken et al., 2007) or Parkin mutations (Muftuoglu et al., 2004) suggesting that the mito­ chondrial dysfunction observed in animal models may be relevant to human disease. Hence, consid­ erable evidence supports a role for PINK1 and Parkin in protecting against cell death due to mitochondrial dysfunction and oxidative stress. Mutations and multiplications in SNCA may also promote mitochondrial dysfunction leading to neuronal death. Mitochondrial pathology fol­ lowing MPTP exposure is exacerbated in a-synu­ clein transgenic mice (Song et al., 2004), and

neuronal cells expressing mutant a-synuclein showed a selective increase in mitochondrial dys­ function and apoptotic cell death when treated with a proteasome inhibitor (Tanaka et al., 2001). Inhibition of the mitochondrial respiratory chain complex I, which is caused by pesticides and certain other environmental toxins, commonly leads to the accumulation of a-synuclein-positive inclusions suggesting that a-synuclein aggregation may be a consequence of mitochondrial dysfunc­ tion (Betarbet et al., 2000; Manning-Bog et al., 2002). Interestingly, a-synuclein knock-out mice are resistant to the toxic effects of MPTP on neu­ rons while a-synuclein transgenic mice are more sensitive indicating that a-synuclein might be necessary for neuronal toxicity associated with impaired complex I activity (Dauer et al., 2002; Song et al., 2004). Taken together, this evidence suggests that a-synuclein, likely in aggregate form, may have a toxic role both in causing mitochon­ drial dysfunction and in the deleterious effects resulting from it. Future studies will hopefully elucidate the nature of the relationship between a-synuclein and mitochondrial dysfunction.

Ubiquitin–proteasome system impairment Compelling evidence from genetic studies links UPS dysfunction to development of PD. Mutations in Parkin associated with the disease are widely believed to cause impairment of UPS function with consequent accumulation of poten­ tially cytotoxic proteins that may result in death of dopamine neurons (Chung et al., 2001a; Moore et al., 2005). In support of this, proteasome inhi­ bitors are found to cause a number of phenotypes that closely recapitulate those in PD when injected into rats (McNaught et al., 2004). Furthermore, genetic knock-out of a 26 proteasomal subunit in mice impairs ubiquitin-mediated protein degrada­ tion and leads to intraneuronal Lewy-like inclu­ sion formation and substantial degeneration in the nigrostriatal pathway (Bedford et al., 2008). The accumulation of aggregated proteins such as

34

a-synuclein in nigral neuron Lewy bodies in sporadic PD also indicates mishandling of protein turnover perhaps due to impaired UPS function. As previously mentioned, Lewy bodies are often absent in certain familial forms of PD suggesting that they may not be neuropathological in PD but are, instead, a hallmark of protein aggregation and therefore still supportive of a role for protein mis­ handling in PD pathology. Aggregated a-synu­ clein has been shown to strongly bind to and inhibit the 26S proteasome in vitro (Snyder et al., 2003), and over-expression of mutant a-synuclein in cells was observed to decrease proteasome function and cause selective toxicity to catechola­ minergic neurons (Petrucelli et al., 2002). Interest­ ingly, co-expression of Parkin was reported to reduce sensitivity of a-synuclein-expressing cells to proteasome inhibitors suggesting that Parkin protects against mutant a-synuclein-mediated toxicity. Similarly, over-expression of the molecu­ lar chaperone Hsp70 in flies (Auluck et al., 2002) or transgenic expression of the yeast chaperone Hsp104 in rats (Lo Bianco et al., 2008) prevents degeneration of dopamine neurons caused by expression of mutant a-synuclein indicating that chaperones function may protect against PD pathology related to a-synuclein. Consistent with this, Lewy bodies examined from post-mortem human brain were found to contain molecular chaperones (Auluck et al., 2002), which may indi­ cate that in the PD brain, these fail to effectively prevent protein aggregation at physiological levels. Further investigations assessing the ability of molecular chaperones to prevent neuronal toxicity associated with a-synuclein aggregation will hopefully lead to new therapeutic strategies for PD.

Conclusion Although the majority of PD is sporadic, the discovery of rare familial forms of PD and subsequent identification of disease-causing mutations have provided extremely valuable

tools to begin to understand the cellular net­ work of dysfunction that ultimately results in neuronal demise and manifestation of disease phenotypes. Through the generation of cellular and small animal models expressing PD-linked genetic mutations, new insights into the mechanisms of disease pathogenesis have been achieved. Further study will hopefully lead to new disease-modifying treatments for PD that will provide more than temporary symptomatic relief.

Acknowledgements This work was supported by grants from National Institutes of Health, National Institute of Neurological Disorders and Stroke P50 NS38377, NS054207. T.M.D. is the Leonard and Madlyn Abramson Professor in Neurode­ generative diseases.

Abbreviations PD SNARE MEF2D LRRK2 eIF4E 4E-BP ERM MAPK MAPKK JNK PINK1 TRAP1 MPTP

Parkinson’s Disease soluble NSF attachment protein receptors myocyte enhancer factor 2D leucine-rich repeat kinase 2 eukaryotic initiation factor 4E eukaryotic initiation factor 4E binding protein ezrin/radixin/meosin mitogen-activated protein kinase mitogen-activated protein kinase kinase c-Jun N-terminal kinase PTEN-induced putative kinase 1 TNF-receptor-associated protein 1 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine

35

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41 locus for autosomal recessive early-onset Parkinsonism, PARK6, on human chromosome 1p35-p36. American Journal of Human Genetics, 68(4), 895–900. Valente, E. M., Brancati, F., Ferraris, A., Graham, E. A., Davis, M. B., Breteler, M. M., et al. (2002). PARK6-linked Parkinsonism occurs in several European families. Annals of Neurology, 51(1), 14–18. Van Den Eeden, S. K., Tanner, C. M., Bernstein, A. L., Fross, R. D., Leimpeter, A., Bloch, D. A., et al. (2003). Incidence of Parkinson’s disease: Variation by age, gender, and race/ethni­ city. American Journal of Epidemiology, 157(11), 1015–1022. Vilarino-Guell, C., Soto, A. I., Lincoln, S. J., Ben Yahmed, S., Kefi, M., Heckman, M. G., et al. (2009). ATP13A2 variabil­ ity in Parkinson disease. Human Mutation, 30(3), 406–410. Wang, D., Qian, L., Xiong, H., Liu, J., Neckameyer, W. S., Oldham, S., et al. (2006). Antioxidants protect PINK1­ dependent dopaminergic neurons in Drosophila. Proceed­ ings of the National Academy of Sciences of the United States of America, 103(36), 13520–13525. Ward, C. D., Duvoisin, R. C., Ince, S. E., Nutt, J. D., Eldridge, R., & Calne, D. B. (1983). Parkinson’s disease in 65 pairs of twins and in a set of quadruplets. Neurology, 33(7), 815–824. Webber, P. J., & West, A. B. (2009). LRRK2 in Parkinson’s disease: Function in cells and neurodegeneration. The FEBS Journal, 276(22), 6436–6444. West, A. B., Moore, D. J., Choi, C., Andrabi, S. A., Li, X., Dikeman, D., et al. (2007). Parkinson’s disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity. Human Molecular Genetics, 16 (2), 223–232. Whitworth, A. J., Theodore, D. A., Greene, J. C., Benes, H., Wes, P. D., & Pallanck, L. J. (2005). Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson’s disease. Proceedings of the National Academy of Sciences of the United States of America, 102(22), 8024–8029. Wood-Kaczmar, A., Gandhi, S., Yao, Z., Abramov, A. Y., Miljan, E. A., Keen, G., et al. (2008). PINK1 is necessary for long term survival and mitochondrial function in human dopaminergic neurons. PLoS One, 3(6), e2455. Xu, J., Kao, S. Y., Lee, F. J., Song, W., Jin, L. W., & Yankner, B. A. (2002). Dopamine-dependent neurotoxicity of alpha­ synuclein: A mechanism for selective neurodegeneration in Parkinson disease. Nature Medicine, 8(6), 600–606.

Yang, Y., Gehrke, S., Imai, Y., Huang, Z., Ouyang, Y., Wang, J. W., et al. (2006). Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactiva­ tion of Drosophila Pink1 is rescued by Parkin. Proceedings of the National Academy of Sciences of the United States of America, 103(28), 10793–10798. Yang, Q., She, H., Gearing, M., Colla, E., Lee, M., Shacka, J. J., et al. (2009). Regulation of neuronal survival factor MEF2D by chaperone-mediated autophagy. Science, 323(5910), 124–127. Yasuda, T., Miyachi, S., Kitagawa, R., Wada, K., Nihira, T., Ren, Y. R., et al. (2007). Neuronal specificity of alpha-synuclein toxicity and effect of Parkin co-expression in primates. Neuroscience, 144(2), 743–753. Zarranz, J. J., Alegre, J., Gomez-Esteban, J. C., Lezcano, E., Ros, R., Ampuero, I., et al. (2004). The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body demen­ tia. Annals of Neurology, 55(2), 164–173. Zhang, Y., Gao, J., Chung, K. K., Huang, H., Dawson, V. L., & Dawson, T. M. (2000). Parkin functions as an E2-dependent ubiquitin- protein ligase and promotes the degradation of the synaptic vesicle-associated protein, CDCrel-1. Proceedings of the National Academy of Sciences of the United States of America, 97(24), 13354–13359. Zhou, W., & Freed, C. R. (2005). DJ-1 up-regulates glutathione synthesis during oxidative stress and inhibits A53T alpha­ synuclein toxicity. The Journal of Biological Chemistry, 280(52), 43150–43158. Zhou, C., Huang, Y., Shao, Y., May, J., Prou, D., Perier, C., et al. (2008). The kinase domain of mitochondrial PINK1 faces the cytoplasm. Proceedings of the National Academy of Sciences of the United States of America, 105(33), 12022– 12027. Zimprich, A., Biskup, S., Leitner, P., Lichtner, P., Farrer, M., Lincoln, S., et al. (2004a). Mutations in LRRK2 cause auto­ somal-dominant Parkinsonism with pleomorphic pathology. Neuron, 44(4), 601–607. Zimprich, A., Muller-Myhsok, B., Farrer, M., Leitner, P., Sharma, M., Hulihan, M., et al. (2004b). The PARK8 locus in autosomal dominant Parkinsonism: Confirmation of linkage and further delineation of the diseasecontaining interval. American Journal of Human Genetics, 74(1), 11–19.

A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 3

Unravelling the role of defective genes Mark R. Cookson
Cell Biology and Gene Expression Unit, Laboratory of Neurogenetics, National Institute on Aging, Bethesda, MD, USA

Abstract: Several genes that cause familial forms of Parkinson’s disease (PD) or similar disorders have been found in recent years. The aim of this review is to cover two broad aspects of the logic of genetics. The first aspect is the recognition that PD can have a genetic basis, either for Mendelian families where genes can be identified because mutations segregate with disease or in populations where more common variants are associated with disease. There are several causal genes for both dominant and recessive forms of parkinsonism, some of which overlap with sporadic PD and some of which have more complex phenotypes. Several of the dominant loci have also been reliably identified as risk factors for sporadic PD. The second topic is how the study of multiple mutations in any given gene can help understand the role that the protein under investigation plays in PD. Examples will be given of both recessive and dominant genes for parkinsonism, showing how the analysis of multiple gene mutations can be a powerful approach for dissecting out which function(s) are important for the disease process. Keywords: Genetics; LRRK2; Synuclein; Parkin; PINK1; DJ-1

jection neurons in the substantia nigra that under­ lies the equally characteristic movement disorder seen clinically in patients. Furthermore, and as will be discussed here, some of the same genes act as risk factors for sporadic disease, suggesting that sporadic and inherited PD share common patho­ genic mechanisms. The focus of this review is on how to take the increasing amounts of genetic data and use it to understand how genetic variants influence protein function. However, it is important to first revisit the genetics of PD and related disorders and to outline briefly how genetic variants can be assigned to be causal.

Introduction Our understanding of the underlying cause of Parkinson’s disease (PD) has been revolutionized in recent years by the recognition that there are genetic diseases that overlap phenotypically with this common disorder. Although most cases of PD are not inherited, there are many families known worldwide with Mendelian inheritance of diseases who have the characteristic loss of dopamine pro


Corresponding author. Tel.: þ1 301 451 3870; Fax: þ1 301 451 7295 E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83003-1

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The genetic basis of parkinsonism There are two accepted tests for whether a gene variant can be considered causal for a given phe­ notype. Either a gene is inherited in a manner that shows segregation with a given trait, usually in a dominant or recessive Mendelian fashion, or a genetic variant shows association with a pheno­ type in a population. Genes that show segregation tend to be associated with stronger effects on protein function than those that show association, which tends to be subtler.

Mendelian genes for PD show segregation Of the Mendelian variants in PD, there are several well-characterized genes, two of which show dominant inheritance. In these cases, because we expect to see disease from a single mutated allele, there is often generation-to-generation transmis­ sion of the trait and the disease segregates, or tracks with mutation, for all of the people. A slight issue is penetrance, that is what proportion of people with the dominant mutation express the disease. PD is an age-related disorder and the dominant mutations show age-dependent penetrance that, in some cases, seems to be incom­ pletely penetrant even at old age. The first gene discovered for PD was SNCA, which codes for the a-synuclein (SNCA) protein, which is a small (14.4 kDa) protein with repeats towards the N-terminus and an acidic ‘tail’ region at the C-terminus. There are now three point muta­ tions, A53T (Polymeropoulos et al., 1997), A30P (Kruger et al., 1998) and E46K (Zarranz et al., 2004), all in the repeat region. There are also tri­ plications (Singleton et al., 2003) and duplications (Chartier-Harlin et al., 2004; Ibanez et al., 2004) of the entire gene locus reported in different families. All of these variants, whether point mutations or multiplications, show dominant inheritance and segregate with a Lewy body phenotype that can be similar to either PD or diffuse Lewy body disease (DLBD). Given that SNCA is a major component

of Lewy bodies (Spillantini et al., 1997), these data support the general argument that we can define diseases with protein deposition by their patholo­ gical outcomes (Hardy, 2005). Penetrance is age dependent and generally complete for A53T, E46K and the triplications but appears to be slightly lower in A30P and in duplication families. The latter mutations also appear to give a slightly milder, more brain stem-restricted form of PD than the former, which tends to be more like DLBD. Overall, these data show that SNCA mutations are a rare but convincing cause of PD/DLBD. The second dominant cause of PD is the much more recently discovered gene leucine-rich repeat kinase 2 (LRRK2), which encodes the LRRK2 protein. LRRK2 is a large multi-domain protein, and there are mutations that segregate with disease in three regions; R1441C and R1441G in the ROC domain [for Ras of complex proteins, a guanosine triphosphate (GTP)-binding region], Y1699C in the COR domain (for C-terminal of ROC) and G2019S and I2020T in the kinase domain (Di Fonzo et al., 2005; Funayama et al., 2005; Gilks et al., 2005; Kachergus et al., 2005; Nichols et al., 2005; Paisan-Ruiz et al., 2004; Zimprich et al., 2004). All of these variants show good evidence for segregation in multiple families and are convin­ cingly causal. There are some non-penetrant cases particularly reported for G2019S, which is the most common mutation found to date. Specifically, there are case reports including a healthy, older (>90 years) individual with the G2019S mutation who was free of detectable neurological symptoms upon examination (Kay et al., 2005). This type of case is important as it tells us why G2019S can be found in apparently sporadic PD; presumably the index patient had one parent with a mutation but the parent never developed PD during their lifetime. Overall, the evidence strongly supports the patho­ genicity of LRRK2 mutations, with the important note that there is age-dependent and probably decreased penetrance. Another interesting observation about LRRK2 mutations is that while clinically the disease is

45

generally similar to sporadic PD (Haugarvoll and Wszolek, 2009), the pathological outcomes can be quite variable, as originally emphasized in one of the first cloning papers (Zimprich et al., 2004). Although most cases examined to date have Lewy bodies containing SNCA, some have instead just dopaminergic neuron degeneration and some have protein aggregation that can include the pro­ tein tau (Cookson et al., 2008). This is true even within families, where different pathologies are associated with the same mutation. This is perhaps surprising as it implies that the pathological out­ come for some has a complex relationship to the gene mutation, unlike the example of SNCA discussed above. One way to resolve this apparent contradiction is to place LRRK2 genetically upstream of depos­ ited proteins such as SNCA or tau, implying that the same initial mutation might then result in dif­ ferent pathological outcomes depending on the course the disease takes. There is some experi­ mental evidence for this (see below), and it is a reasonable interpretation of the available data, although it would then be confusing that the same mutation produces similar clinical outcomes. Another thought is that perhaps the final protein deposition (Lewy bodies, tau inclusions, etc.) is only tangentially related to the clinical phenotype. We might even extend this idea to suggest that while proteins such as SNCA and tau are involved in the pathological process of LRRK2, their deposition into Lewy bodies or tau inclusions is not required for the disease process. This is an extension of the argument that while Lewy bodies are strongly associated with PD, they may be ancillary to some aspects of the disease process. By extension, the toxic protein species might not be the Lewy body itself but some unidentified version of SNCA or tau, perhaps a relatively soluble oligomeric species (Cookson, 2005). Dominant mutations in SNCA and LRRK2 therefore account for a number of different cases and show the required segregation with disease in multiple families. There is therefore strong genetic evidence that these are causal genes for PD and

related pathologies. There are also recessive mutations in three genes, parkin (Kitada et al., 1998), DJ-1 (Bonifati et al., 2003) and PINK1 (PTEN-induced novel kinase 1) (Valente et al., 2004), that show convincing segregation with early-onset disease in multiple families. Because these are recessive genes, it is common for each parent to contribute one mutant allele so that there are affected offspring of unaffected parents. In some cases, especially where there are consan­ guineous marriages (first cousins or similar), the two mutant alleles will be the same, although compound heterozygotes have been reported for all three recessive parkinsonism genes. All sub­ jects who have two mutant alleles are clinically affected and therefore show segregation under a recessive model, although again there is an agerelated expression of the phenotype. Mutations in parkin, DJ-1 and PINK1 include gene rearrange­ ments (deletion and duplications of whole exons), truncations and point mutations. Deletion and truncation mutations are simple to interpret as loss-of-function alleles and duplication events often disrupt the protein-coding frame, thus effec­ tively removing full-length protein. Point muta­ tions can include those that destabilize the protein for DJ-1 (Miller et al., 2003) and PINK1 (Beilina et al., 2005), thus mimicking loss of function. One area of controversy is the status of people with heterozygous mutations in parkin or PINK1 who have late-onset, typical PD in contrast to early-onset recessive disease seen with patients with two mutant alleles. In most of these cases, there is insufficient evidence to say that these mutations segregate in a dominant fashion – par­ ents who would have contributed the mutant allele are not affected with PD and siblings, etc., are not affected at rates higher than chance alone. Two alternative hypotheses are that single parkin mutations might act as risk factors for sporadic PD (Klein et al., 2007) or that the presence of PD in some carriers of recessive mutations might be a coincidence, which could occur relatively fre­ quently in a common disease such as PD.

46

Some of the variants reported might not be pathogenic but rather rare polymorphisms again found at random in patients with a common spora­ dic disease, for example PD. A good example of this is mutations in Htra2/omi which were nomi­ nated as a gene for PD based on the occurrence of heterozygous mutations in four cases and not in 500 controls (Strauss et al., 2005). However, subsequent sequencing approaches revealed the nominated mutation (and other variants) in con­ trols (Simon-Sanchez and Singleton, 2008) and a recent study failed to provide support for associa­ tion of Omi variants with PD (Kruger et al., 2009). Therefore, even though the original data were correct, Htra2/Omi is not a gene for PD. One clue that the originally nominated mutations were not causal was that the four cases with PD were apparently sporadic, and so there was rather little support for pathogenicity by segregation. In these cases, it is important to sequence a large number of controls to check that the variant is not a rare but benign version of the same gene.

Risk factor genes show association Some genes do not segregate with disease in families but show association with the given phe­ notype, that is, is over- or under-represented in cases versus controls. Because by definition risk variants are present in both cases and controls, assigning pathogenicity is in essence a statistical estimate of the effect. Replication of any apparent initial association in multiple studies is therefore extremely important. A good example of a highly replicated association is ApoE4 variant and Alzheimer’s disease, which is consistently near the top of systematic analyses of association studies (see http://www.alzgene.org/). This is because ApoE4 has a strong effect, raising the risk of Alzheimer’s disease by about fourfold, and is a common allele and thus is easy to replicate across studies even with modest numbers of samples (in the 100s). There are a number of genes that are nomi­ nated as showing association with PD, and for

reasons of brevity we cannot review all of them here (http://www.pdgene.org/ is a useful resource for the interested reader). As an illustrative exam­ ple, we might consider the data on association of SNCA variants with PD. After SNCA had been shown to be a gene for dominant Lewy body dis­ ease, several groups examined whether common variation around the SNCA locus was associated with sporadic PD with both negative (Parsian et al., 1998) and positive results reported (Kruger et al., 1999). With time several additional data sets were collected and collectively supported an asso­ ciation of variants both within the promoter region and towards the 30 -end of SNCA with PD [reviewed in Tan (2007)]. However, the size of effect of risk variants in SNCA is modest, perhaps raising lifetime risk of PD by about 25–30%. Two other genes stood out from these analyses, includ­ ing variation around the microtubule-associated protein tau (MAPT) /tau gene and around LRRK2 (Tan, 2007). One of the limitations of association studies is that one has a pre-conceived hypothesis, that a given gene is involved in PD, that there is suffi­ cient genetic variation around that gene to be measurable in a given population and that the size of effect is sufficiently strong to be identified in a given number of samples. While this undoubt­ edly yields insight and can helpfully exclude genes that are not of strong effect, in the last few years methods have been developed to interrogate the genome in a less-biased way, using genome-wide association studies (GWASs). In GWAS, large numbers of common variants are genotyped in large numbers (typically several 1000s) of controls and cases with the given phenotype. Because the genes are not pre-specified, GWAS has the poten­ tial to identify novel risk loci for PD. Two recent studies illustrate the power of this approach: one performed in Caucasian PD patients and controls (Simon-Sanchez et al., 2009) and one in Asian populations (Satake et al., 2009). With a few thousand cases in both studies, each was powered to detect modest asso­ ciations, in the range of an ~25% alteration in risk

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for PD which seems reasonable given the data above from prior association studies. Interestingly, in both studies the top ‘hits’ were in and around SNCA/a-synuclein. In the study of people from European ancestry, MAPT/tau also gave a strong signal (Simon-Sanchez et al., 2009), although this was not seen in the study of people with Asian ancestry (Satake et al., 2009) as the tau gene dif­ fers between these two populations (Stefansson et al., 2005). Both studies also nominated a weaker signal around LRRK2, stronger in the Asian population probably because there is a relatively common variant in LRRK2 (G2385R) that is more frequent in Asian populations and that shows robust association with PD (e.g. Farrer et al., 2007). These GWAS studies therefore nominate genes that we might have expected for PD based on the genetics of Mendelian forms, that is SNCA and LRRK2. But there are a number of surprises. Firstly, new loci were also nominated, including one that has been given the designation PARK16 that contains several candidate genes. Secondly, there was a relatively strong signal for tau at least in Caucasian populations. Although this had been nominated as a risk gene for PD, because most cases of PD do not have tau deposition it seemed unlikely that MAPT would have as strong of an effect as SNCA, but on GWAS the two are close to equal. Thirdly, it was also interesting that the recessive genes were not nominated by GWAS. This does not mean that parkin, DJ-1 and PINK1 are not genes for PD but rather that the effects of rare variants in these genes are not strong enough at the population level to be measurable in a GWAS design. Collectively, the evidence from segregating var­ iants has revealed genes of strong effect in rare families and the evidence from association studies show weaker effects in the commoner sporadic form of PD. That these two sets of genetic approaches produce candidates that overlap (SNCA, LRRK2 and perhaps MAPT) and in at least one case are also associated with the charac­ teristic protein deposition seen in PD (SNCA in

Lewy bodies) suggests that familial and sporadic PD may share common pathogenic mechanisms. The next step is then to understand the effects of variation in the nominated genes, using a variety of different models to attempt to put genes in biologically meaningful pathways. As this litera­ ture is huge, not all papers on SNCA, LRRK2, tau, parkin, DJ-1 and PINK1 can be reviewed here. Instead, the general principles of how one can take genetic information will be discussed using examples from some of the recent literature on this set of proteins. For clarity, these will be separated into genes for dominant PD/Lewy body disease and recessive parkinsonism. One very important general argument that will be illustrated is that the human genetic data for any given muta­ tion takes priority over supportive arguments for or against pathogenicity from molecular, cell or animal models. As will be discussed, it is critical that independent pieces of weaker data, each of which are ambiguous by themselves, are not allowed to support each other like two drunks standing against each other at the end of the night.

Mutations in recessive genes decrease protein function Recessive genes usually cause a loss of protein function, and we can be reasonably certain that this is the case for parkin, pink1 and DJ-1 as all three have mutations that segregate with disease under a recessive model that are large deletions. For example, for DJ-1 one of the first reported mutations was a deletion of the entire protein open-reading frame (Bonifati et al., 2003). There­ fore, we can reasonably assume the understanding that the recessive genes require identifying the normal function of the proteins involved and describing what happens when that function is lost. Therefore, knockout or knockdown models are useful in defining phenotypes related to lossof-function genes. An additional approach that can be useful is to use a wide range of different recessive mutations, other than those that are

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simply unstable or large deletions, and show that they all lack a given property, either a biochemical activity or a phenotype such as protection against toxic stress. In this way, we can be more confident that the identified function or phenotype is relevant for human disease. An example of using knockouts to define path­ ways comes from the work on the Drosophila melanogaster homologues of PINK1 and parkin. In the fly, loss-of-function alleles of either gene result in a series of age-related phenotypes includ­ ing male sterility and decreased ability to fly (Clark et al., 2006; Greene et al., 2003; Park et al., 2006). In turn, both of these phenotypes are related to dysfunction in mitochondria. The male sterility seems to be a consequence of failure of spermatids to individualize during spermato­ genesis, which is dependent on transformation of mitochondria (Riparbelli and Callaini, 2007), while the flight defects relate to swollen mitochon­ dria in the musculature and apoptosis of muscle cells (Greene et al., 2003). The mitochondrial phenotypes were perhaps expected for PINK1, which had already been shown to be a mitochondrially directed kinase (Beilina et al., 2005; Valente et al., 2004) with the kinase domain facing the cytoplasm on the outer mitochondrial membrane (Zhou et al., 2008). How­ ever, the mitochondrial phenotypes were very intri­ guing for parkin, which had been suggested previously to be present largely in the cytoplasm, at least under basal conditions (Cookson et al., 2003). Parkin is a protein ubiquitin E3 ligase, responsible for the addition of ubiquitin to substrate proteins, but none of the reported substrates are known mitochondrial proteins themselves. Further­ more, while mice deficient in parkin or PINK1 do not have dramatic phenotypes, they do have impair­ ment of mitochondrial function (Gautier et al., 2008; Palacino et al., 2004). Skin fibroblasts from human cases with parkin (Mortiboys et al., 2008) or PINK1 mutations (Exner et al., 2007) also have mitochon­ drial impairment. Therefore, PINK1 and parkin deficiency result in mitochondrial dysfunction across a number of

different species but the reasons for this are unclear, especially for parkin. Part of the answer appears to be that parkin can be a mitochondrial protein, but only under specific circumstances. If cells in culture expressing parkin are exposed to carbonyl cyanide m-chlorophenylhydrazone, which allows protons to equalize across mitochon­ drial membrane and depolarizes the organelle, then parkin can be selectively recruited to the damaged mitochondria (Narendra et al., 2008). Once recruited, parkin then promotes the removal of the depolarized mitochondria by autophagy. Presumably, in the absence of parkin, damaged mitochondria will slowly accumulate in energyrich tissues. Another surprise was that the phenotype of PINK1-deficient flies could be overcome by increasing the expression of parkin, but not the other way around (Clark et al., 2006; Park et al., 2006). Allied to the similar phenotypes caused by loss of PINK1 or parkin function in humans, these results suggest a common pathway with PINK1 genetically upstream of parkin. This work has been extended into mammalian systems by showing that recruitment of parkin to depolarized mitochondria is PINK1 dependent (Geisler et al., 2010; Narendra et al., 2010; Vives-Bauza et al., 2010), although this does not quite explain how parkin is able to rescue PINK1 deficiency in flies if recruitment to mitochondria is required for function. Returning to the theme of this chapter, we can now ask how mutations in these two genes influ­ ence these functional measures. Using mitochon­ drial recruitment of parkin as a measure of activity in cells, all recessive versions of PINK1 were shown to be non-functional, even those that are stable and expressed at the same level as wild-type protein (Narendra et al., 2010). The only excep­ tion is G411S, a variant that has been found in the heterozygous state rather than a homozygous version expected for a recessive allele. It is there­ fore ambiguous whether G411S is pathogenic. Similarly, recessive versions of parkin either are not recruited to the mitochondrial surface or fail

49

to trigger clearance of mitochondria by autophagy after depolarization (Narendra et al., 2010). Taken together, these various studies have identified a series of phenotypes that result from PINK1 or parkin deficiency and show that authen­ tic recessive mutations are non-functional in these assays. For PINK1, it is also reported that the kinase activity is important for function in these assays or in assays of neuroprotection, as artificial kinase dead versions do not substitute for wildtype protein (Dagda et al., 2009; Haque et al., 2008; Petit et al., 2005; Sandebring et al., 2009). However, there are still a series of unanswered questions related to this putative mitochondrial nexus for recessive parkinsonism. Both PINK1 and parkin are enzymes, being a kinase and an E3 ligase, respectively, so it is critical to under­ stand their substrates, specifically which substrates are responsible for maintaining mitochondrial function and integrity in various systems. There are some reports of a direct phosphorylation of parkin by PINK1 (Kim et al., 2008; Sha et al., 2010) but also negative reports (Vives-Bauza et al., 2010), leaving the most direct possible connection ambiguous. The problem of direct substrates is critical for the development of more direct assays for PINK1 and parkin function. Another unresolved question is the role of the third gene for recessive parkinsonism, DJ-1. DJ-1 appears to play a role in the control of mitochon­ drial function, particularly under oxidative circum­ stances (Blackinton et al., 2009; Canet-Aviles et al., 2004; Dodson and Guo, 2007; Hayashi et al., 2009; Junn et al., 2009; Krebiehl et al., 2010; Li et al., 2005; Ved et al., 2005; Zhang et al., 2005). Thus, it seems reasonable that DJ-1 may play similar physiological roles to PINK1/ parkin, although DJ-1 cannot substitute for loss of PINK1 like parkin (Exner et al., 2007) suggest­ ing it is either upstream of PINK1/parkin or in a parallel pathway. Finally, it is worth considering why recessive parkinsonism cases have restricted neuronal loss in humans, specifically dopamine neurons of the substantia nigra. All three genes for recessive

parkinsonism are widely expressed in most cell types and tissues, so limited expression to one group of neurons cannot explain why there is specific cell loss. Furthermore, the mitochondrial phenotype in the flight muscles and spermatids of Drosophila says that phenotypes of PINK1 or parkin deficiency are probably not due to dopa­ mine metabolism or neuronal activity per se, with the caveat that this is a different species, so there may be fundamental aspects of the biology that are not conserved. One possible candidate for sensitivity to loss of recessive parkinsonism genes is adenosine triphosphate (ATP) utilization by mitochondria under aerobic conditions. There is evidence that flight muscles in Drosophila are particularly sensitive to superoxide radicals gener­ ated by mitochondria (Godenschwege et al., 2009). The sensitivity of dopamine neurons to toxins such as rotenone and 1-methyl-4-phenyl­ 1,2,3,6-tetrahydropyridine (MPTP) that inhibit ATP production and result in reactive oxygen species (ROS) production may also hint at that there may be similar reasons for apparently dis­ parate phenotypes across species, although this remains speculative and difficult to test if mouse models lack robust phenotypes. These various data show that understanding the recessive nature of inheritance in early-onset par­ kinsonism helps us set up models that are instruc­ tive to understanding normal function and, from there, to show how mutations might lead to disease.

Mutations in SNCA and LRRK2 alter protein function If this logic is appealingly simple for recessive mutations, the situation for dominant genes is much more complex because here we cannot be sure if normal function of the proteins is at all relevant to the disease process. This is because dominant mutations can have mechanisms such as gain of novel function that are unrelated to the normal role of the protein, as shown for

50

superoxide dismutase mutations relevant for familial amyotrophic lateral sclerosis (Bruijn et al., 2004). However, clues to pathogenic mechanisms can be obtained by again considering what makes mutations similar to each other. Perhaps the best example of this comes from studies of SNCA protein chemistry in vitro. Like other proteins that are deposited in neurodegen­ erative diseases, SNCA can acquire a beta-sheet­ like structure in some conditions and aggregate into higher order aggregated species (Cookson, 2005). Interestingly in the context of mutations that increase protein expression without changing amino acid sequence, such as the duplication and triplication alleles, protein aggregation is a concentration-dependent phenomenon (Giasson et al., 1999; Wood et al., 1999) and therefore simply having too much protein may trigger aggre­ gation and mimic the effects of point mutations. Both the A53T (Narhi et al., 1999) and the E46K mutations (Greenbaum et al., 2005) increase the potential for SNCA to aggregate in these in vitro models. Interestingly, A30P can actually slow the formation of mature fibrils, the end product of aggregation reactions that may represent the deposited species in Lewy bodies. The shared property of A30P and A53T is the increased formation of oligomers, which are relatively soluble, partially aggregated species formed on the pathway to fibril formation (Conway et al., 2000). Therefore, if we follow the logic that shared properties of mutations are more likely to repre­ sent authentic pathogenic mechanisms then oligo­ mer formation is a good candidate for a toxic event to mediate the toxic effects of SNCA. There is some support for this concept from cell culture models where soluble oligomers can be identified (Xu et al., 2002) and where oligomers can be toxic when applied to the outside of cell membranes (Danzer et al., 2007), possibly through a pore-like mechanism (Kostka et al., 2008). Although this has not been verified using in vivo models, it therefore seems reasonable from the shared behaviour of mutant proteins

in vitro that oligomer formation is toxic to neurons. However, this logic is a little uncertain in part because it relies on the behaviour of the A30P mutation that is found only in one small family and at apparently decreased penetrance. Therefore, interpretation of these mutations requires some caution. This is particularly com­ plex when there are also clear dosage effects and the wild-type protein can be toxic in humans. Another example of the complexity of under­ standing dominant mutations is LRRK2. LRRK2 is a complex protein, but as it contains two pos­ sible enzymatic activities, a kinase domain and a GTP-binding region that contain dominant pathogenic mutations, it seems reasonable to examine which of these contributes to patho­ genicity. Several studies, admittedly using simple in vitro systems, suggest that all mutations in LRRK2 are toxic when expressed at high levels in cultured cells (Greggio et al., 2006, 2007; Iaccarino et al., 2007; Jorgensen et al., 2009; MacLeod et al., 2006; Smith et al., 2005, 2006). At this first approximation, this toxicity appears to be similar irrespective of whether mutations are in the GTP-binding region (e.g. R1441C/G), in the kinase domain (G2019S and I2020T) or in the intervening COR sequence (Y1699C). It is therefore interesting to ask whether these mutations really share similar mechanisms at a biochemical and cellular level. One obvious experiment is to measure how different pathogenic mutations affect kinase activ­ ity. Although there is some variation from study to study, the overall picture is that while G2019S in the kinase domain increases kinase activity by about twofold, the remaining mutations have no significant effect (Greggio and Cookson, 2009). Therefore, altered kinase activity is not a consis­ tent effect of mutations in this domain. But the acute toxicity of mutant LRRK2 is dependent on kinase activity (Greggio et al., 2006; Smith et al., 2006). How can we reconcile the similar effects of different mutations if they are in distinct domains of the same protein and if they have differential effects on kinase activity?

51

One idea is that the assays that most groups have used are not measuring the correct substrate. Several laboratories initially measured kinase activity with autophosphorylation, which many kinases will perform in vitro but may be a conse­ quence of high concentrations of enzymes in the test tube. Therefore, autophosphorylation may not be a true physiological activity and results may be biased by using the wrong readout. Although several alternate substrates to autopho­ sphorylation have been proposed, to this point none are proven to be physiological either [reviewed in Taymans and Cookson (2010)]. And in any case, when kinase activity of LRRK2 is measured with heterologous substrates, then the results are largely similar as for autophosphoryla­ tion (Greggio and Cookson, 2009) suggesting that the effects are general to mutations and not dependent on the precise assay conditions. Mutations in the ROC region, which has measurable but weak GTPase activity (Lewis et al., 2007; Li et al., 2007), tend to have lower GTPase activity. It has been suggested that GTP binding to LRRK2 or its homologue LRRK1 can stimulate kinase activity (Korr et al., 2006; Smith et al., 2006). Therefore, one might predict that there are circumstances where LRRK2 might have increased kinase activity for mutations out­ side of the kinase domain, if the GTP-bound state of LRRK2 is the more active and more toxic version and if mutations outside of the kinase domain slow turnover from GTP to GDP. However, there is little evidence yet that this hap­ pens and the basic data that GTP stimulates kinase activity of LRRK2 has been challenged recently (Liu et al., 2010). An alternative view is that the kinase activity of LRRK2 might regulate GTP binding and/or GTPase activity. Support for this idea comes from three recent studies identifying that LRRK2 can phosphorylate its own ROC/GTPase domain (Gloeckner et al., 2010; Greggio et al., 2009; Kamikawaji et al., 2009), leading to the pro­ posal that kinase regulates GTPase activity. This is reasonable if LRRK2 is a dimeric kinase that

autophosphorylates within the dimer, as suggested elsewhere (Greggio et al., 2008; Sen et al., 2009), but only if that activity is physiologically relevant, which is not yet proven. The overall message about LRRK2 is that while it is feasible to measure at least surrogates of the two major enzyme activities for this protein there are still difficulties in resolving both of these into a simple model for pathogenesis with a shared single output for all mutations. Clearly, a major challenge for the field is to identify the authentic outputs of LRRK2 kinase or other activities and to try and model the pathogenesis of the human condition. One area where some recent progress has been made is in understanding the relationship between LRRK2 and other dominant forms of PD. Mouse models have been developed that express mutant forms of LRRK2 in the brain, including a Bacterial Artificial Chromosome (BAC)-driven R1441G line (Li et al., 2009) and a transgenic G2019S cDNA mouse (Lin et al., 2009). The first animal model is especially interesting because although phenotypes in the lines were generally mild, there was evidence of accumulation of tau in axons (Li et al., 2009). The second animal model showed that there is an additive effect of expres­ sing mutant forms of LRRK2 and SNCA (Lin et al., 2009). Furthermore, knockout of LRRK2 limits the toxic effects of mutant SNCA suggesting that the effects are specific and not simply due to over-expression of two toxic proteins in the same cells. These results are important because they show that there are causal relationships between the two genes implicated in the genetics of PD, SNCA and LRRK2 and further suggest a role for tau in the same pathogenic pathway. Although the models are imperfect – none have frank degeneration of dopaminergic neurons in the sub­ stantia nigra – they reinforce the concept that not only should we examine multiple mutations in the same gene but we should also examine the inter­ actions between genes that produce similar phenotypes in patients. By extension, this leads

52

to the much more difficult question of asking how genes that show association with PD affect life­ time risk of disease.

Risk variants found in association studies likely have subtle mechanisms As discussed above, recent GWAS studies have reinforced two previously nominated genes that appear to increase lifetime risk of PD, SNCA/ a-synuclein and MAPT/tau, with several other genes of similar effect size being present in the human genome notably LRRK2 and the PARK16 locus. A significant challenge is to understand why these different genes influence disease risk, particularly when with association studies it is not always clear if the nominated variant (usually a single-nucleotide polymorphism, SNP) is actually causal for disease. SNPs are inherited in relatively large linkage disequilibrium blocks (as is the case for PARK16) and knowing which gene is the causal variant is therefore difficult. Furthermore, not all SNPs change protein sequence, so for many it is difficult to determine which is most likely to have a biological effect. Occasionally, there are hints as to ways in which genes might affect risk. For example, the nomi­ nated MAPT risk variants appear to increase tau mRNA expression (Simon-Sanchez et al., 2009). This suggests that having more tau without it being deposited may be an interesting mechanism by which these variants contribute to disease, but this hypothesis requires further work to under­ stand the interactions of tau and SNCA, given that the latter is the most pathologically relevant species. It is reasonable to think that SNCA risk alleles might increase expression of that protein, especially as multiplication mutations around the SNCA locus are causal for PD, but this remains to be proven. In total, these data show that for genes that change risk of PD over a lifetime, the effects are probably subtle and may in some cases be related to altered mRNA or protein expression

levels. However, there are additional important questions that need to be resolved. Both SNCA and tau are expressed in all neurons and yet show association with PD where there is prefer­ ential vulnerability of dopamine neurons. This is not an absolutely selective effect as SNCA can accumulate in other brain areas (e.g. the cortex in DLBD) and tau is associated with frontotem­ poral dementia, the association with parkinsonism is still striking. LRRK2 expression is actually higher in areas that are targeted by nigral neurons than in the ventral midbrain itself (Galter, 2006 #120) at least at the mRNA level, and thus selec­ tivity here shows an inverse correlation with where the gene is expressed. As discussed for recessive parkinsonism, the reasons for selectivity are not immediately obvious for any of the domi­ nant and risk factor genes. One might speculate that some of the same factors (ROS generation from mitochondrial metabolism) might be involved, but this is extremely speculative. Clearly, understanding why gene mutations or expression differences results in PD is a critical question for the future.

Summary The rapid pace of discovery in the genetics of PD has led to a huge amount of data to sort through that will present a challenge for biological under­ standing over the next few years. Two of the key ideas enunciated here are that understanding how multiple different mutations in the same gene cause disease and, by extension, how multiple genes for the same phenotype work are critical for developing a general pathogenic framework for PD. Importantly, at least some of the genetic influences on PD are shared between rare familial cases and sporadic disease making it feasible to suppose that pathogenic events may be shared between the two sets of aetiologies. This in turn suggests that a further understanding of genetic effects might be helpful in developing new ideas about the pathogenesis of PD and eventually

53

for the treatment of this disorder. Finally, the reasons for the preferential effects of mutations in widely expressed proteins on dopamine neu­ rons remain difficult to identify. This, along with the strong effects of aging on PD and related phenotypes, remains a critical next step for the field in trying to understand the pathophysiology of PD. Acknowledgements This research was supported by the Intramural Research Program of the National Institute of Health, National Institute on Aging.

Abbreviations COR DLBD GWAS LRRK2 MAPT PD PINK1 ROC SNCA SNP

C-terminal of ROC domain diffuse Lewy body disease genome-wide association study leucine-rich repeat kinase 2 microtubule-associated protein tau Parkinson’s disease PTEN-induced novel kinase 1 Ras of complex proteins domain a-synuclein (gene name) single-nucleotide polymorphism

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A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright � 2010 Elsevier B.V. All rights reserved.

CHAPTER 4

What causes the death of dopaminergic neurons in Parkinson’s disease? D. James Surmeier
, Jaime N. Guzman, Javier Sanchez-Padilla and Joshua A. Goldberg Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

Abstract: The factors governing neuronal loss in Parkinson’s disease (PD) are the subject of continuing speculation and experimental study. In recent years, factors that act on most or all cell types (pan-cellular factors), particularly genetic mutations and environmental toxins, have dominated public discussions of disease aetiology. Although there is compelling evidence supporting an association between disease risk and these factors, the pattern of neuronal pathology and cell loss is difficult to explain without cell-specific factors. This chapter focuses on recent studies showing that the neurons at greatest risk in PD – substantia nigra pars compacta (SNc) dopamine (DA) neurons – have a distinctive physiological phenotype that could contribute to their vulnerability. The opening of L-type calcium channels during autonomous pacemaking results in sustained calcium entry into the cytoplasm of SNc DA neurons, resulting in elevated mitochondrial oxidant stress and susceptibility to toxins used to create animal models of PD. This cell-specific stress could increase the negative consequences of pan-cellular factors that broadly challenge either mitochondrial or proteostatic competence. The availability of well-tolerated, orally deliverable antagonists for L-type calcium channels points to a novel neuroprotective strategy that could complement current attempts to boost mitochondrial function in the early stages of the disease.

Pan-cellular risk factors in Parkinson’s disease

non-neuronal cell types. The four best-documented pan-cellular factors are age, genetic mutations, environmental toxins and inflammation. The strongest risk factor in Parkinson’s disease (PD) is age (Calne and Langston, 1983; de Lau and Breteler, 2006). Disease incidence rises exponentially above the age of 65. Because improve­ ments in health care are increasing life expectancy, the number of PD patients is expected to grow dramatically in the coming years, reaching over 2 million in the United States by 2030

Studies over the past decade have made great progress in identifying factors that increase disease risk. The vast majority of these are pan-cellular factors; that is, factors that in principle have a broad, negative impact on neuronal and


Corresponding author. Tel.: 312-503-4904; Fax: 312-503-5101;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83004-3

59

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(Dorsey et al., 2007). Why age is such a strong risk factor is unknown, but it is widely speculated that declining mitochondrial function is a key factor (Boumezbeur et al., 2010; Schapira, 2008). In the last decade, perhaps the greatest single advance in the PD field has been the identification of genes that increase disease risk (Gasser, 2009). At present, seven genes have been clearly linked to familial forms of PD or Parkinsonism (Gasser, 2009; Lees et al., 2009). Although these still account for less than 10% of all the cases of PD, in some ethnic populations genetic mutations appear to account for a much larger fraction of cases (Lees et al., 2009). Unfortunately, most of the PD-associated genes are of unknown or poorly understood function. However, this gap is rapidly closing and there are common themes that are beginning to emerge. One of these themes is mitochondrial dysfunc­ tion. Three of the genes associated with a recessive, early-onset form of the disease (DJ-1, PINK1, Parkin) are directly linked to mitochondrial func­ tion, providing a potential connection with changes associated with aging (Schapira, 2008). DJ-1 is a mitochondrially enriched, redox-sensitive protein, giving it the capacity to signal oxidative challenges and potentially coordinate a variety of mitochon­ drial oxidative defence mechanisms (AndresMateos et al., 2007; Kahle et al., 2009). Parkin and PTEN-induced putative kinase 1 or PINK1 also have mitochondrial roles. Fruit flies with functional deletions of Parkin have fragmented and apoptotic mitochondria (Greene et al., 2003); knockout mice have a less dramatic but a clear mitochondrial phenotype (including decreased mitochondrial (respiratory) function, decreased metabolic drive and increased lipid and protein phosphorylation) (Palacino et al., 2004). PINK1 deletion leads to a similar phenotype in Drosophila as does Parkin deletion – fragmented cristae and apoptotic mito­ chondria; this phenotype can be rescued by Parkin over-expression, suggesting involvement in some common biochemical pathway (Clark et al., 2006; Park et al., 2006). Although found both in cytosolic and in mitochondrial preparations, PINK1 has an

N-terminus mitochondrial targeting sequence (Exner et al., 2007). Although the functions of the other genes pro­ minently linked to PD (SNCA, LRRK2) remain poorly defined, proteostatic dysfunction resulting in Lewy body (LB) formation is commonly thought to be an essential component of the dis­ ease aetiology (Sulzer, 2007). The third pan-cellular factor that has been identi­ fied is environmental toxin exposure. The proposi­ tion that toxins, particularly those that target mitochondria, could be a factor in PD has long been part of the mindset of the field given the ability of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri­ dine (MPTP) and rotenone to reproduce key aspects of the disease phenotype (Betarbet et al., 2000; Przedborski et al., 2004). Recent epidemiological studies have found convincing support for a link between pesticide exposure and the risk of develop­ ing PD (Kamel et al., 2007; Tanner et al., 2009). A fourth pan-cellular factor in PD is inflamma­ tion (Hartmann et al. 2003; Hirsch and Hunot, 2009; Hunot and Hirsch, 2003). In toxin models of PD, inflammation and resultant oxidant stress are important modulators of cell loss (Hunot et al., 2004; Teismann et al., 2003). In the later stages of the human disease, there are clear signs of micro­ glial activation and inflammation that could contribute to progression (Tansey and Goldberg, 2010). Recent work has shown how extrinsic oxida­ tive stress, like that created by inflammation, could result in neuronal death in a cell with high cytosolic calcium levels. Reactive oxygen species (ROS)­ mediated activation of protein kinase C beta phos­ phorylates 66-kD isoform of the growth factor adapter Shc (p66shc), promoting transport into mitochondria where it alters calcium responses and promotes apoptosis (Pinton et al., 2007). In the last year, the proposition that a fifth pancellular factor – a viral or prion-like infection – is causative in PD has been advanced (Hawkes et al., 2007; Olanow and Prusiner, 2009). In the absence of a direct demonstration of an infectious agent, there are two main pieces of evidence that have been used to argue for this type of process.

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The first is apparent staging of LB pathology in PD (Braak et al., 2004); the Braak hypothesis asserts that the pathology progresses from periph­ eral enteric autonomic ganglia to the caudal medullary autonomic cell groups and then ros­ trally into the brain. This apparent progression has been taken as evidence of an infection (Hawkes et al., 2007). However, it is far from clear that there is this sort of progression in the majority of PD patients, as the whole hypothesis turns on the supposition that patients with medul­ lary and ganglionic pathology alone would have developed PD had they lived longer. Moreover, there is considerable variability in the regional pattern of LB pathology, and LBs have an uncer­ tain connection to the pathophysiology underlying the symptoms of the disease (Burke et al., 2008; Jellinger, 2008). The second piece of evidence is derived from grafting embryonic dopamine (DA) neurons into PD patients (Kordower et al., 2008; Li et al., 2008; Mendez et al., 2008). In some of these grafts, DA neurons had LBs. Since these neurons were relatively young, the appearance of LBs has been taken as evidence of the spread of a virus or of a prion-like agent from the host. How­ ever, these data are open to alternative interpreta­ tion. The most obvious of which is that DA neurons are particularly susceptible to the stress of grafting, leading to ‘premature’ proteostatic dysfunction and LB formation. The apparent restriction of LBs to the DA neurons in the graft is certainly consistent with this explanation and not with an infection model. More importantly, at present, there is no compelling evidence that the pattern of neuronal pathology in PD conforms to the predictions of an infection model. The LB pathology in PD does not follow a nearest neigh­ bour rule. Neurons in the nucleus tractus solitar­ ius, for example, show no signs of pathology in PD in spite of being next to neurons in one of the most vulnerable nuclei (dorsal motor nucleus of the vagus [DMV]). There is no evidence that vulner­ ability is predicted by synaptic connectivity either. Arguments made that connectivity is an issue con­ sistently ignore the fact that every major neuronal

population affected with PD is synaptically coupled to a population of neurons that do not display significant pathology. In view of the dearth of hard scientific support, the infection model of PD is difficult to take seriously. Thus, studies of pan-cellular factors in PD have identified several potential processes in the aetiol­ ogy of PD, the most compelling of which are mitochondrial and proteostatic dysfunction. What is left unexplained by these studies is the pattern of neuronal dysfunction and loss in PD.

Pan-cellular risk factors • Age and declining mitochondrial function • Genetic mutations that compromise mito­ chondrial or proteostatic function • Environmental toxins that target mitochondria • Inflammation (late stage?) Cell-specific risk factors in PD Although there are signs of distributed neuro­ pathology in PD (as judged by LB formation) (Braak et al., 2004), the motor symptoms, includ­ ing bradykinesia, rigidity and resting tremor, are clearly linked to the degeneration and death of substantia nigra pars compacta (SNc) DA neurons (Hornykiewicz, 1966; Riederer and Wuketich, 1976). The palliative efficacy of L-DOPA – a DA precursor – is testament to the centrality of these neurons in the motor symptoms of PD. SNc DA neurons constitute a tiny fraction of all the neurons in the brain (<0.0001%), arguing that there must be cell-specific factors at work in PD. What might these factors be? DA itself has long been viewed as a culprit, as oxidation of cytosolic DA (and its metabolites) is damaging (Greena­ myre and Hastings, 2004; Sulzer, 2007). However, there are reasons to doubt this type of cellular stress alone is responsible for the loss of DA neurons in PD. First, there is considerable regio­ nal variability in the vulnerability of DA neurons

62

in PD, with some being devoid of pathological markers (Damier et al., 1999; Ito et al., 1992; Kish et al., 1988; Matzuk and Saper, 1985; Saper et al., 1991). Second, L-DOPA administration (which relieves symptoms by elevating DA levels in PD patients) does not appear to accelerate dis­ ease progression (Fahn, 2005), suggesting that DA is not a significant source of reactive oxidative stress, at least in the short term. Sulzer and collea­ gues have recently reported that calcium entry through L-type channels stimulates DA metabo­ lism in SNc DA neurons, pushing cytosolic DA concentrations into a toxic range with L-DOPA loading (Mosharov et al., 2009). For this mechan­ ism to be relevant to selective vulnerability, one would have to posit that modest elevations in cytosolic DA over decades lead to an accumula­ tion of cellular defects that ultimately produces cell death. If true, treating patients in the early stages of the disease with direct acting agonists, rather than L-DOPA, should lead to a slower progression of the disease. That said, the frank death or phenotypic decline of a variety of nondopaminergic neurons in PD argues that DA itself is not likely to be the principal cell-specific risk factor in the disease. Another distinctive feature of SNc DA neurons, and many of the other neurons that succumb in PD (e.g. locus coeruleus [LC] neurons), is their enor­ mous axonal field. Recent anatomical work has estimated that a typical SNc DA neuron has mean axonal length of 470 000 mm (Matsuda et al., 2009). Each axon supports ~370 000 synapses, orders of magnitude higher than the number supported by cortical pyramidal neurons, for example (Arbuth­ nott and Wickens, 2007). Sustaining such a large field must elevate axonal protein trafficking and proteostatic stress. Given that alpha-synuclein is largely a synaptic protein, its trafficking must be elevated in SNc DA neurons, potentially contribut­ ing to the axonal pathology seen in PD patients (Galvin et al., 1999). Moreover, because synaptic terminals are metabolically demanding specializa­ tions that typically require mitochondria, sustaining this axonal field could create a mitochondrial sink

for these neurons, lowering mitochondrial density in the somatodendritic region and lowering spare oxi­ dative capacity, creating an energy crisis (Nicholls, 2008). In fact, mitochondrial density in the somato­ dendritic region of SNc DA neurons appears to be abnormally low (Liang et al., 2007). Diminished oxidative reserve capacity could increase the pro­ duction of damaging superoxide by mitochondria, contributing to their decline with age. Our work in the last few years has focused on the potential role of physiological phenotype. Unlike the vast majority of neurons in the brain, adult SNc DA neurons are autonomously active, generating regular, broad action poten­ tials (2–4 Hz) in the absence of synaptic input (Fig. 1a–d) (Chan et al., 2007; Grace and Bun­ ney, 1984; Guzman et al., 2009; Nedergaard et al., 1993). This pacemaking activity is believed to be important to maintaining ambient DA levels in regions that are innervated by these neurons, particularly the striatum (Romo and Schultz, 1990). While most neurons rely exclu­ sively on monovalent cation channels to drive pacemaking, SNc DA neurons also engage L-type ion channels that allow calcium to enter the cytoplasm (Bonci et al., 1998; Ping and She­ pard, 1996; Puopolo et al., 2007), leading to oscillations in intracellular calcium concentra­ tions (Chan et al., 2007; Guzman et al., 2009; Wilson and Callaway, 2000) (Fig. 1e). The L-type calcium channels used by SNc DA neu­ rons in pacemaking have a distinctive Cav1.3 pore-forming subunit encoded by Cacna1d (Chan et al., 2007; Striessnig et al., 2006). Cav1.3 calcium channels are relatively rare, con­ stituting only about 10% of the all the L-type calcium channels found in the brain (SinneggerBrauns et al., 2009). Channels with this subunit differ from other L-type calcium channels in that they open at relatively hyperpolarized poten­ tials, allowing them to contribute to the mechan­ isms driving the membrane potential to spike threshold underlying autonomous pacemaking (Chan et al., 2007; Guzman et al., 2009; Puopolo et al., 2007).

63

(a)

(b)

SNc

IR-DIC

SNc DA neuron SNr

cp

Dorsal Medial

(c)

(d) Action potential

DA neuron GABAergic interneuron –40 mV

1 ms (e)

2 sec Voltage

+Low isradipine

40 µm Calcium concentration

1 sec

0.02 ΔG/R

Fig. 1. SNc DA neurons have a distinctive physiology. (a) Representative acute midbrain slice illustrating the SNc DA neurons of the ventral tier region (highlighted in red) selected for electrophysiological recordings. (b) Image depicting a patched SNc DA neuron visualized under infrared differential interference contrast (IR-DIC) microscopy. (c) Whole-cell current clamp recording displaying firing of action potentials for an SNc DA neuron. SNc DA neurons are neuronal pacemakers with firing frequency between 1 and 4 Hz. (d) SNc DA neurons (black trace) display broader action potentials when compared to action potential spikes from medium spiny neurons (green trace). (e) SNc DA neurons pacemaker spikes are coupled to calcium transients sensitive to L-type calcium channel blockers. Shown in the left, a reconstruction of an SNc DA neuron filled with the red dye Alexa594 (50 mM) and the calciumsensitive Fluo4 (200 mM). Shown to the right of the DA neuron, representative pacemaking traces before and after bath application of the L-type calcium channel blocker (5 mM isradipine), and below pacemaking traces, time-matched dendritic calcium transients from a distal dendrite (80 mm away from soma). Calcium transients were abolished by application of isradipine yet the pacemaking firing was insensitive to this calcium channel blocker.

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The sustained engagement of Cav1.3 calcium channels during pacemaking comes at an apparent metabolic cost to SNc DA neurons. Because of its involvement in cellular processes ranging from the regulation of enzyme activity to programmed cell death, calcium is under very tight homeostatic control, with a cytosolic set point near 100 nM – 10 000 times lower than the concentration of cal­ cium in the extracellular space (Berridge et al., 2000; Orrenius et al., 2003; Rizzuto, 2001). Calcium entering neurons is rapidly sequestered or pumped back across the steep plasma membrane concen­ tration gradient; this process requires energy stored in Adenosine-50 -triphosphate (ATP) or in ion gradients that are maintained with ATPdependent pumps, like the Na-K ATPase. In most neurons, calcium channel opening is a rare event, occurring primarily during very brief action potentials. This makes the task and the metabolic cost to the cell readily manageable. But in SNc DA neurons, where Cav1.3 calcium channels are open much of the time, the magni­ tude and the spatial extent of calcium influx are much larger (Wilson and Callaway, 2000). Preli­ minary studies using transgenic mice that express a mitochondrially targeted redox-sensitive variant of green fluorescent protein (mito-roGFP) under control of the tyrosine hydroxylase promoter have revealed that indeed mitochondria in SNc DA neurons have a high basal oxidant stress that is a direct consequence of opening of L-type calcium channels. Furthermore, calcium entry (and presumably the concomitant oxidant stress) increases the vulnerability of SNc DA neurons to toxins [MPTP, 6-hydroxydopamine (6-OHDA), rotenone] used to create animal models of PD (Chan et al., 2007). Another reason to suspect that calcium is an important factor is the inverse correlation between expression of the mobile calcium buffer­ ing protein calbindin and vulnerability in PD, as well as in animal models of the disease (German et al., 1992a). Calbindin expression is high in DA neurons of the ventral tegmental area as well as the dorsal tier of the SNc, both areas that are

relatively resistant in PD. What is less clear is precisely why this should be the case. One possi­ bility is that calbindin reduces calcium entry into the endoplasmic reticulum (ER), holding it for plasma membrane extrusion mechanisms and avoiding ‘double pumping’. The glutamatergic synaptic input to SNc DA neurons could also contribute to their vulnerabil­ ity. In vivo, SNc DA neurons spike in at least two other modes that are created by superimposing synaptic input on the basal pacemaking activity (Tepper et al., 1987). From a functional stand­ point as well as from the standpoint of neurode­ generation, the most interesting of these is the burst mode. Because of its association reward prediction errors (Schultz, 2007), elevated release of DA in the striatum and the induction of long-term synaptic plasticity, this burst has generated a great deal of experimental attention. A bevy of studies have recently focused on the mechanisms underlying the burst. These studies have established the necessity of N-methyl-D­ aspartate (NMDA) receptor activation in burst generation (Blythe et al., 2009; Deister et al., 2009; Zweifel et al., 2009). Because pacemaking keeps the membrane potential of SNc DA neu­ rons in a voltage range where magnesium block of NMDA receptors is ineffective, even modest glutamatergic input is capable of producing sub­ stantial NMDA receptor currents. Could calcium entry through NMDA receptors synergize with that through Cav1.3 channels engaged by pace­ making to create a metabolic tipping point for mitochondria? Excitotoxicity has long been hypothesized to be a factor in the aetiology of PD (Beal, 1998; Greenamyre and O’Brien, 1991; Sonsalla et al., 1998). But the engagement of NMDA receptors and the elevation in cytosolic calcium concentration this brings about has been envisioned to be a relatively late stage event, coming only when cells were unable to maintain a stable, hyperpolarized membrane potential. But SNc DA neurons are pacemakers that do not have a ‘stable’ hyperpolarized membrane potential when they are healthy, meaning that

65

NMDA receptors should be more easily recruited. Another factor in this equation is the intracellular calcium stores. Metabotropic glutamate receptor (mGluR) activation mobilizes these stores (Morikawa et al., 2003), forcing neu­ rons to re-sequester calcium (at the expense of ATP). Because of the sustained calcium entry into SNc DA neurons during pacemaking, these stores are fully charged, which adds to the burden created by mGluR activation. In this way, pace­ making and regular activation of NMDA and mGluR receptors should create a sustained cal­ cium ‘storm’ in SNc DA neurons. Although mito­ chondria appear capable of weathering this storm in the short term, the elevation in oxidant stress created by the need to supply a steady stream of ATP to pumps should slowly increase damage to their DNA and accelerate their aging (Bender et al., 2008; Reeve et al., 2008).

Cell-specific risk factors for SNc DA neurons • Oxidation prone neurotransmitter • Profuse axonal terminal field • Slow, autonomous pacemaking with broad action potentials; engagement of L-type Cav1.3 calcium channels in pacemaking, leading to an elevation in cytosolic calcium concentration • Lack of mobile calcium binding proteins, like calbindin • Depolarized membrane potential that promotes the opening of calcium permeable NMDA receptors Do other types of neuron that succumb in PD share these risk factors? There are a number of regions of the brain that have cell loss paralleling that of the SNc (Braak et al., 2004; Fronczek et al., 2007; German et al., 1992b; Jellinger, 2009; Thannickal et al., 2007). Of the cell-specific risk factors discussed, two standout as common to the other cell types that

succumb in PD: autonomous or spontaneous activity with broad action potentials and a depo­ larized membrane potential that promotes the opening of NMDA receptors. Although the avail­ able data set is fragmented, neurons in the DMV, LC, raphe nuclei (RN), pedunculopontine nucleus (PPN), lateral hypothalamus, tuberomammillary nucleus, basal forebrain (BF) and olfactory bulb and all have these two physiological characteristics, while having widely different transmitters and axonal fields. DMV cholinergic neurons, which are thought to be among the earliest neurons with LBs in PD, are spontaneously active (Travagli and Gillis, 1994); this activity is autonomously generated and depends upon L-type calcium channels (unpub­ lished observations). LC noradrenergic neurons, like SNc DA neurons, have large axonal arbors and are autonomous pacemakers (with broad spikes) that engage L-type calcium channels (Wil­ liams et al., 1984). Serotonergic neurons in the RN have broad spikes and are calcium-dependent autonomous pacemakers (Burlhis and Aghajanian, 1987). This is also true for PPN cholinergic neurons (Takakusaki and Kitai, 1997). Tuberomammillary and lateral hypothalamic (orexin expressing) neurons are spontaneously active (Li et al., 2002; Stevens and Haas, 1996; Yamanaka et al., 2003). Tuberomammillary neurons engage L-type calcium channels in this process (Stevens and Haas, 1996; Taddese and Bean, 2002; Williams et al., 1984) (this question has not been addressed in orexin neurons). BF cholinergic neurons are lost in PD (Braak et al., 2004). These neurons have large axonal terminal fields, are spontaneously active in brain slices and have prominent calcium channel currents (Murchison and Griffith, 1995). Moreover, with aging here are significant and deleterious changes in calcium homeostasis in these neurons (Murchison and Griffith, 2007). DA neurons in the olfactory bulb are a slightly different case from the standpoint of pathology. These neurons are calcium-dependent, autonomous pacemakers (Pignatelli et al., 2005). However, there are no signs of cell loss in the olfactory bulb in spite of deficits in olfaction being a harbinger of the motor

66

Is there an interaction between risk factors?

symptoms in PD (Huisman et al., 2008; Postuma et al., 2006). This does not mean that a reliance upon calcium is a bad thing though, as this region is capable of adult neurogenesis (Pignatelli et al., 2009). Although much needs to be done, the shared physiological characteristics of these seven vulner­ able cell types point to a common mechanism underlying their slow functional decline with age: calcium-mediated stress.

Is there an interaction between pan-cellular and cell-specific processes that produce the pattern of degeneration seen in PD (Fig. 2)? Age is the strongest of the pan-cellular risk factors. One of the oldest and most popular theories of aging is that it is a direct consequence of accumulated mito­ chondrial DNA (mtDNA) and organelle damage

Pan-cellular factors Genetic mutations

Inflamation

Aging

Environmental

toxins

Cell-specific factors

Sparse axon branching Calcium binding Brief spikes Low protein Non-reactive Intermittent transmitter activity Hyperpolarized membrane Type A

Risk

High

Profuse axon branching Calcium Reactive transmitter loading Pacemaking

Long spikes

NMDA/mGluR activation Type Z Mitochondrial dysfunction

Proteostatic decline/ degeneration

Parkinson’s disease Fig. 2. Cell death is attributable to a combination of cell-specific factors and pan-cellular factors. The interaction of pan-cellular factors (top) and cell-specific factors (middle) is required to account for the loss of SNc DA neurons in PD. The impact of pan-cellular factors on cellular vulnerability is envisioned to depend upon neuronal phenotype, which is determined by a constellation of features including pacemaking, calcium loading and axonal branching. For the purposes of illustration a low-risk phenotype and a high-risk phenotype (e.g. SNc DA neuron) are illustrated. These factors are envisioned as working together to trigger mitochondrial dysfunction, which then leads to proteostatic decline and cell death underlying Parkinson’s disease.

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produced by ROS and related reactive molecules generated by the electron transport chain (ETC) in the course of oxidative phosphorylation (Harman, 2003; Wallace, 2005). A corollary of this hypothesis is that the rate of aging is directly related to meta­ bolic rate. There is no obvious reason not to extend this organismal postulate to individual cells. The reliance of SNc DA neurons on a metabolically expensive strategy to generate autonomous activity that taxes mitochondria should mean that they age more rapidly than other types of neuron – creating a positive interaction between cell-specific and pancellular risk factors. This perspective predicts that there should be functional impairment or loss of SNc DA neurons with normal aging. Stereological estimates of normal aging-related cell death in humans argue that SNc DA neurons are at a higher risk than many other types of neurons (Stark and Pakkenberg, 2004). In mammals with significantly shorter lifespans, loss of SNc DA neurons with age has not been seen reliably, but there is a clear decline in phenotypic markers with age that match that seen in PD, as well as an increased susceptibility to toxins (Backman et al., 2000; Collier et al., 2007; Ishikawa et al., 1996; Kanaan et al., 2008; McCor­ mack et al., 2004). There is also an aging-related decline in SNc mitochondrial function (Beal, 1995), some of which could easily be attributed to the accumulation of mtDNA mutations with normal aging (Bender et al., 2006). There could be a positive interaction between cell-specific mitochondrial stress and the other pan-cellular risk factors. As with aging, an inter­ action of this sort could help create a tipping point in the pathogenesis of PD that would result in a selective pattern of degeneration. Consider the loss of DJ-1 function that compromise mito­ chondrial oxidant defences. In most neurons, oxidant defence engagement is likely to be mild, episodic and readily endured even without a fully functional defence system. But in cell types, like SNc DA neurons, where oxidant stress appears to be sustained, compromising oxidant defences could come at higher long-term cost. Mutations that diminish proteostatic competence

(e.g. alpha-synuclein over-expression) could also exert their primary effects on cellular viability through increasing ATP utilization. It would be of considerable interest to see if over-expression of alpha-synuclein increased mitochondrial oxi­ dant stress. Furthermore, broadly acting environ­ mental toxins that partially compromise mitochondrial function should have a bigger impact on cell types that have high mitochondrial demands. Lastly, inflammation in the late stages of the disease should significantly increase the production damaging reactive oxygen species in and around surviving neurons. If these neurons are already over-producing ROS because of cellspecific factors, their oxidant defences could be overwhelmed, leading to apoptosis. Can dihydropyridines be therapeutically effective? Given the existence of both cell-specific and pancellular risk factors in PD, how should the devel­ opment of a neuroprotective therapeutic move forward? Current therapeutic strategies are ostensibly targeting the pan-cellular factors (e.g. co-enzyme Q10). Attacking the cell-specific factors is another strategy. Certain risk factors, like dopamine and axonal terminal field, are not malleable, at least not without compromising brain function. One factor that does appear to be a viable target is calcium entry through L-type calcium channels during autonomous or sponta­ neous spiking. These channels are antagonized by dihydropyridines (DHPs) that are approved for human use. DHPs have a very modest side-effect profile and have been used for decades to treat hypertension (Eisenberg et al., 2004). There are two basic questions that have to be answered before moving ahead with this kind of a neuroprotective strategy in the early stages of PD. The first is whether antagonizing L-type calcium channels will significantly impair the ability of SNc DA neurons to perform their duties. The second is whether neuroprotective concentrations of DHP can be achieved in the brain of PD patients.

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Are L-type channels necessary for pacemaking? Several groups over the last 15 years have argued that voltage-dependent calcium channels were necessary (Amini et al., 1999; Mercuri et al., 1994; Nedergaard et al., 1993; Wilson and Callaway, 2000). Considering the sensitivity of pace­ making to DHPs, it has been inferred that these channels were L-type channels. In support of this view, SNc DA neurons robustly express L-type channels having a Cav1.3 pore-forming subunit with the kind of gating properties necessary to drive a sub-threshold membrane potential oscilla­ tion thought to underlie pacemaking (Chan et al., 2007; Guzman et al., 2009; Koschak et al., 2001). However, the concentrations of DHPs necessary to slow or stop pacemaking are more than three orders of magnitude higher than the equilibrium binding constant of most DHPs for Cav1.3 chan­ nels – raising basic questions about the necessity of these channels for pacemaking. As a first step towards understanding what concentration of DHP is sufficient to antagonize L-type calcium channels in SNc DA neurons, a modulated receptor model was constructed using the framework proposed by Bean (1984). In this model, the channel was assumed to have high- and low-affinity states governed by voltage-dependent inactivation (Fig. 3a). The macroscopic balance between these states was governed by voltagedependent inactivation of the Cav1.3 calcium channel. Estimates of this parameter were taken from the work of Koschak et al. (2001) in which channels were heterologously expressed in a cell type that was readily voltage clamped (Fig. 3b). Because isradipine has a relatively high affinity for the Cav1.3 calcium channels thought to under­ lie pacemaking, our initial calculations modelled its actions (Sinnegger-Brauns et al., 2009). The apparent dissociation constant (Kapp) of isradi­ pine as a function of membrane voltage was com­ puted using the formula proposed by Bean (1984) by assuming that the dissociation constant (KD) for high-affinity state was equal to that estimated from equilibrium binding studies (0.48 nM) and

the dissociation constant for the low-affinity state was a 1000-fold higher (480 nM) (SinneggerBrauns et al., 2009) (Fig. 3c). Next, an all-points histogram of membrane potential during pace­ making was generated (Fig. 3d); this distribution had two modes: one near –60 mV and another near –40 mV. Considering this result, the relation­ ship between isradipine concentration and the fraction of Cav1.3 channels available (not antag­ onized) was computed at –50, –60 mV and (for comparison) –90 mV (Fig. 3e). This calculation showed at either –50 or –60 mV, more than 90% of the Cav1.3 channels should be antagonized by 100 nM isradipine, whereas at –90 mV only about 30% of the channels should be antagonized. For the purposes of comparison, the dose–response curve for another commonly used, but less potent, DHP (nifedipine) was calculated at a potential of –60 mV. Considering this calculation, at equili­ brium 1 mM nifedipine should antagonize more than 90% of the Cav1.3 channel population dur­ ing pacemaking (Fig. 3f). These calculations strongly argue that at equilibrium, sub-micromo­ lar concentrations of isradipine and other DHPs should effectively suppress Cav1.3 calcium chan­ nel currents and disrupt pacemaking that depends upon them. These calculations also show that serum concentrations of isradipine found in patients taking the medication for hypertension (~5 nM) should antagonize roughly half of the Cav1.3 channels in SNc DA neurons if we assume equilibration across the blood–brain barrier (BBB). Based upon these results, we re-examined the role of Cav1.3 channels in pacemaking. The pro­ blem is that we needed a measure of Cav1.3 chan­ nel function that was independent of pacemaking. We turned to calcium imaging using two-photon laser scanning microscopy, allowing us to monitor pacemaking neurons deep in a brain slice from the mesencephalon (Fig. 1e, left). In these dual recordings, the dendritic calcium concentration oscillates in phase with somatic spiking. Bath application of 200 nM isradipine for 20 min (to allow an equilibrium to be achieved 50–100 mm

69

below the surface of the slice) eliminated the den­ dritic calcium oscillation, but had no impact on pacemaking rate – providing a clear dissociation between Cav1.3 channel opening and pacemaking (Fig. 1e, right). Does this mean that Cav1.3 channels are comple­ tely superfluous? Modelling the pacemaking process revealed that there is a rich interplay of ionic con­ ductances that lead to autonomous spiking. Many of the conductances play similar roles, so that deficien­ cies in one can be compensated for to maintain the correct spiking rate. For example, when Cav1.3 channels are blocked in our model, outward cur­ rents through small conductance calcium activatedpotassium (SK) channels decline and the trajectory of the after hyperpolarization changes leading to stronger engagement of cationic hyperpolarization activated cyclic nucleotide (HCN) channels. The net effect on pacemaking is minimal. Similarly, if HCN channels are blocked by themselves, Cav1.3 current increases to compensate. However, if both HCN and Cav1.3 channels are blocked, pacemaking stops because the cell cannot generate enough inward current near spike threshold. This behaviour of the model was verified experimentally in SNc DA neurons. The takeaway message is that SNc DA neurons are well designed, with a robust network of ion channels to support pacemaking. It is a fail-safe system because pacemaking is so important to the func­ tioning of the basal ganglia. Our contention that Cav1.3 channels are not necessary for pacemaking has been challenged in a recent paper (Putzier et al., 2009). The crux of their argument is that they can experimentally restart pacemaking that has been halted with high concen­ trations of DHP by using ‘dynamic’ current clamp to reintroduce Cav1.3 channels into the soma. The basic problem with this argument is that there is nothing unique about the inward conductance cre­ ated by the dynamic clamp; introducing a Nav1 channel or an HCN channel would produce the same outcome. So, in the end all the authors demon­ strate that pacemaking is robust in the sense that it can be generated in several ways.

Epidemiological support for cell-specific risk factors Could antagonizing L-type calcium channels pre­ vent or slow PD in a normal lifespan? Calcium channel antagonists (CCAs), including the DHPs used in animal studies, are commonly used in clinical practice to treat hypertension, creating a potential database to be mined. A case–control study of hypertensive patients found a significant reduction in the observed risk of PD with CCA use, but not with medications that reduce blood pressure in other ways (Becker et al., 2008). More recently, a large Danish data set has been exam­ ined (Ritz et al., 2010). The authors agreed with the main conclusions of the Becker et al. study but extended their findings by showing that only DHPs that cross the BBB are associated with reduced PD risk (~30%). Given the short period of treatment in many cases (~2 years), variable dosing and low relative affinity of DHPs for Cav1.3 calcium channels (compared to Cav1.2 channels) (Eisenberg et al., 2004; Kupsch et al., 1996; Mannhold, 1995), this is a surprisingly strong association and lends further credence to the proposition that a BBB permeable and potent Cav1.3 antagonist could be a very effective neuro­ protective agent. That said, these studies are not a substitute for a controlled clinical trial. In the absence of a selec­ tive Cav1.3 CCA, the DHP isradipine is the most attractive drug for such a trial. Isradipine has a relatively higher affinity for Cav1.3 calcium chan­ nels than the other known DHP and has good brain bioavailability (Sinnegger-Brauns et al., 2009). At the doses used to treat hypertension, isradipine has relatively minor side-effects (Fitton and Benfield, 1990). The question is whether it will prove neuroprotective at doses tolerated by the general population. Pharmacokinetic studies by our group have found that serum concentra­ tions of isradipine achieved in mice that are pro­ tected (~1–2 ng/ml) against MPTP and 6-OHDA toxicity are very close to those achieved in humans with a very well-tolerated daily dose (10 mg/day,

(a)

(b)

Cav1.3 channel antagonism by DHPs

1

Inactivation Cav1.3 channels

available

voltage

C voltage

0.8

I

DHP Kc = 500 nM

DHP Ki = 0.5 nM

k1 k–1

C*

Fraction available

C

I

I*

0.6

0.4

0.2 Inact = 1–exp(–(Vm–Vh)/Vc)

unavailable 0

(c) –6

Vh = –42.7 mV, Vc = 6.6 mV –90

–70 –50 –30 Voltage (mV)

–10

(d) Kc=500 nM

Kapp = 1/[c/Kc+(1–c)/Ki]

2

20 mV Count (X1000)

Kapp(Log)

–7

–8

–9

Ki=0.5 nM

1.5

1

–40

0.5

2 sec

Isradipine –10

–90

–70

–50 –30 Voltage (mV)

0 –80 –60 –40 –20 0 Voltage (mV)

–10 (f)

(e) 1

1 @–60 mV

Isradipine 0.8

0.8 5 nM

0.6

@–90 mV

Fraction available

Fraction available

20

~50% @–60 mV 0.4

@–50 mV

0.2

0 –10

Fig. 3. (Continued)

~78%

–9

–8 –7 Drug (Log)

–6

–5

Nifedipine

0.6

0.4 Isradipine 0.2

0

–9

–8 –7 Drug (Log)

–6

–5

71

Dynacirc CR). As shown above, these isradipine concentrations should antagonize around half of the Cav1.3 channels in a pacemaking neuron, suggesting that neuroprotection is achievable. It is also worth considering how DHPs might be used in combination with other drugs that are being tested in clinical neuroprotection trials for PD. Although early trials with creatine, co-enzyme Q10, and other antioxidant supplements have been disappointing (Hung and Schwarzschild, 2007), they share the hypothesis that oxidative stress exacerbates the symptoms and progression of PD. Co-enzyme Q10 is an electron acceptor for complexes I and II that appears compromised in PD patients (Shults, 2005) and is neuroprotective in animal models of PD (Beal, 1998). Creatine is a substrate for ATP production that can both improve mitochondrial efficiency and reduce oxidative stress by buffering fluctuations in cellular energy produc­ tion (Klivenyi et al., 1999). Both approaches are aimed at improving mitochondrial function rather than attacking the source of stress on mitochondria. Rasagiline or deprenyl also could prove to have neuroprotective effects by virtue not of its ability to inhibit monoamine oxidase B, but by their ability to its ability to induce the expression of antioxidant defences (Magyar and Szende, 2004). Because their sites of action differ within the chain of events leading to oxidative stress and mitochondrial dysfunction, a combination therapy could prove more effective than any one therapy alone.

Unanswered questions There are a number of questions concerning the role of cell-specific risk factors in the neuronal pathology seen in PD. Answering these questions not only could provide novel therapeutic strate­ gies for preventing or slowing the progression of the disease but could also help to create strategies for ‘successful aging’ generally. One major question is whether activity-depen­ dent calcium entry into neurons creates a signifi­ cant mitochondrial oxidant stress. In spite of its plausibility, there is no direct evidence that plasma membrane calcium influx elevates mitochondrial oxidative phosphorylation and the production of superoxide. Limiting plasma membrane calcium influx certainly diminishes the sensitivity of SNc DA neurons to mitochondrial toxins, but this effect could be indirect. The development of redox-sensitive optical probes (Desireddi et al., 2010; Hanson et al., 2004; Wang et al., 2008) and two-photon laser scanning microscopy to allow imaging of mitochondria in situ puts this question within reach. Being able to directly assess mito­ chondrial function, living SNc DA neurons could shed light on the intriguing observation that mito­ chondrial uncoupling proteins (UCPs) enhance their resistance to toxins (Andrews et al., 2005, 2006). Mitochondrial UCPs are hypothesized to form part of a negative feedback loop to reduce the production of superoxides by the electron

Fig. 3. Nanomolar concentrations of isradipine should produce a significant antagonism of Cav1.3 calcium channels. (a) Modulated receptor model of dihydropyridine drug action. C stands for a closed state of the channel; C
stands for a closed, drug-bound state. The dissociation constant (Kc) for this reaction is assumed to be 500 nM. I stands for the inactivated state of the channel. Transitions between C and I states are controlled by membrane voltage. I
stands for the inactivated, drug-bound state; the dissociation constant (Ki) for this reaction is assumed to be 0.5 nM. (b) Steady-state inactivation of Cav1.3 channels as a function of membrane voltage; this describes the transition of channels from the C to I states in (a). (c) Plot of the apparent dissociation constant as a function of transmembrane voltage computed from the equation Kapp = 1/[C/Kcþ(1–C)/Ki], where C is the fraction of the channels in the closed state (from (b)) and Kc and Ki are from (a). (d) Recording of an SNc DA neuron and an all-points histogram of membrane potential showing that these neurons reside at potentials more depolarized than –60 mV. (e) Plot of the fraction of available channels as a function of drug concentration for three different membrane potentials. The plots reveal that with a holding potential of –60 mV, roughly half the channels are antagonized by 5 nM isradipine; only a small percentage of the channels will be antagonized by this drug concentration if the membrane potential is at –90 mV (as would be the case in a striatal medium spiny neuron). (f) Plot of the fraction of Cav1.3 channels available as a function of isradipine concentration (green line) at –60 mV or of a lower affinity antagonist nifedipine (red line).

72

transport chain (Brand et al., 2004). There are five known UCPs (UCP1–5); UCP2, 4 and 5 are robustly expressed in the SNc (Andrews et al., 2005). While UCP2 has been implicated in the response to MPTP, understanding the role of these proteins in regulating physiological calcium stress could point to novel therapeutic strategies. A related albeit more difficult question is whether the role of mitochondria in intracellular calcium buffering contributes to neuronal apopto­ sis or necrosis in slowly progressing neurodegen­ erative diseases, like PD. Although mitochondria do not normally flux calcium from the cytoplasm at physiological concentrations, calcium released from the ER through inositol trisphosphate or ryanodine receptors can enter mitochondria at points of apposition between the organelles, which form functional calcium microdomains in which calcium concentrations can rise into the micromolar range (Csordas et al., 2006; Rizzuto and Pozzan, 2006). Through these junctions, ‘dumping’ of ER calcium stores into mitochondria could trigger apoptosis in marginally competent mitochondria (Hajnoczky et al., 2003). However, the vast majority of the studies demonstrating the existence of close interactions between mitochon­ dria and the ER have been performed in cell lines; none have been performed in SNc dopaminergic neurons where the functional relationship between these organelles could be quite different. That said, mechanisms like this seem to be in play in Alzheimer’s disease (Bezprozvanny, 2009). A second major set of questions is how genetic mutations associated with PD interact with cellspecific risk factors like calcium entry to trigger neuronal pathology and death. It is easy to ima­ gine that a negative dominant mutation of a gene like DJ-1 could exacerbate the basal oxidative stress in an SNc DA neuron and accelerate mito­ chondrial and proteostatic collapse, leaving neu­ rons lacking the same basal stress largely unaffected. But these types of interaction between mutations and cellular phenotype have not been pursued. All too often, the effects of genetic muta­ tions are pursued solely in cell lines, immature

neurons or in neurons that are not vulnerable to the disease. A third set of questions is whether the nondopaminergic neurons that are vulnerable in PD share a common cellular phenotype that is thera­ peutically manipulable. As mentioned above, there is evidence that a large subset of the neurons that die or functionally decline are spontaneously active, have broad spikes and flux lots of calcium. However, the phenotypic characterization must be explored more systematically in those cell types at greatest risk (DMV, LC, BF cholinergic neurons, dorsal raphe, etc.). Moreover, this should be done in situ (either with in vivo recording or in brain slices from adult mice) where the behaviour of the neurons is as close as possible to that found in humans. Conclusions Although pan-cellular risk factors dominate cur­ rent thinking about the aetiology of PD, there are compelling reasons to believe that cell-specific factors are important as well. From a theoretical standpoint, it is very difficult to explain the pattern of neuronal pathology in PD without these factors. While transmitter or anatomical phenotype might contribute to the vulnerability of SNc DA neu­ rons, the trait with the clearest mechanistic path to cellular aging and degeneration is the engage­ ment of L-type calcium channels in the generation of autonomous spiking. The sustained entry of calcium undoubtedly taxes the ATP-dependent pumps responsible for keeping its concentration low, and in so doing creates a burden on mito­ chondrial oxidative phosphorylation. An inevita­ ble consequence of oxidative phosphorylation is the production of superoxide capable of damaging DNA and proteins. Although this metabolic stress is not sufficient to disable SNc DA neurons in the short run, it is possible that in the long run it exacerbates the normal aging-related decline in mitochondrial function, resulting in persistent energy shortages that compromise proteostatic

73

competence. Because L-type channels are not necessary for SNc DA neurons to do their job, L-type channel antagonists seem to be a viable neuroprotective strategy. These drugs are well tolerated and safe and their use is associated with a diminished risk of PD. Because this physio­ logical phenotype is not unique to SNc DA neu­ rons but appears to be shared by many of the neurons that succumb in PD, these antagonists could confer protection well beyond the SNc.

ETC ER 6-OHDA HCN LH LRRK2 LB L-DOPA LC MPTP mGluR Na-K ATPase NMDA PD PPN PINK-1 RN ROS

SNc SNCA TTX UCP VTA

small conductance calcium activated-potassium channel substantia nigra pars compacta gene name for alpha-synuclein tetrodotoxin uncoupling proteins ventral tegmental area

References

Abbreviations ATP BF BBB CCAs DA DHP DMV

SK

adenosine triphosphate basal forebrain blood brain barrier calcium channel antagonists dopamine dihydropyridines dorsal motor nucleus of the vagus electron transport chain endoplasmic reticulum 6-hydroxydopamine hyperpolarization activated cyclic nucleotide channel lateral hypothalamus leucine rich repeat kinase 2 Lewy bodies levodopa or L-3,4-dihydroxyphenylalanine locus coeruleus 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine neurotoxin metabotropic glutamate receptors sodium/potassium ATPse N-methyl-D-aspartate Parkinson’s Disease pedunculopontine nucleus PTEN-induced putative kinase 1 raphe nuclei reactive oxygen species

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A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 5

Intracellular signalling pathways in dopamine cell death and axonal degeneration Robert E. Burke
Departments of Neurology, Pathology and Cell Biology, Columbia University, New York, NY, USA

Abstract: The pathways of programmed cell death (PCD) are now understood in extraordinary detail at the molecular level. Although much evidence suggests that they are likely to play a role in Parkinson’s disease (PD), the precise nature of that role remains unknown. Two pathways of cell death that are especially well characterized are cyclin-dependent kinase 5-mediated phosphorylation of myocyte enhancer factor 2 and the mitogen-activated protein kinase signalling cascade. Although blockade of these pathways in animals has achieved a truly remarkable degree of neuroprotection of the neuron cell soma, it has not achieved protection of axons. Thus, there is a need to explore beyond the canonical pathways of PCD and investigate mechanisms of axon destruction. We also need to move beyond the narrow classic concept that the mechanisms of PCD are activated exclusively ‘downstream’, following cellular injury. Studies in the genetics of PD suggest that in some forms of the disease, activation may be an early ‘upstream’ event. Additionally, recent observations suggest that cell death in some contexts may not be initiated by injury, but instead by a failure of intrinsic cell survival signalling. These new points of view offer new opportunities for molecular targeting. Keywords: Apoptosis; Programmed cell death; LRRK2; DJ-1; Akt; PTEN; GDNF

The concept expressed in the title, that there are cell-autonomous molecular pathways intrinsic to neurons that, if activated, will lead to their destruction, is a relatively recent one in the molecular era of Parkinson’s disease (PD) research. This era can perhaps best be said to have begun just over 50 years ago when Carlsson and his colleagues (1957)

first reported the ability of DOPA to reverse the akinetic effect of reserpine. The birth of the concept of programmed cell death (PCD) as a cell­ autonomous, ordered molecular pathway of death is more difficult to place in time, because early descriptions of developmental cell death appeared many decades ago (Glucksmann, 1951). However, the molecular basis of these death events was not known until the landmark studies of H. Robert Horvitz and his colleagues in the late 1980s and early 1990s (Ellis et al., 1991). There soon followed




Corresponding author. Tel.: þ1-212-305-7374; Fax: þ1-212-305-5450; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83005-5

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an explosion of research on the molecular path­ ways of PCD (Johnson and Deckwerth, 1993; Raff et al., 1993; Thompson, 1995), and although debate may continue today about the precise role of PCD in PD, there appears to be an emerging consensus over the past 5–10 years that it is quite likely to be an important component of the degen­ erative process (reviewed in Burke (2008). Although the concept of PCD is a relatively recent one, there is nevertheless some basis for concern that new and future generations of neu­ roscientists may take the concept for granted and not realize what a paradigm shift it created in thinking about therapeutic approaches to neuro­ protection. Before the concept of PCD was accepted, it was deemed essential to identify the primary cause of PD, whether it might be an environmental toxin or other agent, before inter­ ventional steps could be envisioned. However, with the emergence of the concept of PCD, it was realized that even in the absence of knowl­ edge of primary causes (as remains the case today for sporadic PD), effective therapeutic interven­ tion, based on blockade of PCD, may be possible. Since the groundbreaking work of Horvitz and colleagues, the field of cell death research has amassed an enormous amount of highly detailed and complex information about the many, diverse pathways of PCD. One of the purposes of this review is to take stock of the progress that has been made and to evaluate our prospects for fulfilling the origi­ nal promise of the PCD concept, to achieve diseasealtering therapies for PD. Clearly, we have not yet achieved this promise, and one of our goals is to ask what impediments remain, and what we have learned that will guide our future efforts. Another goal of this review is to assess how ideas about the relationship between PCD and the degenerative process of PD have evolved over recent years. The original concept was that the primary, unknown cause, or causes, of PD would induce cellular dysfunction that at some point would trigger the mechanisms of PCD, which would then operate ‘downstream’ to bring about

the demise of the neuron. While this simple ‘sequential’ model may yet hold true, ideas about the relationships between early neuron dysfunction and PCD in PD have evolved to suggest the possi­ bility of a more ‘upstream’ role. This possibility is suggested by a number of new genetic discoveries in PD that indicate that disease-causing mutations may act by disrupting normal cellular regulation of PCD. Another new point of view is that rather than death pathways being activated secondary to some form of cellular injury, they instead may be the cellular ‘default mode’ and become activated due to the failure of normal suppression by survival signalling. Such a possibility is suggested by some of the gene discoveries, by a number of recent observations on the mechanisms of action of some neurotoxins and by some of the effects described following neurotrophic deprivation in mature mice.

PCD in dopamine neurons: major pathways of neuron destruction As we have stated, enormous growth in our knowledge of pathways of PCD has shown that they are numerous, diverse and complexly inter­ related (Burke, 2008). Adding to this complexity at the molecular level is that the utilization of these diverse paths can change depending on the cellular context in the same cell. For example, within post-mitotic dopamine neurons of the sub­ stantia nigra pars compacta (SNpc), while endo­ plasmic reticulum stress-related induction of PCD plays an essential role in neurotoxin-induced PCD, it does not in naturally occurring cell death (Silva et al., 2005b). Given the breadth and com­ plexity of the topic, this overview will necessarily be selective, and we have chosen to highlight only two key pathways of death that are especially well substantiated by a diverse and extensive array of experimental observations. We will limit this review to cell death mediators that are cell autonomous to dopaminergic neurons. The role of

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non-cell-autonomous mediators will be taken up by Dr. Etienne Hirsch in this volume.

Cyclin-dependent kinase-5 and myocyte enhancer factor 2 transcription factors Cyclin-dependent kinase 5 (Cdk5) is a serine/threo­ nine protein kinase that is structurally related to other Cdks known for their role in regulating the cell cycle (Lew and Wang, 1995; Lew et al., 1992; Meyerson et al., 1992). Unlike these other kinases, however, Cdk5, which is predominantly expressed in neurons (Meyerson et al., 1992), is most highly expressed after mitosis is complete (Tsai et al., 1993). In addition, unlike other Cdks, Cdk5 is not dependent on association with cyclins for activation; instead, it is activated by a brain-specific proteins, p35 (Lew et al., 1994; Tsai et al., 1994) and p39 (Tang et al., 1995). It has been suggested that Cdk5 and p35 play a role in neural differentiation because targeted disruption of the genes results in abnormal develop­ ment (Chae et al., 1997; Ohshima et al., 1996). In addition to this role in neural development, Cdk5 has been implicated in apoptosis (Ahuja et al., 1997; Zhang et al., 1997). In brain, we observed high levels of Cdk5 expression in apoptotic profiles in naturally occurring cell death in the substantia nigra (SN) (Henchcliffe and Burke, 1997). We subse­ quently found that both Cdk5 and p35 are generally expressed in neuronal apoptosis in the SN, induced by diverse forms of injury (Neystat et al., 2001). In all of these contexts, Cdk5 protein was identified within the nucleus. Smith and colleagues (2003) provided evidence of a functional role for Cdk5 in mediating death of dopamine neurons in the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyri­ dine (MPTP) model (Smith et al., 2003). The use of the chronic MPTP model in this investigation is notable and important, because clear morphologic evidence of apoptosis is observed (Tatton and Kish, 1997), unlike the widely used acute model, where apoptosis does not occur (Jackson-Lewis et al., 1995). In the chronic model, MPTP induces an

increase in the activity of Cdk5, and toxicity is reduced by Cdk5 blockade, either by pharmacolo­ gic inhibition or by use of viral vector-mediated expression of a dominant-negative mutant. Mechanistic insight into the role of Cdk5 in med­ iating neurotoxin-induced PCD was first provided by Gong and colleagues (2003), who observed that Cdk5 localized to the nucleus phosphorylates and thereby decreases the activity of the pro-survival transcription factor, myocyte enhancer factor 2 (MEF2). MEF2 was first identified as a regulator of muscle gene expression (Potthoff and Olson, 2007), but subsequent work identified more widespread cellular expression and roles in diverse cellular func­ tions, including proliferation, morphogenesis, differ­ entiation and cell survival. MEF2 proteins (four isoforms have been identified) are expressed in brain, and one of the first activities of MEF2 to be identified in neurons was a role as a mediator of survival due to calcium influx induced by neuronal activity (Mao et al., 1999). In a model of glutamateinduced neurotoxicity, Tang and colleagues (2005) observed that injury was associated with diminished protein levels of MEF2, due to caspase cleavage which, in turn, was dependent on MEF2 phosphor­ ylation by Cdk5. Blockade of Cdk5 activity by means of pharmacologic inhibition or by transduction with a dominant-negative Cdk5 mutant afforded neuropro­ tection from both glutamate and hydrogen peroxide­ induced neurotoxicity (Tang et al., 2005). Closely related observations have been made in the chronic MPTP model. Crocker and colleagues (Crocker et al., 2003) had previously shown that in this model MPTP induces activation of the cal­ cium-dependent proteases, the calpains. In a sub­ sequent investigation, this group determined that activation of the calpains induces cleavage of the Cdk5-interacting protein p35 to its more active, cleaved form, p25 (Smith et al., 2006). The impor­ tance of this activation was determined by inves­ tigations of p35 null mice, which revealed a marked resistance to MPTP-induced dopamine neuron loss. As previously demonstrated by Tang and colleagues (2005) for glutamate toxicity,

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Smith and associates (2006) demonstrated that MPTP, by activation of Cdk5, induced the phos­ phorylation and subsequent inactivation of MEF2. Blockade of MEF2 phosphorylation occurred in p35 null mice, and neuroprotection was afforded by transduction of SN dopamine neurons with a phosphorylation-resistant mutant form of MEF2. Thus, these investigators have revealed an important serves of molecular events involving cal­ pain and then Cdk5 activation, leading to MEF2 phosphorylation and inactivation by caspase clea­ vage. It is important to note, however, that although these molecular processes can be success­ fully blocked, with protection of neuron cell bodies, such blockade does not afford protection of axons. Neither Cdk5 blockade (Smith et al., 2003) nor calpain inhibition (Crocker et al., 2003) provided protection at the striatal axon level. This inability of approaches based on blockade of apoptosis to pro­ vide protection at the axon level is a recurring theme, as discussed further below.

The mixed lineage kinase -c-jun N-terminal kinase signalling cascade The earliest evidence that the mitogen-activated protein kinase (MAPK) cascade plays an impor­ tant role in PCD in neurons derived from studies in vitro. These studies have previously been reviewed (Silva et al., 2005a), so it will suffice to mention only the critical highlights. Early investi­ gations demonstrated upregulation of c-jun pro­ tein (Ham et al., 1995) and mRNA (Estus et al., 1994) in sympathetic neurons following nerve growth factor (NGF) withdrawal. Treatment of these neurons with a dominant-negative form of c-jun protected them from NGF withdrawalinduced cell death, whereas over-expression of wild-type c-jun protein resulted in significant induction of apoptosis even in the presence of NGF (Ham et al., 1995). Similarly, microinjection of neutralizing antibodies for c-jun protein signifi­ cantly reduced neuronal death following NGF withdrawal (Estus et al., 1994).

The importance of these early findings has been confirmed by numerous observations in the in vivo context. The earliest studies specifically within the SN in models of death induced by 6-hydroxydopamine (6OHDA) (Jenkins et al., 1993) and by axotomy (Leah et al., 1993) noted substantial and sustained increases in c-jun expres­ sion, but these changes were interpreted largely in relation to a possible role in regenerative responses. With subsequent increased awareness of apoptosis as a distinct morphology of PCD (Kerr et al., 1995), it became clear that c-jun expression could be correlated at the cellular level with this form of cell death in living animals. This was true in the context of natural cell death and induced death in the central nervous system (Ferrer et al., 1996a; 1996b). Oo et al. (1999) demonstrated in a post-natal model of apoptosis in the SNpc, induced by early target deprivation, that c-jun and c-jun N-terminal kinase (JNK) expression could be correlated at a cellular level with apoptotic morphology. Thus, these morpho­ logic studies of apoptotic cell death suggested a clear correlation with c-jun expression. The first principal evidence for a functional role for JNK/c-jun signalling in cell death in living animals derived from studies in JNK null animals. Yang and co-workers (1997b) showed that JNK3 null mice are resistant to kainic acid-induced hip­ pocampal neuron apoptosis. While this study demonstrated a clear role for this JNK isoform in mediating cell death, it remained an open question whether c-jun itself was the relevant substrate for this effect, as other substrates exist. To address this issue, Behrens and colleagues (1999) created mice by homologous recombination in which the endogenous c-jun gene was replaced by an altered gene in which the serines at positions 63 and 73 were replaced by non-phosphorylatable alanines. Mice homozygous for this mutant form of c-jun were also resistant to apoptosis. Thus, the phosphorylation of c-jun by JNK appears to be necessary for apoptosis in this model. A functional role for c-jun in mediating death specifically within dopamine neurons has been

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supported by many studies. Crocker et al. (2001) have demonstrated in an axotomy model that ade­ novirus-mediated expression of a c-jun dominantnegative construct prevents the loss of dopamine neurons. A functional role for JNK/c-jun signal­ ling is also supported by the demonstration that gene transfer of the JNK binding domain of JIP-1 (which inhibits JNK activation) protects dopamine neurons from chronic MPTP toxicity (Xia et al., 2001). In view of this evidence that phosphoryla­ tion of c-jun plays a role, and given that JNK is the dominant kinase for c-jun (Kyriakis and Avruch, 2001), it would be predicted that JNK isoforms would also play a role in the death of these neu­ rons. Hunot and co-investigators have shown in a model of acute MPTP toxicity that both JNK2 and JNK3 homozygous null animals are resistant; and compound mutant JNK2 and JNK3 homozygous nulls were even more protected (Hunot et al., 2004). These results, however, are difficult to interpret in specific relation to PCD, because, as mentioned earlier, apoptosis does not occur in the acute MPTP model (Jackson-Lewis et al., 1995). In an alternate model of neurotoxin-mediated degeneration of dopamine neurons, that induced by intra-striatal injection of 6OHDA (Sauer and Oertel, 1994), in which definitive evidence of apop­ tosis has been observed (Marti et al., 2002), clear protective effects of inhibition of the MAPK signal­ ling cascade have been observed. Chen and collea­ gues demonstrated that transduction of dopamine neurons of the SN by use of adeno-associated virus (AAV) vectors to express dominant-negative forms of the dual leucine zipper kinases (DLKs) provides a degree of protection of that correlates precisely with the efficiency of transduction and the degree of inhibition of c-jun phosphorylation (Chen et al., 2008). In this investigation, although there was remarkable preservation of dopamine neuron cell bodies, there was no protection of their axon projections. In view of the extensive evidence implicating the MAPK cascade in mediat­ ing PCD in dopamine neurons, and yet the impor­ tant role also of other death signalling pathways, such as the calpain-Cdk5-MEF2 pathway outlined

above, the question arises as to whether phosphor­ ylation of c-jun by the JNKs is essential for death to occur. To address this issue, we examined the effect of single or combined JNK2 and JNK3 null muta­ tions on apoptotic death in the 6OHDA model. We observed that while single null mutations substan­ tially inhibit apoptosis in the acute period following toxin injury, neither provides a lasting protection of dopamine neurons; at 4 weeks following injury, there is no detectable neuroprotection (Ries et al., 2008). However, combined homozygous null muta­ tion of JNK2 and JNK3 completely abrogated apoptotic neuron death in this model and provided a virtually complete protection at 4 weeks after lesion. In marked contrast to this striking protec­ tion at the level of dopamine neuron cell bodies, there was no protection of their axonal projections (Fig. 1). Thus, a recurring theme in experimental studies of neuroprotection in animal models of dopami­ nergic neurodegeneration is that while many approaches based on blockade of PCD have pro­ vided robust protection at the cell body level in diverse models, most have fallen short in their ability to protect dopaminergic axonal projections.

Intracellular signalling pathways for dopaminergic axonal degeneration These observations, that successful protection of cell bodies by blockade of PCD does not also provide protection of axons, should come as no surprise. The concept that important mediators of PCD, such as the caspases, do not play a role in axon degeneration received considerable support from investigations by Finn and colleagues (2000) who noted that caspase-3 is not activated in a variety of models of axon degeneration. These negative results are not universal, because in other contexts, particularly developmental con­ texts, a role for caspases has been identified (Buki et al., 2000; Cowan et al., 2001; El-Khodor and Burke, 2002; Nikolaev et al., 2009; Srinivasan et al., 1998). Nevertheless, it remains true that

84 (a)

10 6.OHDA

4 JNK2/3 Null

Apoptosis in SNpc

5 μm

6

Control

Wildtype

(b)

8

2 0 WT

JNK2

JNK3 JNK2/3

(c)

EXP

JNK2/3 Null

Wildtype

CON

Anterior Fig. 1. Resistance of neuron cell bodies, but not axons, to degeneration in JNK null mice. (a) The intra-striatal 6OHDA neurotoxin model induces apoptosis in SN dopamine neurons in wild-type mice. A typical example of an apoptotic profile, with characteristic chromatin clumps, is demonstrated by thionin counterstain in the inset. The homozygous jnk2 and jnk3 single null mutations suppressed apoptosis by 95 and 98%, respectively, and the homozygous jnk2/3 double null mutations completely abrogated apoptosis. (b) The homozygous jnk2/3 double null mutations provided virtually complete protection of SN dopamine neurons. Among wild-type (WT) mice, there was a 63% loss of dopamine neurons, typical for this model, whereas among jnk2/3 nulls, there was only a 4% decrease. Low-power photomicrographs of representative TH-immunostained SN sections from wild-type (top) and jnk2/3 nulls (bottom) following unilateral 6OHDA injection. (c) Homozygous jnk2/3 double null mice are not resistant to retrograde degeneration of nigrostriatal dopaminergic axons induced by intra-striatal 6OHDA. Following 6OHDA, there is a virtually complete loss of TH-positive fibre staining in homozygous jnk2/3 null mice, as in wild-type, throughout the striatum.

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experimental measures intended to block apopto­ sis in adult models of neuron degeneration often prevent cell body but not axonal degeneration, not only in various neurotoxin models of parkinson­ ism, as we have described (Chen et al., 2008; Eberhardt et al., 2000; Hayley et al., 2004; Ries et al., 2008; Silva et al., 2005b), but also in a genetic model of motor neuron disease (Sagot et al., 1995).

Axon degeneration and PD This recognition of the distinction between the canonical pathways of PCD and the molecular pathways of axonal destruction, which remain largely unknown, has important implications for neuroprotective therapeutics for PD. We have recently reviewed the evidence to suggest that neurodegeneration in PD begins not in the cell soma, but in the axons (Cheng et al., 2010). Briefly, there is compelling and consistent evi­ dence that at the time of first appearance of motor signs, there is about a 30% loss of total SN neurons in comparison to age-matched con­ trols. On the other hand, multiple lines of evi­ dence suggest that, at first appearance of motor signs, there has been a 50–60% loss of striatal dopaminergic terminals. This assessment is in keeping with observations that, at the time of death, depending on disease duration, while there has been 60–80% loss of SN dopamine neurons (Fearnley and Lees, 1991; Pakkenberg et al., 1991), there has been a much more pro­ found loss of striatal dopaminergic markers (Bernheimer et al., 1973; Kish et al., 1988; Scherman et al., 1989). The concept that axonal involvement is an early feature of PD is also supported by recent observa­ tions made in a bacterial artificial chromosome (BAC) transgenic model based on the expression of hLRRK2(R1441G) (Li et al., 2009). These mice show the development of an age-related hypokine­ sia by 9–10 months that is reversible by treatment with levodopa. There is no loss of mesencephalic

dopamine neurons, but pathology is observed in dopaminergic axons. On tyrosine hydroxylase (TH) immunostaining, the axons are fragmented, they are associated with axonal spheroids, and they form dystrophic neurites (Li et al., 2009). These abnormal axonal features are also observed by staining for abnormally phosphorylated tau. These observations are in keeping with evidence that LRRK2 plays an important role in the regulation of neurite growth and integrity. MacLeod and col­ leagues (2006) have reported that mutant forms of LRRK2 induce decreases of neurite length in pri­ mary neuron culture. Similar observations were made for the LRRK2(G2019S) mutant in neuronally differentiated neuroblastoma cells (Plowey et al., 2008) and in primary neurons derived from transgenic mice (Parisiadou et al., 2009). The mole­ cular basis of these effects is not known, but of potential interest in this regard is the identification of moesin, and the closely allied proteins ezrin and radixin, as possible LRRK2 substrates (Jaleel et al., 2007). These proteins have been implicated in the regulation of neurite outgrowth (Paglini et al., 1998). The ability of LRRK2 to regulate the phos­ phorylation status of these proteins, and the closely correlated degree of neurite growth, has been observed in primary cultures (Parisiadou et al., 2009). Thus, based on analysis of the predominant site of pathology in PD at its onset, and evidence from autosomal dominant genetic forms of the disease, it is reasonable to hypothesize that axon dysfunction may be an early feature of PD. Such a possibility may be relevant to understanding why, to date, clinical trials of anti-apoptotic approaches in PD have failed. This point can perhaps be illustrated by consideration of the failure of the PRECEPT neuroprotection trial in PD (The Parkinson Study Group PRECEPT Investigators, 2007). This trial examined the ability of a mixed lineage kinase inhi­ bitor, CEP-1347, to forestall disease progression in early PD. The rationale for the trial was that block­ ade of the MAPK signalling pathway by a variety of means, including administration of CEP-1347, had been shown to block apoptosis and provide

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neuroprotection in a variety of PD models (reviewed in Silva et al., 2005a). While there are of course many possible reasons why the trial failed, as previously reviewed (Waldmeier et al., 2006), an important possibility is that although blockade of MAPK signalling blocks apoptosis, it does not pro­ tect axons in the mature brain (Chen et al., 2008; Ries et al., 2008). If the brunt of the pathology is at the level of the axons and their terminals through­ out the course of the disease, and given also that the terminals are, of course, the principal site of dopa­ mine release, it would follow that it is the progres­ sive degeneration of axons and their terminals, and not neuron loss, that is the primary determinant of clinical progression. If such is the case, then block­ ade of apoptosis cannot be expected to forestall the clinical progression of the disease.

Inhibition of axon degeneration: is it feasible? Unlike the remarkably detailed knowledge that we have acquired of the many molecular pathways of PCD, we know exceedingly little of the mechanisms of axon degeneration. In the face of so little information, what hope do we have that we will be able to define these pathways and ultimately provide neuroprotection, just as we have been able to achieve for the cell soma? There is much work to be done, but the feasibility of this goal is suggested by observations in the unique Wallerian degeneration slow (WldS) mutant mouse. The most striking evidence that axons can sur­ vive irrespective of destruction of the neuronal soma derives from observations made in the WldS mouse (Coleman and Perry, 2002). This mutation arose spontaneously in C57Bl/6 mice, and it was demonstrated to cause delayed Waller­ ian degeneration in peripheral nerve after axot­ omy (Lunn et al., 1989). The mutation was identified as an 85-kb tandem triplication that results in a novel chimeric mRNA that encodes for the N-terminal 70 amino acids of ubiquitina­ tion factor E4B (Ube4B), followed by the

complete coding sequence for the nicotinamide adenine dinucleotide (NAD) synthesizing enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT) (Conforti et al., 2000; Mack et al., 2001). It was shown by Deckwerth and Johnson (1994) that axons of sympathetic ganglion neurons derived from WldS mice survive following with­ drawal of NGF in spite of induction of apoptosis in the cell soma. The WldS mutation protects axons of many different types of neurons, in diverse species, from a wide variety of injuries, including toxic peripheral neuropathies (Wang et al., 2001) and genetic neuropathies [Mi et al. (2005) and see Coleman (2005) and Luo and O’Leary (2005) for reviews]. These observations suggest that with a deeper understanding of the mechanisms underlying the WldS phenotype, it may be possible to target the molecular pathways of axon degeneration with therapeutic benefit. It is now clear that enzymatic activity of NMNAT is necessary, but not suffi­ cient, for axon protection (Araki et al., 2004; Conforti et al., 2009; MacDonald et al., 2006; Sasaki et al., 2009). In addition to its enzymatic activity, NMNAT appears to require correct cel­ lular targeting. Interestingly, the full protection phenotype can be observed in experiments with NMNAT3, a mitochondrially targeted isoform (Avery et al., 2009; Sasaki et al., 2006; Yahata et al., 2009). The possibility that the axon protection pro­ vided by WldS may be effective within the nigros­ triatal dopaminergic system is supported by observations made in neurotoxin models of Par­ kinsonism. The WldS mutation protects dopami­ nergic axons, but not cell bodies, from medial forebrain bundle injection of 6OHDA (Sajadi et al., 2004) and injection of MPTP (Hasbani and O’Malley, 2006). We have characterized the WldS phenotype in four models of nigrostriatal axon injury: two that utilize 6OHDA or axotomy to induce anterograde degeneration and two that use these methods to induce retrograde degenera­ tion. Our observations confirm the promise of exploiting WldS -related mechanisms for axon

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protection, but they also present a new layer of complexity. We find that for both 6OHDA and axotomy, WldS provides protection from antero­ grade, but not retrograde degeneration (Cheng and Burke, 2010). We observe this protection as preserved immunostaining for TH in axons and striatum, and by structural integrity visualized by green fluorescent protein (GFP) in TH-GFP mice. Therefore, while WldS offers the promise of an approach to axon protection, it reveals fundamen­ tally different processes underlying antero- and retrograde degeneration in this system, and the need to explore these mechanisms more deeply.

PCD in dopamine neurons: is it only downstream? The prevailing concept of the role for PCD in PD has been that its mediators are ‘downstream’ effectors of more proximate and specific causes related to genetic or environmental factors. How­ ever, recent studies of four genes that cause inher­ ited forms of Parkinsonism suggest that there may be more intimate and upstream relationships with the mediators of PCD. Mutations in three of these genes, parkin, PINK1, and DJ-1, cause autosomal recessive forms of Parkinsonism, and several lines of evidence suggest that the disease-causing lossof-function mutations may result in an increased propensity for neurons to die. Mutations in a fourth gene, LRRK2, cause autosomal dominant forms of Parkinsonism, and recent work indicates that they may lead to activation of the extrinsic pathway of PCD. Much of this evidence has recently been reviewed (Burke, 2008), so for our purposes here, we will highlight only two exam­ ples of these recent developments. Mutations in the gene for DJ-1 (PARK7) cause an autosomal recessive early-onset form of famil­ ial PD. Bonifati and colleagues first localized the gene for PARK7 in families from Italy and the Netherlands to chromosome 1p36 (Bonifati et al., 2003). They subsequently determined that in the Dutch family a deletion mutation affects the coding region of DJ-1, and in the Italian

family a L166P mutation is present and likely to result in loss of function (Bonifati et al., 2003). Human DJ-1 was first identified as an oncogene (Nagakubo et al., 1997), and later determined to be H2O2-responsive, suggesting that it may function as an antioxidant protein (Mitsumoto and Nakagawa, 2001). Studies in vivo have suggested that one function of DJ-1 may be to negatively regulate apoptotic pathways. In a Drosophila model, Yang and col­ leagues (2005) have shown that knockdown of the Drosophila DJ-1 homologue in dopaminergic neurons by a transgenic RNAi approach results in a progressive decline in their number and diminished dopamine content. As would be pre­ dicted from a number of in vitro studies, DJ-1 knockdown in neurons resulted in increased sensitivity to oxidative stress due to H2O2 exposure. These investigators determined that the neurodegeneration phenotype induced by RNAi knockdown of DJ-1 could be suppressed by co-expression of phosphatidylinositol-3-kinase (PI3K), an upstream mediator of Akt kinase sig­ nalling. Conversely, the degenerative phenotype was exacerbated by co-expression of PTEN, a negative regulator of PI3K/Akt signalling. These results are complimented by those of Kim et al. (2005), who demonstrated, also in Drosophila, that DJ-1 serves as a suppresser of PTEN. They also demonstrated in mammalian cells that increased expression of DJ-1 results in increased phosphorylation and activation of Akt, with enhanced cell survival (Kim et al., 2005). Thus, converging lines of evidence suggest that DJ-1 positively regulates the anti-apoptotic Akt kinase pathway. The mechanism by which DJ-1 may achieve regulation of the Akt signalling pathway is unknown. Clements et al. (2006) have shown that DJ-1 stabilizes Nrf2 protein, a key regulator of antioxidant responses. Protein stabilization is achieved by the ability of DJ-1 to prevent the association of a cytosolic inhibitor, Keap1, with Nrf2, thereby preventing Nrf2 ubiquitination and degradation via the proteasome pathway. The

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(a) Extracellular Fas

N

Intracellular

CRD1 CRD2 CRD3

TM

death domain

C

Ligand Binding

(b) Fasl

DD

FADD

DD

Fas death domain

precise mechanism by which DJ-1 prevents Keap1 association with Nrf2, and how, in turn, this effect may be related to activation of the Akt pathway, if at all, is unknown. An alternative possible mechanism by which DJ-1 may regulate PI3K/ Akt signalling is suggested by the observations of van der Brug and colleagues, who identified DJ-1 as an RNA-binding protein (van der Brug et al., 2008). One of the classes of RNAs bound by DJ-1 was members of the PI3K/Akt cascade. Another example of the close, upstream rela­ tionship between a disease-causing gene and the pathways of PCD is the recent demonstration by Ho and colleagues (2009) that LRRK2 interacts with mediators of the extrinsic pathway of PCD. The molecular features of cell death signalling through the extrinsic pathway are less widely stu­ died among neuroscientists than those of the intrinsic pathway, so we will briefly review some of the highlights. PCD is initiated through the extrinsic pathway by the binding of extracellular ligands to receptors (‘death receptors’) which belong to the tumour necrosis factor (TNF) super­ family (Locksley et al., 2001). Members of this family are characterized by a homotrimeric struc­ ture and extracellular cysteine-rich domains that interact with ligands that also form homotrimeric assemblies (Fig. 2). For the purposes of this review, we will restrict our attention to a single member of the TNF superfamily: Fas (also known as Apo-1 or CD95). Fas, like other members of the TNF superfamily, is characterized by the pre­ sence of intracellular death domains (DDs), which mediate homotypic interactions with other pro­ teins containing DDs, which, in turn, mediate PCD signalling (Fig. 2). Following the binding of the Fas ligand (FasL) to the Fas receptor, signal­ ling is mediated by a variety of pathways (Choi and Benveniste, 2004), but two in particular have been implicated in cell death. In the first, a homo­ typic interaction occurs between the intracellular DD of Fas and the DD of FADD (Fas-associated protein with DD). FADD contains at its N-termi­ nus a death effector domain (DED) (Tibbetts et al., 2003), which mediates an interaction with

or

Daxx

Inactive caspase-8

ASK-1

Bid tBid

Active caspase-8

JNK

Fig. 2. Mediation of the extrinsic pathway of PCD by Fas. (a) The protein domain structure of Fas. The receptor has cysteinerich domains (CRD) in the ligand binding region, characteristic of members of the TNF superfamily of receptors. There is a DD in the intracellular portion of the receptor. (b) Fas death signalling pathways. See text for details.

a similar DED in pro-caspase-8 (Fig. 2). This interaction permits auto-cleavage, and activation, of caspase-8 by induced proximity. This complex, comprised of ligand, receptor, FADD and pro­ caspase-8 is referred to as the death-inducing sig­ nalling complex (DISC) (Wajant, 2003). The clea­ vage and activation of caspase-8 mediates death by two distinct mechanisms. In some cells, the induced caspase-8 activation is sufficient to med­ iate cleavage of downstream caspases, such as caspase-3, leading to cell death (Fig. 2). In other cells caspase-8 cleaves a BH3 domain-only mem­ ber of the Bcl-2 family, Bid. This truncated form (tBid) can induce mitochondrial release of cyto­ chrome c and other mitochondrial mediators of cell death (Fig. 2). An alternate mechanism for

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mediation of cell death by Fas is initiated by an interaction between the DD of Fas and that of the protein Daxx (Yang et al., 1997a). Unlike FADD, Daxx does not contain a DED. This interaction between Fas and Daxx results in the activation of apoptosis signalling kinase-1, a MAP kinase kinase (Ichijo et al., 1997), which, in turn, activates JNK. Daxx has been suggested to interact with DJ-1 (Junn et al., 2005). Ho and colleagues have demonstrated that LRRK2 protein interacts with FADD and that disease-causing mutations enhance this interac­ tion. The functional significance of this interaction was demonstrated by the ability of a dominantnegative form of FADD to block LRRK2-induced cell death. Further exploration of relationships with other members of this death pathway revealed that in the presence of FADD, LRRK2 interacts with caspase-8, and siRNA-based reduc­ tion of caspase-8 diminished LRRK2-induced cell death. The disease relevance of these observations was demonstrated by the finding of a cleaved, activated form of caspase-8 in brains of patients with PD due to the LRRK2 mutations.

PCD in dopamine neurons: is it the default mode? Another aspect of the classic concept of the rela­ tionship between neurodegeneration in PD and the processes of PCD that is currently being revised is the notion that initiation of PCD in the disease always requires an initial cellular injury. The hypothesis outlined above for DJ-1, that it normally serves as a positive regulator of PI3K/ Akt signalling, suggests an alternate scenario, that even in the absence of cellular injury, if survival signalling pathways fail in the mature brain, then the processes of PCD become activated. This con­ cept of an ongoing balance within the cell between the processes of cell death and those of cell survi­ val is widely accepted in the context of neuron development. According to classic neurotrophic theory, an immature neuron must achieve target contact and thereby attain neurotrophic support

(Oppenheim, 1991). If it fails, survival signalling is not maintained, and it undergoes PCD. However, once target contact is achieved, and the neuron safely survives the developmental cell death per­ iod, whether or not it remains dependent on tar­ get-derived or alternate sources of trophic support has been unknown. For SN dopamine neurons, developmental target dependence lasts only 2 weeks (Kelly and Burke, 1996). Numerous studies have shown that following extensive striatal target lesion in adulthood, there is no loss of SN dopa­ mine neurons (Krammer, 1980; Lundberg et al., 1994; Stefanis and Burke, 1996). In spite of this lack of evidence for a need for target-derived support for mature SN dopamine neurons, several recent studies have clearly demonstrated that these neurons continue to depend on neurotrophic support, and, presum­ ably, survival signalling, in the mature brain. Pascual and colleagues (2008) have shown that following conditional deletion of glial cell linederived neurotrophic factor (GDNF) in mature mice, there is a progressive loss of SN dopamine neurons, such that by 7 months after the deletion, there is a 70% reduction in TH-positive neurons. A similar observation was made by Kramer and colleagues following conditional deletion of the Ret tyrosine kinase in dopamine neurons during development. While the deletion had no effect on the post-natal number of dopamine neurons, it resulted in a 38% loss of dopamine neurons by 2 years of age. This neuron loss was accompanied by loss of striatal dopaminergic innervation and gliosis, particularly in the dorso-lateral quadrant (Kramer et al., 2007). Similar observations have been made for the orphan nuclear receptor Nurr1. Kadkhodaei and colleagues (2009) have shown that conditional deletion of Nurr1 in SN dopamine neurons in mature mice induces a 50% loss of THpositive neuronal profiles and decreased immu­ noreactivity in the striatum . In the absence of signs of neuronal degeneration, this loss was attributed to a partial loss of phenotype. The observations of Pascual et al. (2008) and Kramer et al. (2007) strongly suggest that survival

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signalling pathways that are activated by GDNF are likely to play a role in maintaining the viability of mature dopamine neurons. The signalling pathways utilized by GDNF to support neuron survival are diverse and complex, so this brief overview will touch only upon the highlights. Although the best characterized pathways are those that are activated following GDNF–GFRa1 activation of the Ret tyr­ osine kinase (Fig. 3) [reviewed in Airaksinen and Saarma (2002)], there is considerable evidence that in association with cell membrane lipid rafts, GDNF–GFRa1 may also signal independently of Ret, by activation of Src-family kinases, with result­ ing activation of cyclic-AMP-response element binding protein (CREB) (Poteryaev et al., 1999; Trupp et al., 1999). Nevertheless, the greater abundance of evidence implicates Ret-dependent NON-RET

RET GDNF

GDNF GFRα1

raft

c-Ret GFRα1

P

SRC family

kinase

PLCγ

SHC

PI3K

CREB RAS

AKT

ERK Fig. 3. GDNF signalling pathways. In some cellular contexts, GDNF signals independently of RET via interactions in cell membrane rafts with as yet unknown transmembrane proteins, leading to activation of Src-family kinases [see Trupp et al. (1999) and reviewed in Airaksinen and Saarma (2002)]. More often, GDNF signals through GFRa1 interactions with Ret. These interactions result in signalling through several candidate pathways, including PLCg, Ras-ERKs and PI3K­ Akt [modified from Trupp et al. (1999)].

signalling, and a variety of downstream pathways, including PLCg (Trupp et al., 1999) and MEK-ERK1/2 (Cavanaugh et al., 2006; Ugarte et al., 2003), have been implicated in survival signal­ ling (Fig. 3). The most abundant evidence has iden­ tified Ret-dependent PI3K/Akt signalling as having a central role in mediating GDNF neuron survival responses (Besset et al., 2000; Chen et al., 2001; Coulpier and Ibanez, 2004; De Vita et al., 2000; Encinas et al., 2001; Perez-Garcia et al., 2004; Pong et al., 1998; Sawada et al., 2000; Soler et al., 1999; Ugarte et al., 2003). A number of these investiga­ tions, while finding evidence for a role for PI3K/Akt in mediating survival, failed to find such evidence for ERK1/2 (Besset et al., 2000; Chen et al., 2001; Soler et al., 1999). Especially interesting, in a study of survival signalling mediated at the axon terminal and by retrograde axon transport found that Akt, but not ERK1/2, played a role (Coulpier and Iba­ nez, 2004). Nevertheless, PI3K/Akt signalling does not account for all instances of GDNF-mediated survival, and it is clear that cellular context is impor­ tant. In post-natal mouse sympathetic neurons, GDNF-mediated, Ret-dependent survival requires inhibitor of kappa B kinase rather than PI3K (Encinas et al., 2008). Remarkably little is known about the activity of survival signalling pathways in SN dopamine neu­ rons in vivo. We have shown that a constitutively active form of Akt has marked neurotrophic effects, including an increase in neuron size, expres­ sion of phenotypic markers and sprouting, in both mature and aged mice (Ries et al., 2006). In addi­ tion, Akt is able to provide marked neuroprotec­ tion against 6OHDA-induced apoptosis. However, less is known about the normal physiologic role played by endogenous Akt. During post-natal development, it clearly regulates the magnitude of the developmental cell death event. Transduction of post-natal SN dopamine neurons with a consti­ tutively active form increases the number of these neurons that survive developmental cell death (Ries et al., 2009). Conversely, transduction of these neurons with a dominant-negative form results in an induction of apoptosis and a diminished

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number of neurons that survive into adulthood. However, whether Akt plays a role in maintenance of survival in the adult setting is unknown. The possibility that Akt may play such a role is suggested by the recent findings of Greene and colleagues on the mechanism of action of dopami­ nergic neurotoxins. In a serial analysis of gene expression screen of genes upregulated by 6OHDA in a cellular model, they identified RTP801 (REDD1), a gene implicated in apoptosis in neurons (Malagelada et al., 2006). They deter­ mined that RTP801 is induced in the MPTP model and in PD post-mortem SN. The functional signif­ icance of its expression in cellular models was demonstrated by an ability of a shRNA approach to provide neuroprotection. Their investigations further showed that RTP801 acts by a general suppression of the mTor kinase, which, in turn, results in dephosphorylation of Akt (Malagelada et al., 2008). The disease relevance of this effect was suggested by their finding that phospho-Akt, but not total Akt, is diminished in PD brain (Mala­ gelada et al., 2008). More recently they have shown that partial and selective suppression of mTor, paradoxically, is protective in both in vitro and in vivo models of neurotoxicity for the reason that it prevents induction of RTP801. This inhibi­ tion of RTP801 provides protection by preventing the general inhibition of mTor activity that results in Akt dephosphorylation (Malagelada et al., 2010). The disease relevance of these observations is supported by the recent demonstration that phospho-Akt is normally expressed at high levels in dopamine neurons of the SN, and that it is diminished in PD brain (Timmons et al., 2009). Collectively, these results suggest that an early molecular event in PD may be the loss of survival signalling provided by phosphorylated Akt.

therapies for neurodegenerative disorders like PD has remained. The relevance of the concept of PCD to the pathogenesis and treatment of these disorders has not only stood the test of time, it has evolved and generalized. Just as we have been able to define the canonical pathways of PCD, we now can seek to define the pathways of pro­ grammed axon degeneration. We now realize that the pathways of PCD may not be confined to a late role of destroying cells after the damage has already been done; they may be intimately involved from the beginning. And finally, we have come to appreciate that even in the adult context, the pathways of PCD are just one side of the survival equation; on the other side, and equally important, are the pathways of cell survival. All of these new views of the concept of PCD offer a multitude of opportunities for the development of new approaches to neuroprotec­ tive therapeutics.

Summary and conclusions

GDNF

Since the emergence of a molecular understanding of PCD about 20 years ago, its potential to provide a scientific basis for effective neuroprotective

JNK MAPK

Acknowledgements This work was supported by NS26836, NS38370, the RJG Foundation and the Parkinson’s disease Foundation.

Abbreviations 6OHDA Cdk5 DD DED DISC DLK FADD

6-hydroxydopamine Cyclin-dependent kinase 5 death domain death effector domain death-inducing signalling complex dual leucine zipper kinase Fas-associated protein with DD glial cell line-derived neurotrophic factor c-jun N-terminal kinase mitogen-activated protein kinase

92

MEF2 NAD NMNAT PCD PD PI3K SN SNpc TH TNF WldS

myocyte enhancer factor 2 transcription factors nicotinamide adenine dinucleotide nicotinamide mononucleotide adenylyltransferase programmed cell death Parkinson’s disease phosphatidylinositol-3-kinase substantia nigra substantia nigra pars compacta tyrosine hydroxylase tumor necrosis factor Wallerian degeneration slow

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against axonal degeneration: a model of gene therapy for peripheral neuropathy. Annals of Neurology, 50, 773–779. Xia, X. G., Harding, T., Weller, M., Bieneman, A., Uney, J. B., & Schulz, J. B. (2001). Gene transfer of the JNK interacting protein-1 protects dopaminergic neurons in the MPTP model of parkinson’s disease. Proceedings of the National Academy of Sciences of the United States of America, 98, 10433–10438. Yahata, N., Yuasa, S., & Araki, T. (2009). Nicotinamide mono­ nucleotide adenylyltransferase expression in mitochondrial matrix delays Wallerian degeneration. Journal of Neu­ roscience, 29, 6276–6284. Yang, Y., Gehrke, S., Haque, M. E., Imai, Y., Kosek, J., Yang, L., et al. (2005). Inactivation of drosophila DJ-1 leads to impair­ ments of oxidative stress response and phosphatidylinositol 3­ kinase/akt signaling. Proceedings of the National Academy of Sciences of the United States of America, 102, 13670–13675. Yang, X., Khosravi-Far, R., Chang, H. Y., & Baltimore, D. (1997a). Daxx, a novel fas-binding protein that activates JNK and apoptosis. Cell, 89, 1067–1076. Yang, D. D., Kuan, C. Y., Whitmarsh, A. J., Rincon, M., Zheng, T. S., Davis, R. J., et al. (1997b). Absence of excito­ toxicity-induced apoptosis in the hippocampus of mice lack­ ing the jnk3 gene. Nature, 389, 865–870. Zhang, Q., Ahuja, H. S., Zakeri, Z. F., & Wolgemuth, D. J. (1997). Cyclin-dependent kinase 5 is associated with apopto­ tic cell death during development and tissue remodeling. Developmental Biology, 183, 222–233.

A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 6

Control of mitochondrial integrity in Parkinson’s disease Cristofol Vives-Bauza, Maja Tocilescu, Rosa L.A. deVries, Dana M. Alessi,

Vernice Jackson-Lewis and Serge Przedborski


Departments of Neurology, Pathology and Cell Biology, and the Center for Motor Neuron Biology and Disease,

Columbia University, New York, NY, USA

Abstract: Parkinson’s disease (PD) is the most common neurodegenerative movement disorder associated with a loss of dopaminergic neurons. The role of mitochondria in the aetiology of PD has been questioned for decades, mostly from the perspective of bioenergetic failure. For decades, a deficit in mitochondrial respiration was thought to be a key factor in PD neurodegeneration. However, excluding a few exceptions where a clinical picture of parkinsonism is associated with a mitochondrial DNA mutation, preclinical and clinical studies have failed to identify any genetic mutations in the genes encoding for the electron transport chain complexes in PD patients. More recently, it has been discovered that mutations in the genes encoding for Parkin, PINK1 (PTEN-induced putative kinase-1) and DJ-1 are associated with familial forms of PD and with mitochondrial alterations, including morphological abnormalities. These results have led many researchers to revisit the question of mitochondrial biology as a primary mechanism in PD pathogenesis, this time from an angle of perturbation in mitochondrial dynamics and not from the angle of a deficit in respiration. Keywords: Parkinson’s disease; Mitochondria; Fusion; Fission; Mitophagy; PINK1; Parkin

Defects in mitochondrial metabolism are frequently involved in diseases of aging. Of these, Parkinson’s disease is the second most common disorder of the aging brain after Alzheimer’s disease and is clinically characterized by progressive resting tremor, rigidity, slowness of voluntary movements, bradykinesia, gait disturbance, poor

balance and dementia (Dauer and Przedborski, 2003). Pathologically, PD is characterized by the presence of proteinaceous inclusions called Lewy bodies, and the biochemical hallmark is a profound deficit in brain dopamine due to the degeneration of dopaminergic neurons (DA) in the substantia nigra pars compacta (SNpc) of the midbrain. Although the loss of nigral DA and terminals is responsible for the movement disor­ der part of PD, it is important to stress that PD neurodegeneration is not restricted to the


Corresponding author. Tel.: (212) 342-4119; Fax: (212) 342-4512;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83006-7

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dopaminergic systems as widespread neuronal loss can be detected in other catecholaminergic and non-catecholaminergic nuclei (Braak et al., 2003). The role of mitochondria in PD pathogenesis has been discussed extensively (see review Zhou et al., 2008). The fact that mitochondrial complex I inhibitors, like rotenone and MPTP (1-methyl-4­ phenyl-1,2,3,6-tetrahydropyridine), cause dopami­ nergic cell death in the SNpc and mimic a PD phenotype (Betarbet et al., 2000; Langston and Ballard, 1983; Liou et al., 1996; Przedborski and Vila, 2003; Ricaurte et al., 1986), pointed researchers towards oxidative stress and/or energy imbalance as causative of PD. Although this dilemma is still unresolved, in recent years, with the discovery of mutations in genes that are directly associated with mitochondrial function, research has focused on novel roles for mitochon­ dria in PD pathogenesis that imply that defects in mitochondrial dynamics and mitochondrial autop­ hagy (mitophagy) exist. Mitochondria are highly dynamic organelles that undergo constant changes in morphology and distribution in order to perform the correct cellular assignment at the appropriate time and at the appropriate location. In addition to their canonical role in supplying cellular energy, mito­ chondria play vital roles in calcium homeostasis and in reactive oxygen generation. They also have a central role in multifaceted cell death (see review in Smith et al., 2008). Mitochondria actively divide (fission), fuse with one another (fusion) and undergo regulated turnover (mito­ phagy). In neurons, mitochondria are actively transported throughout axons and dendrites (Hollenbeck and Saxton, 2005). These active pro­ cesses regulate mitochondrial function by facili­ tating mitochondrial recruitment to critical subcellular compartments, by content exchange between mitochondria, by mitochondrial shape control, by mitochondrial communication with the cytosol and by mitochondrial quality control. As a result, mitochondria can readily adapt to changes in cellular requirements, whether they

be due to physiological or environmental needs. Given the complex topology and energy require­ ments of the nervous system, it is not so surpris­ ing that defects in mitochondrial dynamics can lead to neurological disease. Strikingly, several proteins, which are encoded by genes that have been mutated in neurodegenerative diseases, are involved in mitochondrial dynamics, which sug­ gests that this is particularly important for the integrity of the nervous system. In order to explain why mitochondrial integrity is becoming particularly important in the aetiology of PD, we detail below the molecular mechanisms that govern mitochondrial dynamics, mitochondrial trafficking and specific turnover (mitophagy), since it will help the reader to understand the emerging evidence relating PD proteins to these molecular mechanisms.

Mitochondrial fusion and fission machinery Mitochondrial fusion and fission are catalyzed by specific machineries; a central role is played by dynamin-related proteins that are conserved between fungi and vertebrates and are predicted to hydrolize guanosine triphosphate (GTP) to exert their function. For mitochondrial fusion, key required proteins include the Mitofusins 1 and 2 (Mfn1 and Mfn2, homologues of yeast Fzo1p) (Chen et al., 2003; Santel and Fuller, 2001) dynamin-related proteins anchored to the outer mitochondrial membrane (OMM) involved in OMM fusion, and the optic atrophy protein 1 (Opa1, homologue of yeast Mgm1p) (Alexander et al., 2000; Delettre et al., 2000), a dynamin-related protein of the inter­ membrane space that associates with membranes and contributes to inner mitochondrial membrane (IMM) fusion. These proteins can physically inter­ act (Guillery et al., 2008) and appear to act in concert to modulate mitochondrial fusion. This process depends on the energy released by the hydrolysis of GTP (Meeusen et al., 2004).

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Although fusions of the OMM and IMM are nor­ mally highly synchronized, they can be uncoupled. For example, in mammalian cells, dissipation of mitochondrial membrane potential (D�m) appears to selectively inhibit IMM fusion (Malka et al., 2005; Twig et al., 2008a). Mutations in Mfn2 cause Charcot-Marie-Tooth type 2A, a peripheral neuropathy affecting sensory and motor neurons of the distal extremities (Zuchner et al., 2004). Mutations in Opa1 are the pre-dominant cause of autosomal dominant optic atrophy (DOA), a degeneration of retinal ganglia cells that leads to optic nerve atrophy (Alexander et al., 2000; Delettre et al., 2000). For mitochondrial fission, dynamin-related pro­ tein 1 (Drp1), a large dynamin-related protein (Dnm1p in yeast) (Smirnova et al., 2001) needs to move from the cytosol to the mitochondria. Drp1 is essential for mitochondrial division in all tested phyla (Shaw and Nunnari, 2002; Smirnova et al., 2001). In yeast, Drp1 recruitment is clearly dependent on the OMM protein Fis1 (Bleazard et al., 1999). In mammalian cells, however, knock­ down (KO) of Fis1 blocks mitochondrial fission without affecting Drp1 localization to mitochon­ dria (Lee et al., 2004). Drp1 activity is regulated by phosphorylation mediated by different kinases, such as cAMP-dependent protein kinase (Chang and Blackstone, 2007; Cribbs and Strack, 2007; Taguchi et al., 2007), Ca2þ/calmodulin-dependent protein kinase I alpha (Han et al., 2008) and cyclin-dependent kinase (Cdk1/cyclin B) (Taguchi et al., 2007). Mutations in several serine residues of its GTPase effector domain translate into elon­ gated mitochondria (Chang and Blackstone, 2007; Cribbs and Strack, 2007; Han et al., 2008; Taguchi et al., 2007). Drp-1 can also be modulated by ubiquitination via the mitochondrial RING finger ubiquitin ligase MARCH-5 (Nakamura et al., 2006; Yonashiro et al., 2006) and by SUMOyla­ tion, mediated by conjugation with the small ubiquitin-like modifier-1 (SUMO1) (Harder et al., 2004). Aside from Drp-1 and Fis1, novel proteins like the mitochondrial fission factor (Mff), a tailanchored OMM protein, have been associated

with mitochondrial fission in mammals. Downre­ gulation of Mff induces mitochondrial elongation and resistance to carbonyl cyanide m-chlorophe­ nylhydrazone-induced fragmentation (GandreBabbe and van der Bliek, 2008). Remarkably, Mff is part of a novel membrane complex of ~200 kDa that differs from the Fis1 containing complex, suggesting that Mff and Fis1 mediate different molecular steps in mitochondrial fission (Gandre-Babbe and van der Bliek, 2008). The potential role of Mff in Drp1 recruitment to mito­ chondria remains to be evaluated. So far, no inherited diseases have been associated with mutations in fission-related genes. In fact, only an isolated case of neonatal lethality with abnor­ mal brain development has been attributed to a defect in Drp1 (Waterham et al., 2007). The mechanisms by which aberrant fusion and/ or fission can affect mitochondrial function and, consequently, cellular homeostasis are not yet understood. Fusion may protect mitochondrial function by allowing mitochondria to repartition their contents, enabling protein complementa­ tion, equal distribution of metabolites and mtDNA repair. Indeed, fusion may function in a coordinated manner to maintain and protect mtDNA. Maintenance of mtDNA cannot take place without mitochondrial fusion (Rapaport et al., 1998), and fusion of mitochondria has been directly implicated in preventing the accu­ mulation of damaged mtDNA (Ono et al., 2001). More recently, mitochondrial fission has been shown to be critical to mtDNA maintenance (Parone et al., 2008). Since mtDNA mutations accumulate in the brain with age (Corral-Deb­ rinski et al., 1992), these functions may be of particular importance to neurons and to neuro­ degenerative diseases. The involvement of mtDNA damage in the aetiology of PD is still controversial, but several studies have suggested an association between mtDNA mutations and PD (Parker and Parks, 2005; Winkler-Stuck et al., 2005). Moreover, fission may be important for the facilitation of the equal segregation of the

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mitochondria into daughter cells during cell division and for enhancing the distribution of mitochondria along cytoskeletal tracks. In addi­ tion, fission may help in the isolation of damaged mitochondrial segments and thus promote their autophagy, as discussed below (Twig et al., 2008a). When these protective mechanisms fail, mitochondrial fission can also promote apoptosis, an important topic that has been extensively reviewed (Suen et al., 2008). In neurons, the mitochondrial fission/fusion machinery is critically involved in the formation of synapses and dendritic spines. Inhibition of mito­ chondrial fission translates into a loss of mitochon­ dria from dendritic spines resulting in a reduction of synapse formation, whereas an excess of fission increases synapse formation (Li et al., 2004). In addition, upon depletion of Drp1, mitochondria cannot be directed to the synapses, leading to synaptic dysfunction (Verstreken et al., 2005). The study of PD genes causative of the familial forms has provided the strongest evidence sup­ porting a direct role of mitochondrial dynamics in the aetiology of PD. So far, five proteins have been implicated in the pathogenesis of PD, a­ synuclein, Parkin, PTEN-induced putative kinase-1 (PINK1), DJ-1 and leucine-rich repeat kinase 2 (LRRK2) (Bonifati, 2007). Emerging evi­ dence suggests that the pathogenic mechanisms involving mutations in the genes that encode for these proteins may have common threats. Of spe­ cial interest for this review is the case of PINK1 and Parkin. Recent studies in Drosophila have provided compelling evidence that PINK1 and Parkin act in a linear pathway to control mitochon­ drial morphology in indirect flight muscles and DA neurons (Clark et al., 2006; Yang et al., 2006). PINK1 (PARK6) is a nuclearly encoded gene that translates for a mitochondrially targeted ser­ ine/threonine kinase. Mutations in the PINK1 gene are associated with the autosomal recessive inheritance of PD (Valente et al., 2004), directly linking a mitochondrial protein to PD pathogen­ esis. The role of PINK1 in the regulation of the mitochondrial fission and fusion machinery has

recently been proposed in a series of articles (Deng et al., 2008; Park et al., 2009; Poole et al., 2008; Yang et al., 2008). Drosophila PINK1 (dPINK1) mutant flies and dPINK1 KO by RNAi exhibit susceptibility to stress, decreased cellular adenosine-50 -triphosphate (ATP) levels, reduced mtDNA content and altered mitochon­ drial morphology in both flight muscles and DA. The mitochondrial abnormalities consist of mito­ chondrial aggregates and swollen or enlarged mitochondria with disorganized cristae. Parkin, the product of the PARK2 gene, is a cytosolic ubiquitin E3 ligase protein that plays a role in the ubiquitin-dependent proteasome path­ way (Shimura et al., 2000). Mutant or KO Parkin flies share strikingly similar phenotypes (Greene et al., 2003; Pesah et al., 2004; Wang et al., 2007) to those of dPINK1 KO and mutant flies. Due to this genetic redundancy, different studies tested whether the ectopic expression of Parkin could influence the dPINK1 phenotype (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). Con­ sensus results were obtained proving that Parkin over-expression completely rescued the effect of the loss of PINK1 activity (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). Interestingly, PINK1 over-expression did not rescue the Parkin-null phenotype, suggesting that PINK1 lies upstream of Parkin in a common pathway. Also, the phenotype of double PINK1/Parkin knockouts resembled that of single mutants, suggesting that PINK1 and Parkin do not act in parallel (Clark et al., 2006; Park et al., 2006). The role of the PINK1/Parkin pathway promot­ ing fission and/or inhibiting fusion of mitochondria is supported by different studies in Drosophila (Deng et al., 2008; Park et al., 2009; Poole et al., 2008; Yang et al., 2008). Over-expression of the mitochondrial fission protein Drp1 rescued the PINK1 or Parkin mutant phenotypes. Moreover, the loss of fusion by mutant or depleted Opa1 or Mfn also partially rescued either Parkin or PINK1 mutant phenotypes. In contrast, over-expression of Opa1 or Mfn alone, presumably to upregulate mito­ chondrial fusion, resulted in mitochondrial swelling

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and did not rescue the PINK1 mutant phenotype (Park et al., 2009). However, Parkin over-expres­ sion did rescue the Opa1 over-expression pheno­ type. Interestingly, Yang et al. showed that the PINK1/Parkin interaction with mitochondrial fusion and fission also occurs in DA (Yang et al., 2008). Together, these findings illustrate a role for the PINK1/Parkin pathway promoting fission and/ or inhibiting fusion in Drosophila muscle and neu­ ronal mitochondria. Whether a similar PINK1/Parkin relationship can be said of the mammalian system is still debatable. Silencing of PINK1 in HeLa cells resulted in mito­ chondrial fragmentation, disorganized cristae and functional deficiencies. Similar results were observed in neuroblastoma cells with siRNA KO of PINK1 and Parkin (Park et al., 2009), in SH-SY5Y cells and in primary cells cultured from patients carrying PINK1 mutations (Dagda et al., 2009; Exner et al., 2007; Wood-Kaczmar et al., 2008). As in Drosophila, these phenotypes could be rescued by Parkin over-expression (Exner et al., 2007). By contrary, Mortiboys et al. (2008) recently demonstrated that cultured fibroblasts from patients carrying Parkin mutations exhibited mitochondrial morphological abnormalities, being longer and more branched than controls. In addition, over-expression of PINK1 in COS-7 cells resulted in punctuate mitochondria, whereas suppression of PINK1 with shRNA resulted in long, tubular mitochondria, a phenotype inhibited by the over-expression of the fission proteins hFis1 or Drp1 (Yang et al., 2008). Conversely, no gross mitochondrial morphological defects were found in PINK1 KO mice (Kitada et al., 2007), even though functional effects were seen (Gautier et al., 2008). This suggests that mitochondrial morphology changes can result from loss of PINK1 function but may possibly be secondary to other changes. While clearly further studies are necessary to understand these conflicts, it is entirely possible that given the dynamic and interrelated regulation between fission and fusion, PINK1/Parkin effects may differ depending on the cell type and/or cellular conditions.

Moreover, and as will be explained in detail below, PINK1/Parkin in mammals may also be involved in the trafficking and regulation of mito­ chondrial turnover. As Parkin is not specifically a mitochondrial protein and must be translocated to the organelle, its effects on mitochondrial mor­ phology are likely tied to a larger pathway med­ iating mitochondrial maintenance. Recent studies have not only begun to elucidate such a pathway but also have collectively pointed to mitochondrial dynamics as a critical target.

Mitochondrial motility Beyond fission and fusion, another important aspect of mitochondrial dynamics is mitochondrial motility, especially in highly polarized cells such as neurons. Mitochondria are transported along cytoskeletal tracks to areas in the cell where the energy demands are high and/or where calcium buffering is required. Rapid, long-distance trans­ mission of mitochondria is accomplished via the microtubule network, whereas actin serves as tracks for short-range transport of mitochondria to areas where the microtubules are not found (Ligon and Steward, 2000; Morris and Hollen­ beck, 1995; Rube and van der Bliek, 2004). In addition, the integrity of intermediate filaments is an important determinant for the correct locali­ zation of mitochondria as well as for the regula­ tion of mitochondrial function (e.g. apoptosis, energy metabolism) (Toivola et al., 2005). Neurons require specialized mechanisms to regu­ late the transport and retention of mitochondria in the vicinity of active growth cones, nodes of Ranvier and synaptic terminals, where energy production and calcium homeostasis capacity are in high demand (Hollenbeck and Saxton, 2005). Defective transport of axonal mitochondria is implicated in human neurological disorders and neurodegenerative diseases (reviewed in Chan, 2006; Hirokawa and Takemura, 2004). Mitochondria undergo anterograde transport towards the plus end of microtubules from the

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cell soma to peripheral structures and retrograde movement in the opposite direction. In mammals, the kinesin isoforms kinesin-3 (KIF1B) and kine­ sin-1 (KHC, KIF5B) utilize the energy released in ATP hydrolysis to move their specific cargo, the mitochondria, in the anterograde direction (Ligon and Steward, 2000; Nangaku et al., 1994; Stowers et al., 2002; Tanaka et al., 1998). The microtubuleassociated minus end-directed dynein motor is the driving force for the retrograde transport of mito­ chondria to the cell body (Pilling et al., 2006; Varadi et al., 2004). Translocation along actin is suggested to be accomplished by myosin V motors in higher eukaryotes (Langford, 2002). In mam­ mals, two OMM proteins, Miro1 and Miro2 (also known as Rhot T1 and Rhot T2; Fransson et al., 2003), interact with the KHC motor, with the help of protein adaptors, the Milton family proteins. Milton, a fly protein, binds both Miros and KHC to support mitochondrial transport (Glater et al., 2006; Rice and Gelfand, 2006; Stowers et al., 2002). Mammals have two homologues, named Grif-1 and OIP106 (also known as TRAK2 and TRAK1, respectively) (Brickley et al., 2005). Physiological elevations of cytosolic calcium are known to control mitochondrial motility in a vari­ ety of mammalian cells (for review see Graier et al., 2007; Hajnoczky et al., 2006) and Miro serves as a calcium sensor (Macaskill et al., 2009; Saotome et al., 2008; Wang and Schwarz, 2009). PINK1 has recently been shown to form a multiprotein complex with Miro and Milton (Weihofen et al., 2009). Interestingly, abnormal mitochon­ drial morphology, associated with a loss of func­ tional PINK1, was ameliorated by Milton and Miro over-expression (Weihofen et al., 2009). As stated above, a key role of mitochondria is to buffer calcium and to prevent calcium overload in the cytosol. Recently, two independent studies have shown that PINK1 regulates calcium efflux from the mitochondria (Gandhi et al., 2009; Marongiu et al., 2009). Therefore, it could be hypothesized that PINK1, together with Miro and Milton, plays a role in mitochondrial traffick­ ing by regulating mitochondrial calcium efflux.

Signalling through the phosphatidylinositol 3­ kinase (PI3K) pathway has also been implicated in the regulation of mitochondrial movement (Hollenbeck and Saxton, 2005). Interestingly, PINK1 was originally identified by an analysis of expression profiles from cancer cells after the introduction of the exogenous phosphatase and tensin homologue (PTEN), a tumour suppressor that is involved in the regulation of the PI3K signalling pathway (Unoki and Nakamura, 2001). Nevertheless, whether the PI3K pathway is impor­ tant for the regulation of PINK1 and how this might impact on mitochondrial motility remain to be determined. Association of Parkin with mitochondrial trans­ port has also been proposed. Parkin was shown to bind to and stabilize microtubules (Yang et al., 2005). Since mitochondrial transport is dependent on the microtubule system in neuronal processes (Hollenbeck and Saxton, 2005), one could hypothesize that Parkin, or mutations in Parkin, could affect mitochondrial transport throughout the neuron. Although so far there are no studies linking the mitochondrial motility machinery to LRRK2, it is important to emphasize that LRRK2 GTPase domain shares significant sequence similarity with the Rho GTPase domain of Miro (Guo et al., 2005).

Mitophagy Autophagy is a catabolic process involving the degradation of a cell’s own components by engulf­ ment into autophagosomes. The formation of autophagosomes is initiated by class III phosphoi­ nositide 3-kinase and autophagy-related gene 6 (Atg6, also known as Beclin-1). In addition, at least two other systems are involved, that of the ubiquitin-like protein Atg8 (known as LC3 in mammalian cells) and the Atg4 protease on the one hand and the Atg12-Atg5-Atg16 complex on the other hand (Schmid and Munz, 2007). The outer membrane of the autophagosome fuses

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with a lysosome in the cytoplasm to form an autophagolysosome where its contents are degraded via acidic lysosomal hydrolases. During nutrient starvation, autophagy is activated leading to the breakdown of non-vital components and to the release of nutrients, insuring that vital processes can continue. But, autophagy also appears to have a housekeeping role in maintain­ ing quality control by degrading damaged orga­ nelles, cell membranes and protein aggregates. It is thought that mitochondrial autophagy, or mito­ phagy, is the major route by which mitochondria are degraded. In this view, the cell would selec­ tively induce mitophagy to expunge malfunction­ ing mitochondria, thus ridding the cell of troublesome sources of reactive oxygen species, apoptosis-inducing factors or unnecessary meta­ bolic burdens. In mammalian cells, loss of D�m appears to be a common feature of mitophagy, suggesting that damaged mitochondria are targets for autophago­ some removal (Kim et al., 2007; Rodriguez-Enri­ quez et al., 2006; Twig et al., 2008a). A couple of proteins have been identified as markers of mito­ phagy. Ulk1, a serine/threonine kinase that is expressed in autophagosome membranes, seems to be a critical regulator of mitochondrial and ribosomal clearance (Kundu et al., 2008). At the mitochondrial site, Nix, a non-canonical BH3 member of the Bcl2 family of proteins, is a selec­ tive autophagy receptor for binding to LC3/ GABARAP proteins, which are ubiquitin-like modifiers that are required for the growth of autophagosome membranes (Chen et al., 2008; Sandoval et al., 2008; Schweers et al., 2007; Zhang and Ney, 2008). Nix mediates mitochon­ drial clearance after mitochondrial damage and during erythrocyte differentiation (Novak et al., 2010; Sandoval et al., 2008; Schweers et al., 2007). Recent work has linked the mitochondrial fis­ sion/fusion machinery to autophagy, but whether mitophagy occurs as a result is still open to ques­ tion. Mitophagy seems to depend on the presence of fission or loss of fusion because by knocking down Drp-1 or Fis1, or by over-expression of

Opa1, mitophagy is significantly reduced (Naren­ dra et al., 2008; Twig et al., 2008a). In contract, an excess of fission, driven by the over-expression of Drp1 or Fis1, translates into mitochondrial disap­ pearance (Arnoult et al., 2005; Gomes and Scor­ rano, 2008). Consistent with the idea that fission increases mitochondrial autophagy, over-expres­ sion of hFis1 selectively reduced the mitochon­ drial, but not the endoplasmic reticulum (ER) mass. Twig and co-workers tested the role of fis­ sion and fusion in mitophagy by following several individual mitochondria through rounds of fission and fusion. They observed that depolarized mito­ chondria could not re-fuse and were preferentially segregated in LC3-positive structures (Twig et al., 2008a, 2008b). Because depolarization occurred well before autophagy and was maintained, the authors sought an irreversible event that pre­ vented re-fusion, focusing on Opa1. When Opa1 was over-expressed, a reduced fraction of mito­ chondria was found in autophagosomes, implying loss of Opa1 as the signal. Likewise, KO of Fis1 or Drp1 reduced autophagy. Here, mitophagy occurs through macroautophagy and the process is clearly selective in terms of which mitochondria become engulfed, but whether the autophago­ somes contain only mitochondria without surrounding cytoplasm is not reported. In the context of PD, mitophagy has become a potential pathogenic mechanism since Narendra and co-workers found that, in mammalian cells, Parkin is selectively recruited to depolarized mito­ chondria, followed by a stimulation of mitochon­ drial autophagy (Narendra et al., 2008). Within 48 h, the mitochondrial markers Tom20, cytochrome c and TRAP1 were lost. The specificity of mitochon­ drial elimination was remarkable since even peroxi­ somes were left intact. Evidence for the autophagic removal of mitochondria was that mitochondria appeared within green fluorescent protein (GFP)­ LC3-positive structures, and mitochondrial disap­ pearance was prevented in cells treated with the lysosomal inhibitor bafilomycin A1 and in Atg5–/– MEF cells. With these results, this chapter suggests that a specific mammalian protein can signal

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mitophagy. How Parkin is recruited to depolarized mitochondria was a key question. We, and two other groups, have recently shown that Parkin translocation to mitochondria relies on PINK1 expression, even in cells with normal D�m (Geisler et al., 2010; Narendra et al., 2010; Vives-Bauza et al., 2010). We also observed that, once at the mitochondria, Parkin is in close proximity to PINK1, but Parkin does not catalyze PINK1 ubi­ quitination nor does PINK1 phosphorylate Parkin. However, co-over-expression of Parkin and PINK1 collapsed the normal tubular mitochondrial net­ work into large mitochondrial perinuclear clusters, many of which were surrounded by autophagic vacuoles. Overall, our results suggest that Parkin and PINK1 modulate mitochondrial trafficking to the perinuclear region, a sub-cellular area asso­ ciated with autophagy. Interestingly, PD-related mutations in either Parkin or PINK1 impair this process (Geisler et al., 2010; Narendra et al., 2010; Vives-Bauza et al., 2010). Moreover, Geisler and co-workers have found that VDAC1 (voltage­ dependent anion channel 1) is the mitochondrial target for Parkin-mediated lysine 27 polyubiquity­ lation (Geisler et al., 2010). This implies that mito­ chondrial turnover could be involved in the pathogenesis of PD, inducing accumulation of defective mitochondria. How PINK1 translates the mitochondrial depolarization signal to recruit Parkin remains to be elucidated. Further studies will have to evaluate if fission is required to mediate PINK1-dependent recruitment of Parkin to mitochondria. Whether PINK1 influences mito­ chondrial morphology through the direct phosphor­ ylation of Drp1 also remains to be determined. Although the data suggest that mitochondrial fission is required for mitophagy, Gomes and co­ workers found that over-expression of a form of Fis1 (Fisa1), mutated in its ability to cause fission, was at least as efficacious as wt Fis1 in promoting mitochondrial autophagy. The authors concluded that loss of D�m is the common feature enabling the two Fis1 forms to induce mitophagy (Gomes and Scorrano, 2008). In that line, it has been observed that, in neurons induced to undergo

apoptosis, but prevented from dying using caspase inhibitors, mitochondria are fragmented and lose their D�m prior to disappearing (Xue et al., 1999, 2001). Considering the proposed role for a PINK1/ Parkin pathway in the regulation of fission and fusion, it is tempting to speculate that the two path­ ways are linked and that they work in tandem to initiate fission of depolarized mitochondria and subsequently target any resulting dysfunctional mitochondria for mitophagy. This relationship, however, remains to be explored. It is important to emphasize that PINK1 is cleaved and that this cleavage seems to play a crucial role in PINK1 protective function against various stressors (Lin and Kang, 2008; Muqit et al., 2006). To date, the protease responsible for PINK1 cleavage as well as for the PINK1 cleavage site remains to be identified. There are conflicting data suggesting that PINK1 partici­ pates in a pathway with another putative PDrelated protein, Omi/HtrA2 (Plun-Favreau et al., 2008). Omi/HtrA2 is a mitochondrial serine pro­ tease of which mutations affecting its protease activity have been linked to an increased risk of PD (PARK13) (Bogaerts et al., 2008; Strauss et al., 2005), although recent studies have ques­ tioned this finding (Ross et al., 2008; Simon-San­ chez and Singleton, 2008). Plun-Favreau and co­ workers showed that PINK1 and Omi/HtrA2 can interact with each other, and in brain tissue from PD patients carrying PINK1 mutations, Omi/ HtrA2 phosphorylation appeared to be decreased (Plun-Favreau et al., 2007). Phosphorylation of Omi/HtrA2 increased its serine protease activity and was necessary for Omi/HtrA2-mediated pro­ tection of mitochondria when cells were exposed to various toxins (Plun-Favreau et al., 2007). In Drosophila, Omi/HtrA2 interacts genetically with and downstream of PINK1 in a pathway indepen­ dent of Parkin (Whitworth et al., 2008). In that study, over-expression of PINK1 alone or in combination with Omi/HtrA2 resulted in a disor­ ganized eye phenotype, prevented by the downregulation of Omi/HtrA2. But some of these results have been directly contradicted by a

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recently reported genetic loss of function study, which found no strong evidence for the in vivo interaction between Omi/HtrA2 and PINK1 (Yun et al., 2008). Given this conflicting evidence, it is unclear whether this mitochondrial protease will prove to be involved in the PINK1 pathway of mitochondrial dynamics. In that line of argu­ ment, another studied mitochondrial target is the inner mitochondrial membrane protease Rhomboid-7, which has not been previously linked to PD pathogenesis. Rhomboid-7 was demonstrated to be important for normal mito­ chondrial fusion in Drosophila (McQuibban et al., 2006). Over-expression of Rhomboid-7 also enhanced the PINK1 over-expression pheno­ types in Drosophila and has been suggested to function upstream of PINK1 and Parkin (Whitworth et al., 2008). The mammalian homo­ logue, PARL, appears not to be involved in the PINK1-dependent recruitment of Parkin to target mitochondria for mitophagy (Narendra et al., 2010). The relationship of these findings to PD pathogenesis, if any, remains to be studied. But, certainly, this work does provide a step towards further defining the pathway by which PINK1 and Parkin affect mitochondrial dynamics. Another PD-related protein, the LRRK2, has also recently been shown to be involved in autop­ hagy. Mutations in LRRK2 translate into an auto­ somal dominant form of PD. LRRK2 is a multidomain protein that has both a kinase domain and a GTPase domain (Gloeckner et al., 2006; West et al., 2005). In human cells, LRRK2 is specifically located in membrane micro-domains, in multi-vesi­ cular bodies (MVBs) and in autophagic vesicles (AVs) (egre-Abarrategui et al., 2009). Mutations found in PD patients induced autophagic stress characterized by the accumulation of abnormal MVBs and enlarged AVs with high levels of p62. Moreover, silencing of LRRK2 translated into increased autophagic activity and in the prevention of cell death caused by the inhibition of autophagy in starvation conditions (egre-Abarrategui et al., 2009). This data suggest a role for LRRK2 in the endosomal autophagic pathway.

Finding the substrates for PINK1 and LRRK2 kinases, the specific targets for Parkin ubiquitina­ tion and their possible interactions with the autop­ hagic machinery will help to clarify the specific role of PD-related proteins in autophagy. Along this line, it has recently been published that PINK1 promotes autophagy though interacting with beclin1 (Michiorri et al., 2010). Parkin has also been shown to physically interact in vitro and in vivo with SUMO1 to increase its nuclear transport and self-ubiquitination (Um et al., 2006). Downregulation of SUMO1 may inhibit mito­ chondrial fission caused by excess Parkin turnover in the dPINK1/dParkin pathway.

Conclusions The degeneration in PD affects not only DA but many other neurons in the CNS, including popula­ tions in the brain stem, as well as in the sub­ cortical and cortical regions (Braak et al., 2003). The reason why these particular sets of different neurons are selectively vulnerable in PD is not known, but may be key to understanding the underlying mechanisms of neurodegeneration in PD. Remarkably, they share two features: they have long, thin axons, and these axons have little or no myelination (Braak et al., 2004). The high energy requirement of these selectively vulnerable neurons and the long distance between axon term­ inals and cell bodies are likely to provide clues to the underlying mechanisms involved. Neurons with these features are likely to be particularly dependent on proper mitochondrial dynamics. In addition, the DA of the SNpc (degenerate in PD) have a lower mitochondrial mass than neighbour­ ing non-DA or DA of the ventral tegmental area (resistant in PD) (Liang et al., 2007), suggesting the possibility that the vulnerable neurons may be more susceptible to subtle changes in mitochondrial maintenance. It is also generally believed that the degenerative process in PD begins at the terminals rather than the cell body (e.g. see Braak et al., 2004). Whether classic

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apoptotic programmed cell death mechanisms occur in PD is still controversial, there is evidence that axonal/dendritic degenerative mechanisms may be different from the classic mechanisms of programmed cell death (Finn et al., 2000; Raff et al., 2002). Given the importance of mitochon­ drial dynamics for mitochondrial distribution to synapses, synaptic function and overall mitochondrial function, dysfunction in these dynamic processes may be an early step in PD neurodegeneration. As we have shown throughout this review, mito­ chondrial fusion and fission events are tightly interrelated with other mitochondrial mainte­ nance functions such as biogenesis and turnover. Consequently, deregulations in any of these mechanisms can account for a general failure in the control of mitochondrial integrity. There has been a great deal of interest from the PD scientific community in linking the familial associated genes in a common pathogenic path­ way of neurodegeneration. To date, however, a single pathway unifying these proteins has not been fully mapped out. Only PINK1 and Parkin seem to clearly function in the same pathway. In the familial forms of PD, due to mutations in any of these two genes, impaired Parkin recruitment to mitochondria could lead to the accumulation of damaged mitochondria, with a consequent increase in oxidative stress and toxic burden that could lead to a specific kind of cell death of the most susceptible neurons. Nevertheless, there are still lots of questions to be answered to fully char­ acterize the PINK1/Parkin pathway. Future stu­ dies need to address, in detail, the biochemical roles of PINK1/Parkin in terms of substrates for both proteins, as well as their relationship with each other. How PINK1 selectively recruits Parkin to mitochondria? How Parkin selectively targets depolarized mitochondria? Are there third players that modulate PINK1 in response to mito­ chondrial depolarization? These are just a few examples of key questions for future studies in order to be able to use this molecular pathway for therapeutic intervention.

Whether the scenario proposed for familial PDrelated proteins can be extrapolated to the sporadic PD is uncertain. It is possible that other factors, such as toxic exposure, increased oxidative stress, or con­ tributing genetic factors could limit the availability of Parkin and other proteins for proper mitochon­ drial maintenance. In fact, it has been shown that Parkin can be covalently modified and inactivated by dopamine quinone, which may contribute to the increased susceptibility of the DA (LaVoie et al., 2005). Another possibility is that these environmen­ tal factors may directly or indirectly influence mito­ chondrial dynamics per se. In this case, it is not adventurous to think that DA of the SNpc, with lower mitochondrial mass and increased stress in general, may not be able to maintain a distribution of healthy mitochondria and eventually collapse to degeneration, precipitating the PD phenotype.

Acknowledgement The authors are supported by NIH Grants AG021617, NS042269, NS062180, NS064191 and NS38370; U.S. Department of Defense grants (W81XWH-08-1-0522, W81XWH-08-1-0465 and DAMD 17-03-1); the Parkinson Disease Founda­ tion (New York, NY, USA); the Thomas Hartman Foundation For Parkinson’s Research and the MDA/Wings-over-Wall Street. S.P. is the Page and William Black Professor of Neurology.

Abbreviations D�m AVs CaMKIalpha CCCP DA DOA

Mitochondrial membrane potential Autophagic vesicles Ca2þ/calmodulin-dependent protein kinase I alpha carbonyl cyanide m­ chlorophenylhydrazone dopaminergic neurons autosomal dominant optic atrophy

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Drp1 ER GFP GTP IMM LRRK2 mDNA Mff Mfn1 and Mfn2 MPTP OMM Opa1 PD PI3K PINK1 SNpc SUMO1

Dynamin-related protein 1 Endoplasmic reticulum Green fluorescent protein Guanosine triphosphate Inner mitochondrial membrane Leucine-rich repeat kinase 2 Mitochondrial DNA Mitochondrial fission factor Mitofusins 1 and 2 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine Outer mitochondrial membrane optic atrophy protein 1 Parkinson’s disease Phosphatidylinositol 3-kinase PTEN-induced putative kinase 1 substantia nigra pars compacta Ubiquitin-like modifier-1

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A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 7

Role of post-translational modifications in modulating the structure, function and toxicity of a-synuclein: implications for Parkinson’s disease pathogenesis and therapies Abid Oueslati†, Margot Fournier† and Hilal A. Lashuel
Laboratory of Molecular Neurobiology and Neuroproteomics, Brain Mind Institute, The Ecole Polytechnique F�ed�erale de Lausanne (EPFL), Lausanne, Switzerland

Abstract: A better understanding of the molecular and cellular determinants that influence the pathology of Parkinson’s disease (PD) is essential for developing effective diagnostic, preventative and therapeutic strategies to treat this devastating disease. A number of post-translational modifications to a-syn are present within the Lewy bodies in the brains of affected patients and transgenic models of PD and related disorders. However, whether disease-associated a-syn post-translational modifications promote or inhibit a-syn aggregation and neurotoxicity in vivo remains unknown. Herein, we summarize and discuss the major disease-associated post-translational modifications (phosphorylation, truncation and ubiquitination) and present our current understanding of the effect of these modifications on a-syn aggregation and toxicity. Elucidating the molecular mechanisms underlying post-translation modifications of a-syn and the consequences of such modifications on the biochemical, structural, aggregation and toxic properties of the protein is essential for unravelling the molecular basis of its function(s) in health and disease. Furthermore, the identification of the natural enzymes involved in regulating the post-translational modifications of a-synuclein will yield novel and more tractable therapeutic targets to treat PD and related synucleinopathies. Keywords: a-synuclein; post-translationnal modification; phosphorylation; truncation; ubiquitination

Introduction
Corresponding author. Tel.: þ41 21 69 39691, 31812; Fax: þ41 21 693 96 65; Eail: [email protected] †

A better understanding of the molecular and cellular determinants that influence the pathology of Parkinson’s disease (PD) is essential for deve­ loping effective diagnostic, preventative and

These authors contributed equally.

DOI: 10.1016/S0079-6123(10)83007-9

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therapeutic strategies to treat this devastating dis­ ease. Current strategies offer little more than tran­ sient symptomatic relief. Evidence from genetics, pathology, animal modelling, cell culture and in vitro biochemical and biophysical studies supports the hypothesis that a-synuclein (a-syn) plays a cen­ tral role in the pathogenesis of PD and several other neurodegenerative diseases, collectively referred to as ‘synucleinopathies’. Although a-syn has emerged as a viable therapeutic target, the molecular and cellular determinants involved in initiating and/or propagating a-syn aggregation and toxicity and the relationship between these processes and disease progression remain poorly understood. In PD, sev­ eral post-translational modifications of a-syn are associated with the formation of Lewy bodies (LB) in the brains of affected patients and of transgenic (TG) models of PD and other synucleinopathies. However, whether disease-associated a-syn post­ translational modifications such as phosphorylation, truncation, ubiquitination and nitration promote or a

A30P

inhibit a-syn aggregation and neurotoxicity in vivo remains unknown. This understanding is critical to elucidate the role of a-syn in the pathogenesis of PD and to develop therapeutic strategies for PD. There­ fore, characterizing the molecular mechanisms underlying post-translation modifications of a-syn and the consequences of such modifications on the biochemical, structural, aggregation and toxic prop­ erties of the protein is essential for unravelling the molecular basis of its function(s) in health and dis­ ease. Furthermore, identification of the natural enzymes (e.g. kinases and phosphatases) involved in regulating a-syn post-translational modifications will yield novel and more effective therapeutic tar­ gets to treat PD and related synucleinopathies. Structural and biochemical properties of a-syn The sequence of a-syn can be divided into three domains with different features (Fig. 1a). The

E46K A53T

COOH

H2N 1

Amphipathic repeat region

61

Hydrophobic NAC peptide

95

Acidic region

140

b

n Oligomers Monomer-dimer-tetramer

Protofibrils M + PO4 Amyloid fibrils

Fig. 1. a-syn structure and molecular mechanism of its oligomerization and fibrillogenesis. (a) Schematic representation of a-syn, where the N-terminal repeats (KTKEGV) are represented in blue; they partially overlap with the core domain (white). The pathogenic variants associated with familial forms of PD alter the N-terminus of a-syn and enhance its propensity to form protofibrils (A30P) or mature fibrils (E46K and A53T). (b) Illustration of the molecular steps involved in a-syn oligomerization and fibrillogenesis leading to Lewy body formation (adapted from Lashuel and Lansbury, 2006).

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N-terminal region, comprising amino acids 1–60, contains four 11 residue imperfect repeats with a highly conserved hexameric motif (KTKEGV). The central region, comprising residues 61–95, also known as the non-amyloid component region (NAC), is composed of predominantly hydropho­ bic residues and is essential for a-syn fibrillization and LB formation. The C-terminal region (residues 96–149) is highly enriched in acidic (glutamate and aspartate) and proline residues and exists in a dis­ ordered conformation in the monomeric as well as the oligomeric and fibrillar forms of a-syn (Bertini et al., 2007; Wu et al., 2008). The C-terminus of a­ syn has been proposed to function as a solubilizing domain and contributes to a-syn’s thermostability. The C-terminal region of a-syn has been implicated in the majority of a-syn interactions with proteins (Cherny et al., 2004; Fernandez et al., 2004; Giasson et al., 2003; Jensen et al., 1999), metal ions (Brown, 2007; Paik et al., 1999) and other ligands (e.g. dopamine and polyamines) (Hoyer et al., 2004) and contains the majority of post-trans­ lational modification sites. a-Synuclein interactions with the microtubule-associated protein tau, brainspecific p25a and FKBP-type peptidyl-prolyl cis–trans isomerases have all been mapped to the C-terminal region encompassing residues 110–140, suggesting that the C-terminus of a-syn plays a critical role in modulating the stability, structure, aggregation and function of the protein in vivo (Kim et al., 2002). In solution, a-syn (wild type, WT, A30P, E46K and A53T) exists as an ensemble of disordered conformations (Weinreb et al., 1996). However, in the presence of lipids, the N-terminal region adopts an a-helical conformation. Several factors have been shown to contribute to triggering the oligomerization and fibril formation by a-syn, including high protein concentrations, mutations, post-translational modifications and interactions with specific metals (Brown, 2007; Paik et al., 1999) and small molecules (Hoyer et al., 2004). Fibril formation by a-syn proceeds through series of discrete oligomeric intermediates, known as protofibrils of different sizes and morphologies,

including spherical, annular and chain-like struc­ tures (Fig. 1b). The rate of a-synuclein fibrillization is affected by several factors that may be relevant to PD Several post-translational covalent modifications have been described for a-syn, including serine and tyrosine phosphorylation, ubiquitination, nitration, enzymatic cross-linking (e.g. tissue transglutaminase) and C-terminal truncation, some of which correlate well with PD (Fig. 2). In this section we will review the most commonly observed post-translational disease-associated modifications. We will present an overview of the current understanding of their role in disease pathogenesis on the basis of published in vitro and in vivo studies aimed at identifying the sites of modifications, the enzymes involved and the con­ sequences of modulating these modifications on the structure, aggregation, membrane binding and toxicity of a-syn.

Phosphorylation Increasing evidence from pathologic, genetic, ani­ mal model, biochemical and biophysical studies suggests that phosphorylation of a-syn at one or multiple sites may play an important role in regulat­ ing its structure, membrane binding, oligomeriza­ tion, fibril formation, LB formation and neurotoxicity in vivo (Anderson et al., 2006; Fujiwara et al., 2002). The first evidence demon­ strating phosphorylation of a-syn emerged from immunohistochemical and biochemical studies by Iwatsubo and colleagues and was supported later by many studies. Using a phospho-specific antibody and mass spectrometry, they demonstrated that the majority of a-syn within LB and inclusions isolated from brains of patients who died of PD, multiple system atrophy (MSA), dementia with Lewy bodies (DLB) and other synucleinopathies is phosphory­ lated at serine 129 (S129-P) (Anderson et al., 2006;

118

O−

O−

Y125

Y135

C

CH2

S87

CH2

CH2

S129 O

Syn 1−80

O P O−

O−

O− P

O

O

O−

O−

O P O−

α-Synuclein: Post-translational modifications

P

O−

O−

Y133

O−

ubi qui tina tion

n

γ

ing

id Ox

on Syn 1−80

Syn 120−140

N-term NO 2

NO 2

C-term

Y125

C 2

Y135 Y133

Ct

Syn 1−80

NO

ε

C

ati

ink

Ct

HN

Lys (K)

s-l

Syn 1−112

Ct

O

O−

os

Gln (Q)

C

io cat

Cr

Syn 1−120

n Tru

Ub

O P O−

Syn 120−140

O−

Phosphorylation Ub Ub

Fig. 2. Most studied a-syn post-translational modifications reported on the basis of their identification in LB. All the modifications identified thus far, including phosphorylation, truncation, nitration and covalent cross-linking by tissue transglutaminase, occur at and/or involve the C-terminal region of the protein. Only ubiquitination (mono-, di- and tri-) is restricted to the lysine residues in the N-terminal region of the protein comprising residues 1–36.

Fujiwara et al., 2002; Kahle et al., 2000; Okochi et al., 2000; Takahashi et al., 2003). Subsequent cell culture studies demonstrated that a-syn is con­ stitutively phosphorylated at S87 and S129. Since then S129-P has emerged as a defining hallmark of PD and related synucleinopathies (Anderson et al., 2006; Fujiwara et al., 2002; Okochi et al., 2000; Takahashi et al., 2003) and is consistently observed in a-syn inclusion formed in cellular and animal models over-expressing WT or mutant a-syn (Chen and Feany, 2005; Lo Bianco et al., 2002;

Neumann et al., 2002; Takahashi et al., 2003; Yamada et al., 2004). Recent studies from our group demonstrated that a-syn is phosphorylated at S87 in vivo and within LB. The level of S87-P is increased in brains of TG models of synucleinopa­ thies and human brains from Alzheimer’s disease (AD), DLB and MSA patients (Paleologou et al., 2010). A recent report by Feany and colleague demonstrated that a-syn is also phosphorylated at tyrosine 125 (Y125-P) and that phosphorylation at tyrosine residues Y125, Y133 and Y135 suppresses

119

S129-P-induced aggregation and toxicity. These studies demonstrate the presence of multiple phos­ phorylation sites within a-syn and suggest potential cross-talk between the different modified sites.

All potential phosphorylation sites in a-syn are highly conserved A close examination and comparison of the amino acid sequences of all synucleins from humans and other species reveals that the majority of potential a-syn phosphorylation sites (4 serine, 10 threonine, 4 tyrosine residues, Fig. 3b) are highly conserved in all species, suggesting that phosphorylation at one or multiple sites may play important roles in reg­ ulating a-syn function in health and disease.

Considering all a-syn phosphorylation sites identi­ fied in vivo [S129 (Chen and Feany, 2005; Fujiwara et al., 2002), S87 (Paleologou et al., 2010) and Y125 (Chen et al., 2009)] and in vitro [S87 (Kim et al., 2006; Okochi et al., 2000); S129 (Chen and Feany, 2005; Fujiwara et al., 2002; Kim et al., 2006; Okochi et al., 2000; Pronin et al., 2000; Takahashi et al., 2003) and Y125, Y133 and Y136 (Ahn et al., 2002; Ellis et al., 2001; Nakamura et al., 2001; Negro et al., 2002; Takahashi et al., 2003)], it is striking that all these sites cluster at the C-terminal region of a-syn spanning residues 120–140. Only S87 lies in the hydrophobic NAC region of a-syn, which is essential for a-syn aggregation and fibrillogenesis (El-Agnaf et al., 1998). These findings suggest important roles for these modifications in regulat­ ing protein–protein, protein–ligand and protein–

a

b

Fig. 3. a-syn phosphorylation in normal and pathological conditions. (a) Schematic depiction highlighting all the potential phosphorylation sites in a-syn: serine (S), threonine (T) and tyrosine (Y). When phosphorylation of the residue has been reported, the corresponding known kinases are indicated; the sites phosphorylated in human are listed in the upper part of the schema. (b) Multiple sequence alignment of a-syn generated by MUSCLE (version 3.6), in which serine, threonine and tyrosine residues are shown in color; note their high level of conservation trough mammals as only serine 87 is not preserved in rodents.

120

metal interactions, which have been mapped and shown to be mediated by the C-terminal domain of a-syn (see below). Unravelling the role of phosphorylation in modulating the physiological and pathogenic activities of a-syn requires identification of the kinases and phosphatases involved in regulating its phosphorylation in vivo Although the exact kinases and phosphatases responsible for regulating a-syn phosphorylation at S129 in vivo are still not known, a series of in vitro and cell culture-based studies have identified a num­ ber of kinases, which phosphorylate a-syn at S129 and/or S87, including casein kinase I (CKI) (S87 and S129), casein kinase II (CKII) (S129) (Okochi et al., 2000) and the G protein-coupled receptor kinases (GRKs 1, 2, 5 and 6; S129) (Pronin et al., 2000), LRRK2 (leucine-rich repeat kinase 2) (S129) (Qing et al., 2009) and PLKs (polo-like kinases) (S129) (Inglis et al., 2009; Mbefo et al., 2010) (Fig. 3a). Tyrosine phosphorylation has also been reported at Y125 by Fyn (Nakamura et al., 2001), Syk (Negro et al., 2002), Lyn (Negro et al., 2002), cFrg (Negro et al., 2002) and Src tyrosine kinases (Ellis et al., 2001), with Syk (Negro et al., 2002) also phosphorylating at Y133 and Y136. Phosphorylation at S129 and/or S87 alters the conformation of monomeric a-syn and inhibits its fibrillization in vitro Current cellular and in vivo studies aimed at dissect­ ing the functional consequences of phosphorylation at serine, threonine or tyrosine residues mainly rely on genetic approaches to block (e.g. replacing serine/ threonine with alanine, S/T ! A, or tyrosine with phenylalanine, Y ! F) or mimic constitutive phos­ phorylation (e.g. replacement of serine with gluta­ mate or aspartate, S ! E/D). The structural and electrostatic similarities between glutamate/aspar­ tate (net charge of –1) and phosphoserine (net charge of –2) suggest that this type of substitution represents

a reasonable approach to mimic constitutive phos­ phorylation at specific serine residues. However, detailed studies from our group have shown that S ! D/E substitutions do not reproduce all aspects of phosphorylation. For example, structural compar­ isons of the phosphomimics S129D/E with S129-P using nuclear magnetic resonance (NMR) indicate that phosphorylation at S129 and S87 increases the hydrodynamic radius of a-syn to a value comparable to that observed for the WT protein in 8 M urea. In addition, paramagnetic relaxation enhancement of amide protons observed with the help of a spin label attached to residue 18 showed that long-range interactions between the N- and the C-terminal domains of a-syn are attenuated upon phosphoryla­ tion at S129 and S87. On the other hand, mutation of S129 and S87 to glutamate (S129E and S87E) or aspartate (S129D and S87D) does not lead to an extended conformation of monomeric a-syn. This result suggests that mutation of S ! D/E cannot fully mimic the effect of phosphorylation on the structure and dynamics of a-syn. The extent of fibril formation by the phosphomimics S129E/S129D is similar to that of the WT a-syn, whereas phosphor­ ylation at S129 (S129-P) consistently inhibits a-syn fibrillization. On the other hand, S ! D/E substitu­ tion at S87 reproduces the effect of phosphorylation on a-syn aggregation at this residue, that is both substitutions inhibit the fibrillization of a-syn in vitro. However, phosphorylation at S87, but not the S87D mutation, increases the hydrodynamic radius of a-syn and influences its binding to membranes. Together, these studies demonstrate that phosphor­ ylation of a-syn at S129 and/or S87 significantly inhi­ bits its aggregation (Paleologou et al., 2008, 2010; Waxman and Giasson, 2008) and that the S ! D/E substitutions do not reproduce all aspects of phos­ phorylation. Therefore, the results obtained with these mimics should be interpreted with caution. Does phosphorylation promote or prevent a-synuclein aggregation in vivo? To answer this question, one must be able to modulate the level of phosphorylation in vivo.

121

This modulation can be achieved by co-expression of a-syn and the relevant kinase or by comparing the effect of over-expressing the WT protein to the mutants designed to mimic (S129D) or abolish phosphorylation (S129A)(see Table 2). Although in vitro biophysical studies have clearly shown that replacement of serine by glutamate or aspartate does not reproduce all aspects of phosphorylation, the use of these mutants remains the only option for elucidating the role S129 phosphorylation in regulating a-syn aggregation and toxicity in vivo, especially given the lack of knowledge of efficient kinases that phosphorylate a-syn at this residue. In the next sections, we will present an overview of the findings from different in vivo studies aimed at elucidating the consequences of S129 phosphorylation.

GRK2-mediated phosphorylation at S129 or substitution of serine with aspartate (S129D) promotes a-syn oligomerization but does not influence inclusion formation in rodent and Drosophila models of PD Initial studies aimed at investigating phosphoryla­ tion at S129 were carried out in Drosophila, a model in which a-syn expression induces a loss of dopaminergic neurons or retinal degeneration, depending on the promoter used (Feany and Bender, 2000). The co-expression of the Droso­ phila homologue of the kinase GRK2 with a-syn did not prompt or prevent the appearance of pro­ teinase K-resistant inclusions (Chen and Feany, 2005), but it did enhance the formation of a-syn oligomers (Chen et al., 2009) compared to situa­ tions where only a-syn was over-expressed. These results suggest that increasing S129 phosphoryla­ tion does not promote a-syn fibrillization in vivo and argue in favour of the hypothesis that S129 phosphorylation results in the kinetic stabilization and/or accumulation of toxic a-syn oligomers. What remains unclear is whether the overexpression of GRK2 contributes to a-syn toxicity via mechanisms that are independent of a-syn

phosphorylation. Further, the efficiency with which GRK2 phosphorylates a-syn and the turn­ over of S129-P was not investigated in these studies. To overcome the drawbacks associated with kinase over-expression, a complementary approach was used, based on the expression of the S129D or S129A variant. In Drosophila, over-expression of the phosphomimic variant (S129D) yielded results that are comparable to those obtained with GRK2 over-expression: (1) inclusion formation by S129D was similar to that observed for the WT protein and (2) S129D formed more oligomeric forms of a-syn than the WT protein. Similarly, in vitro studies using recombinant proteins demonstrated that fibril for­ mation by the phosphomimics S129D/S129E is similar to that of the WT protein. Together, these studies demonstrate that S ! D mutation at S129 alters the oligomerization, but not fibrillization, of a-syn.

Substitution of serine 129 with alanine (S129A) promotes a-syn inclusion formation and fibrillization in vivo and in vitro, respectively Interestingly, in both the fly and the rodent models, the S129A mutants forms five times more insoluble, proteinase K-resistant inclusions (but fewer oligomers) than WT and S129D a-syn (Chen and Feany, 2005; Chen et al., 2009). In vitro, the S129A mutant forms fibrils more readily and to a greater extent than WT or the S129D mutant. Neither the S ! A nor the S ! E mutation alters the structure or morphology of a-syn fibrils in vitro. Initially, these findings appear to support the toxic hypothesis model, where accelerated fibrillization by this mutant ensures depletion of the toxic oligomers, thus explaining the reduced toxicity observed for S129A in the fly model. However, this correla­ tion between accelerated fibrillization and reduced toxicity did not hold consistently in various rodent models.

122

In rodent models, the effects of S ! D and S ! A substitutions on a-syn aggregation are variable In rodent, over-expression of S129D and S129A a-syn, using AAV-mediated gene transfer in the substantia nigra, led to inconsistent observations, partially due to the different approaches used to characterize and quantify a-syn aggregation. At first, Gorbatyuk et al. (2008) reported that both S129D and S129A form punctate a-syn aggregates in vivo. The authors described a-syn S129D immu­ nostaining in neuronal cytoplasm as heterogeneous, whereas WT or a-syn S129A immunolabelling was more evenly distributed throughout the cell, with­ out providing quantitative evaluation of the level of aggregation. Furthermore, neither the biochemical and structural properties of these aggregates nor the a-syn oligomerization in this model was thor­ oughly investigated, which precludes comparison of the extent and type of a-syn aggregation in other models. On the contrary, a detailed study by Azer­ edo da Silveira et al. (2009) provided experimental observations of aggregation that agreed with data obtained in Drosophila: a-syn S129A forms more b-sheet-rich (thioflavin S-positive), proteinase K-resistant aggregates than WT and S129D a-syn. Finally, a recent study (McFarland et al., 2009) described a-syn-positive deposits in dopaminergic neurons, but revealed similar aggregation patterns for all three proteins (WT, S129D and S129A a-syn). The discrepancy with the previous two models might be due to the fact that McFarland et al. (2009) used a bi-cistronic mRNA to co­ express a-syn and green fluorescent protein (GFP), which may lead to lower expression of a­ syn compared to classical monocistronic RNA. Recent reports suggest neuronal toxicity associated with AAV-mediated over-expression of GFP in the substantia nigra, which could mask the differences in terms of neuronal loss caused by a-syn and the two S129 mutants (Baens et al., 2006; Sawada et al., 2010; Ulusoy et al., 2009). An additional contribut­ ing factor might be the fact the immunohistological analysis was carried out 6 weeks after virus injec­ tion, compared to 8 weeks in the studies by

Azeredo da Silveira et al. (2009) and Gorbatyuk et al. (2008).

Does phosphorylation enhance or protect against a-syn toxicity? The accumulation of S129-P a-syn has been asso­ ciated repeatedly with disease states both in post­ mortem human brains (Fujiwara et al., 2002) and in various models of synucleinopathy (Lo Bianco et al., 2002; Neumann et al., 2002). However, the extent to which does S129 phosphorylation and/or the accumulation of a-syn S129-P represent toxic events in the pathogenesis of PD is not yet fully understood. In Drosophila, the co-expression of a-syn with the GRK2 homologue, as well as over-expression of a-syn S129D, accelerates neu­ ronal loss compared with WT a-syn or S129A alone (Chen and Feany, 2005), suggesting a toxic effect of a-syn S129-P. On the contrary, in a screening performed in yeast, aimed at identifying modulators of a-syn-induced toxicity, PLK2, which was recently shown to be an efficient kinase and a major contributor to S129 phosphorylation in vitro and in vivo, was shown to be protective (Mbefo et al., 2010). This finding was confirmed in two other models, Caenorhabditis elegans and pri­ mary cultures of rat mesencephalic neurons (Gitler et al., 2009). Studies from two independent groups based on the over-expression of the a-syn variants S129D and S129A in rat models support the hypothesis that a-syn S129-P is protective (Azeredo da Silveira et al., 2009; Gorbatyuk et al., 2008). Indeed, over-expression of a-syn S129A in rat substantia nigra was reported to be more toxic than when the WT protein or S129D mutants were over-expressed (Azeredo da Silveira et al., 2009; Gorbatyuk et al., 2008). The results from the two rat models support the hypothesis that phosphorylation of a-syn is pro­ tective. The following findings are consistent with this hypothesis: (1) PLK2 over-expression protects against a-syn-induced toxicity in yeast, C. elegans and primary rat neurons (Gitler et al., 2009) and

123

(2) disease-associated a-syn mutations (E46K and A30P) reduce the level of a-syn phosphorylation at S129 in cell culture and upon in vitro phosphor­ ylation with CK1 and PLK2 (Paleologou and Lashuel, unpublished data). However, a recent study by Hyman and colleagues reported a similar toxicity for the three variants (WT, S129D and S129A) (McFarland et al., 2009). As mentioned above, the results from this study might be biased by co-expression of GFP and a-syn (Baens et al., 2006; Sawada et al., 2010; Ulusoy et al., 2009).

Different mechanisms may be involved in modulating a-syn toxicity in the Drosophila and rodent models of PD Although the increased aggregation of S129A cor­ relates well with its enhanced toxicity in the rat model (Azeredo da Silveira et al., 2009), the oppo­ site was observed in the fly model, where the increased aggregation of S129A, relative to WT and S129D, correlates with protection against a-syn-induced toxicity (Chen and Feany, 2005) (see Table 2). These observations clearly indicate the involvement of different neuropathologic mechanisms between the two models and suggest that a direct correlation between aggregation and toxicity in the fly and rat models may not be possi­ ble, especially if different approaches are used to assess and quantify toxicity and aggregation in the different models. Furthermore, there is no evidence that the aggregation pattern and structure of the aggregates/inclusions formed in the two models are identical. Initial studies by Chen and Feany reported the absence of thioflavin S staining or proteinase K-resistant aggregates in conditions of neuronal loss, where the accumulation of such aggregates occurs and correlates with neuronal loss in the rat model. Furthermore, in the rat model, S129D, which tends to be neuroprotective, formed fewer but larger aggregates than a-syn S129A (Azeredo da Silveira et al., 2009). More recently, Feany and colleagues showed, by western blot, that a-syn-associated toxicity correlates with

the accumulation of soluble high molecular weight oligomeric a-syn species. Therefore, a careful ana­ lysis of data obtained in Drosophila suggests that large aggregates are well handled and do not impair cell survival, while soluble oligomers could be the primary toxic species in flies. Unfortunately, similar biochemical studies have not been carried out in the rat model, so direct comparison of the degree of oligomer formation and accumulation between the two models is not possible. An alternative hypoth­ esis, which could account for the differential toxicity of inclusions between rat and fruit flies, relies on the fact that Drosophila is naturally devoid of a-syn, whereas a rat homologue does exist and bears strong sequence similarity to the human a-syn (>95%). Previous studies have shown that mouse and rat a-syn form fibrils more readily than human a-syn and influence the oligomerization and fibril­ logenesis of human a-syn. Whether the interactions between rat and human a-syn could explain the differences in toxicity between the rat and the fly models remains unknown. However, this hypoth­ esis can be tested by over-expressing a-syn, S129D or S129A in the substantia nigra of one of the a-syn knockout mouse models.

Despite the differences, some consistent observations emerged from both models The effect of phosphorylation using the phosphomimicking approach has yielded different results depending on the host organism (fly vs. rat) and the methods employed to assess and/or quantify aggregation and toxicity (see Table 2). Despite the discrepancy in terms of a-syn toxicity in the different models, comparing the results obtained in the Drosophila and rat models reveal some con­ sistent observations. (1) In addition to blocking phosphorylation, substitution of serine with alanine enhances the aggregation of a-syn in both animal models and in vitro, based on quantitative analysis of a-syn solubility and thioflavin S labelling. (2) In both fly and rat models, S129D exhibits similar aggre­ gation properties as the WT protein (Azeredo da

124

Silveira et al., 2009; Chen and Feany, 2005). Interestingly, both observations are in complete agreement with in vitro biophysical studies using purified recombinant a-syn. These studies showed that a-syn S129A aggregates more rapidly than the WT protein (Paleologou et al., 2008). Although the effects of these two muta­ tions (S ! A and S ! D) on a-syn aggregation are reproducible in these different models (Table 1), subsequent studies raised some con­ cern about their utility to model the phosphory­ lation state of the protein in vivo. A direct comparison of the structural and aggregation properties of monomeric S129D and S129-P, which was prepared by phosphorylating a-syn with CK1 or PLK2, demonstrated that phosphor­ ylation at S129 increases the conformational flex­ ibility of a-syn and strongly inhibits its fibrillization, whereas monomeric S129D exhibits structural and aggregation properties similar to those of the WT protein (Fig. 4c). Observations of intraneuronal deposits are often a good start­ ing point for assessing aggregation, but further analyses to characterize the solubility of struc­ tural properties (using electron microscopy, thioflavin S staining and proteinase K digestion) and oligomerization state (immunoblotting using denaturing and native gel electrophoresis) of a­ syn within these inclusions are crucial to deter­ mine accurately how phosphorylation affects a­ syn aggregation and to elucidate the relationship between a-syn aggregation and toxicity in vivo.

What are the molecular mechanisms underlying phosphorylation-dependent a-syn toxicity? A limited number of studies have attempted to dissect the cellular mechanism associated with phosphorylation-dependent a-syn neurotoxicity in vivo. Using electron microscopy, Azeredo da Silveira et al. (2009) sought to examine subcellu­ lar abnormalities induced by enhancing or block­ ing a-syn phosphorylation. These studies revealed the presence of lysosomal bodies and phagosome-like structures trapped within the aggregates, suggesting an attempt by the neurons to degrade the inclusions by macroautophagy (Azeredo da Silveira et al., 2009). In addition, they reported the association of a-syn S129A with membranous structures, endoplasmic reticu­ lum (ER) and Golgi apparatus, mostly in the vicinity of aggregates; these observations sug­ gested an impairment of ER–Golgi trafficking by non-phosphorylated a-syn. On the contrary, in SH-SY5Y cells, over-expression of a-syn S129A or inhibition of CKII decreases the activa­ tion of ER stress and caspase 3 cleavage. This study also reported that the increase of S129-P a-syn levels, following rotenone treatment, is associated with activation of these pathways (Sugeno et al., 2008). Therefore, whether ER stress is due to a-syn or related to its phosphor­ ylation state remains to be determined. Azeredo da Silveira et al. (2009) suggested that Golgi–ER impairment results from a dysfunction of the

Table 1. Consequences of mutations at position 129 of a-syn on its aggregation and on cell survival AGGREGATION MODEL Rat Rat Rat Drosophila

Gorbatyuk et al., 2008 Azeredo da Silveira et al., 2009 McFarland et al., 2008 Chen and Feany, 2005

SURVIVAL

a-synS129A

a-syn

a-synS129D

a-synS129A

a-syn

a-synS129D

nd ✹✹

nd ✹

nd ✹

†† ††

† †

– †

✹ ✹✹

✹ ✹

✹ ✹

† –

† †

† ††

nd: not determined; ✹: aggregation; †: cell death; –: no cell loss.

125 a

b

c

Fig. 4. The phosphomimics S129D/E or S87E do not reproduce the effect of phosphorylation on the structural properties of monomeric a-syn. (a) Structural comparison of the residue serine, phosphoserine and glutamate illustrating the similarities between the two last species. (b) The hydrodynamic radius of a-syn and a-syn S129D in a phosphate buffer is similar, around 28A (black bar), while the radius increased to 35A after phosphorylation of a-syn either at both S87 and S129 (p-WT) or at S129 only (pS87A) (dashed bars). In all cases, the radii rose to 36A in the presence of urea (white bars) corresponding to a true random-coiled state. (c) Illustration of the conformations populated by a-syn, a-syn S129E and a-syn S129-P (S129-p, S87-p) and of the long-range interactions involved (from Paleologou et al., 2008).

microtubule-mediated transport system, as indi­ cated by the disarrayed neurofilament network when a-syn S129A is over-expressed. In cell lines, phosphorylation at S129 induces microtu­ bule retraction (Kragh et al., 2009). Phosphory­ lated a-syn (S129-P) also co-localizes with activated caspase 9 in different models of synu­ cleinopathy (Fournier et al., 2009; Yamada et al., 2004). It is worth notifying that activated caspase 9 immunostaining is preferentially found in cells positive for S129-P, although this later category of cells represented only a minor subpopulation among neurons accumulating a-syn (Fournier et al., 2009).Over-expression of a-syn S129A or a-syn S129D in rat substantia nigra leads to acti­ vation of caspase 9 (Azeredo da Silveira et al.,

2009), indicating that caspase activation occurs independent of S129 phosphorylation and is likely to be a consequence of a-syn expression.

Novel phosphorylation sites Recent studies from the Feany’s laboratory and our group demonstrated that a-syn in vivo is phosphory­ lated at S87 and Y125. Studies by both groups sug­ gest that modifications at this site correlate with disease and significantly influence the oligomeriza­ tion and fibrillization of a-syn. The levels of S87-P were increased in brains of TG models of synuclei­ nopathies and human brains from AD, Lewy body disease (LBD) and MSA patients. Using antibodies

126

against phosphorylated a-syn (S129-P and S87-P), significant levels of immunoreactivity were detected in the membrane in the LBD, MSA and AD cases but not in normal controls. Given that the remaining potential phosphorylation sites in a-syn are highly conserved, it would not be surprising that these residues also undergo phosphorylation and may play a role in modulating the physiologic and/or pathogenic properties of a-syn. Therefore, further studies, using phospho-specific antibodies targeting the different potential phosphorylation sites, are necessary to map all the phosphorylation sites in a­ syn and elucidate their role in regulating its struc­ ture, aggregation and function(s).

Truncations Truncated a-syn species are present in the normal brain and aggregate with the full-length a-syn in PD and related disorders Biochemical characterization of aggregated a-syn from LBs revealed that it comprises predominantly full-length a-syn, in addition to small amounts of various truncated species with apparent molecular masses of 10–15 kDa (Anderson et al., 2006; Baba et al., 1998; Campbell et al., 2001; Crowther et al., 1998; Li et al., 2005; Liu et al., 2005; Okochi et al., 2000; Spillantini et al., 1998). At least five species were detected using mass spectrometry and repre­ sent C-terminal truncations (Fig. 5) (Anderson et al., 2006). A comparison between LB-derived and cytosolic a-syn forms showed that some trun­ cated species were exclusively observed in the a­ syn derived from LB. The cleavage sites were deter­ mined by tryptic digestion and sequencing using liquid chromatography followed by mass spectro­ metry (LC-MS/MS). Species terminating at D-115 (a-syn-D115), D-119 (a-syn-D119), N-122 (a-syn­ N122), Y-133 (a-syn-Y133) and D135 (a-syn­ D135) were identified (Fig. 5) (Anderson et al., 2006). Three additional truncated forms of a-syn were also identified in samples from PD, DLB and MSA brain tissues: two C-terminal truncated forms

(ending approximately between amino acid resi­ dues 102–125 and 83–110, respectively) and a third N- and C-terminal truncated isoform, which were only detectable in aggregated forms of a-syn (Li et al., 2005; Liu et al., 2005; Tofaris et al., 2003). Interestingly, these isoforms are present in healthy and diseased brains, suggesting that a-syn trunca­ tion occurs under physiologic conditions. The most striking difference is that PD and DLB extracts contained appreciable amounts of truncated a-syn in SDS- and urea-soluble fractions and a significant level of the N- and C-terminal truncated forms (Li et al., 2005; Liu et al., 2005). All together, these observations show that a-syn truncation occurs under normal conditions and suggest that the trun­ cated a-syn may have a normal physiologic role. These findings also suggest that the C-terminal trun­ cated forms that accumulate selectively in LBs or insoluble fractions aggregate more readily and could act as effective seeds to accelerate the aggregation of the full-length protein.

C-terminal truncations promote the fibrillization of a-syn in vitro Among the various post-translational modifications identified to date, the C-terminal deletion variants of a-syn consistently exhibit higher fibrillization pro­ pensity relative to the WT full-length protein. These findings, combined with the observation obtained for C-terminal deletion variants of a-syn in human brains and brains of TG animal models of PD (Li et al., 2005), led to the hypothesis that proteolytic processing of the C-terminus could be responsible for the initiation of a-syn fibrillogenesis in PD, pos­ sibly via a seeding mechanism. Lee and colleagues also reported several naturally occurring C-terminal deletion variants (including amino acids 1–119 or 1–122) in a-syn over-expressing TG mice and pro­ posed that proteolytic processing of the C-terminus of a-syn may play a critical role in the initiation of a-syn aggregation and fibrillogenesis in vivo. The effect of proteolytic cleavage on the aggre­ gation of a-syn has been extensively investigated

127

a Lewy bodies PD whole brain tissue

Neuroxin Proteasome Calpain MMPs

2-14

Cathepsin D

120−125

102−110

122 96−105 115

61

1

9

95 80 83 73 77 78 73 75

54 57 57

119 126−129135 133

Monomers

140 114

97 110

120 114

91−98

122

Fibrils 91−115

119 120

b

1

61

140

95 87

130

120

Fig. 5. Truncation of a-syn in vivo and in vitro. (a) In the upper part of the schema are represented the known sites of truncation of a-syn identified in extracts from LB (black arrows; Anderson et al., 2006) or from brain tissue of PD cases (pink arrows; Li et al., 2005, Liu et al., 2005). Last residue is indicated, or, when not defined, a range is given. Various enzymes able to cleave a-syn monomer or fibrils have been described; their site of digestion is indicated in the lower part of the schema. (b) Representation of the truncated forms studied in TG mice (upper part; Tofaris et al., 2006, Wakamatsu et al., 2008, Daher et al., 2009) and Drosophila (lower part; Periquet et al., 2007).

using recombinant proteins and by over-expres­ sing C-terminal deletion variants in cell lines. In vitro fibrillization studies have consistently shown that truncation of various segments of the C-terminal residues 110–140 enhances the rate of a-syn aggregation and fibrillogenesis. Some fragments (1–110; 1–120) promote nucleation

(Hoyer et al., 2004) and seed the aggregation of full-length a-syn (Li et al., 2005; Murray et al., 2003). Serpell et al. (2000) reported that 1–87 a­ syn aggregates more rapidly than 1–120 a-syn and rat a-syn, which aggregates faster than human a­ syn (Rochet et al., 2000). Similarly, studies by Murray et al. (2003) showed that the C-terminal

128

truncated a-syn variants 1–89, 1–102, 1–110, 1–120 and 1–30 aggregated more rapidly than the fulllength protein with the 1–110 variant showing the most robust enhancement of a-syn fibril forma­ tion. Interestingly, the 1–102 and 1–110 variants, but not 1–120, seed the fibrillization of the fulllength protein in vitro. Together, the results from these studies combined with data from solid-state NMR (Bertini et al., 2007; Wu et al., 2008), and immunogold labelling of a-syn fibrils (Murray et al., 2003) suggest that fibril formation by a-syn is mediated by its N-terminal domain (~1–90), whereas the C-terminal region remains flexible and may play a role in inhibiting the aggregation of monomeric a-syn (Murray et al., 2003). Subse­ quent NMR studies provided further evidence in support of this hypothesis and demonstrated that the C-terminal domain participates in long-range interactions with the N-terminal region of a-syn. These interactions shield the hydrophobic regions within the protein and prevents its self-assembly (Bertoncini et al., 2005; Pawar et al., 2005). Sev­ eral groups have also shown that the C-terminal 20–30 amino acids, which are highly anionic, inhi­ bit fibrillization (Crowther et al., 1998; Kim et al., 2002; Tofaris et al., 2006). This inhibition is mediated by transient interactions between the C-terminal region and the amyloidogenic NAC region. Truncations of residues in this region or a charge-neutralizing effect (e.g. by divalent metals such as Ca2þ and Cu2þ and polyamines) may account for the fibril acceleration by cations, including polyamines and C-terminal deletion mutants of a-syn (Antony et al., 2003; Cohlberg et al., 2002; Goers et al., 2003) and certain metals (Paik et al., 1999; Uversky et al., 2001; Yamin et al., 2003).

Several enzymes have been implicated in the proteolysis of a-syn Although the inhibitory activity of the C-terminal sequence suggests that proteolytic cleavage of this region could promote the fibrillization of a-syn in

vivo, a protease that selectively cleaves this sequence has not been identified. A number of enzymes have been implicated in a-syn cleavage and generation of truncated fragments. Neurosin, a trypsin-like serine protease, was detected in LBs (Iwata et al., 2003; Ogawa et al., 2000). The in vitro cleavage of a-syn by neurosin generates one major fragment 1–80, which does not aggre­ gate in vitro (Iwata et al., 2003) and three minor ones: 1–97, 1–114 and 1–121 (Kasai et al., 2008) (Fig. 5). Interestingly, the phosphorylated form (S129-P) and the disease-associated mutants (A30P and A53T) are more resistant to proteoly­ sis by neurosin (Kasai et al., 2008). The intracellular calcium-dependent protease calpain cleaves monomeric WT or mutant (A30P and A53T) a-syn at several sites within the NAC region to yield fragments that inhibit the aggrega­ tion of the full-length protein (Mishizen-Eberz et al., 2003, 2005) (Fig. 5). The major generated fragments consist of two N-terminally truncated fragments, 58–140 and 84–140, and four C-termin­ ally truncated forms, 1–57, 1–73, 1–75 and 1–83 (Mishizen-Eberz et al., 2003). Furthermore, using N-terminal sequencing and an antibody against the N-terminal truncated a-syn, Dufty et al. (2007) reported another calpain cleavage site between the residues 9 and 10. In the fibrillar state, calpain-mediated cleavage occurs exclu­ sively within the C-terminal region (residues 114 and 122) (Mishizen-Eberz et al., 2005), probably due to this region remaining flexible and exposed to proteases. Calpain-cleaved a-syn species were found in LB and Lewy neurites in diseased brain and co-localize with activated calpain, suggesting a link between a-syn proteolysis by this enzyme and a-syn aggregation and pathology (Dufty et al., 2007). Recently, a lysosomal enzyme, cathepsin D was reported to generate two forms of C-terminally truncated a-syn detectable at 12 and 10 kDa, respectively (Sevlever et al., 2008; Takahashi et al., 2007). Using antibodies to different a-syn epitopes, the authors demonstrated that these fragments end approximately between the

129

residues 91–98 and 91–115, respectively (Sevlever et al., 2008; Takahashi et al., 2007). Sevlever et al. (2008) also demonstrated that cathepsin-gener­ ated a-syn fragments are the major component of oligomeric a-syn species formed under oxida­ tive stress. The formation of these correlated with an increase of CKII expression and a-syn phos­ phorylation in cell culture. These observations suggest a possible relationship among a-syn trun­ cation, phosphorylation and oligomerization when cells are exposed to oxidative stress. Although ubiquitin-mediated degradation of a­ syn has been proposed by several studies, a-syn was reported to undergo proteolytic cleavage by the proteasome, in the absence of ubiquitination (Tofaris et al., 2001). The caspase-like activity of the 20S proteasome generates four a-syn frag­ ments corresponding to 1–73, 1–83, 1–110 and 1–119 (Lewis et al., 2010; Liu et al., 2005). In vitro aggregation assay showed that the 1–110 and 1–120 a-syn fragments aggregate more rapidly than the full-length and can seed the aggregation and formation of hybrid protofibrils of truncated and non-truncated a-syn (Lewis et al., 2010; Liu et al., 2005). The PD-linked mutations do not significantly affect the cleavage of a-syn by the proteasome (Lewis et al., 2010); however, the fragments containing the mutation A53T aggre­ gated more rapidly than the truncated WT and full-length A53T and were shown to seed and accelerate the aggregation of the full-length pro­ tein (Liu et al., 2005). The matrix metalloproteases (MMPs, e.g. MMP-1 and MMP-3), a family of zinc-dependent endopeptidases, also cleave a-syn and generate several C-terminally truncated fragments in vitro (1–54, 1–57, 1–79 and 1–78) (Levin et al., 2009; Sung et al., 2005). Levin and collaborators showed that a-syn in vitro aggregation is increased after a limited proteolysis by MMPs. However, higher MMP concentrations resulted in an inhibition of a-syn aggregation (Levin et al., 2009), most likely due to increased MMP-mediated cleavage within the NAC region, which is essential for a-syn oli­ gomerization and fibril formation.

Truncation (in vivo studies) To explore in vivo the effect of C-terminal trunca­ tions on the aggregation and toxicity of a-syn, TG flies and mice over-expressing various truncated forms of a-syn have been generated (see Table 2). In Drosophila, two different truncations have been investigated: a-syn 1–87, corresponding to a non­ natural truncation with the entire acidic C-terminal domain was deleted and a pathological truncation, a-syn 1–120, with the last C-terminal amino acids removed (Periquet et al., 2007). The 1–87 a-syn variant did not aggregate or induce toxicity when expressed in Drosophila, although expression at levels similar to that of the WT a-syn could not be reached, which may account for these observations. Conversely, the pan neuronal expression of a-syn 1–120 resulted in the appearance in the Drosophila brain, of more abundant oligomers and a-syn­ positive, proteinase K-resistant inclusions as com­ pared to the full-length protein expressed at similar levels. The formation of inclusions by a-syn 1–120 was accompanied by a loss of dopaminergic neu­ rons, which occurred slightly faster for the trun­ cated than for the full-length a-syn. Three different TG mice have been produced, expressing truncated variants of a-syn under the control of the TH or nestin promoter. The first TG mice were generated in a strain devoid of endogenous a-syn; the expression level of a-syn 1–120 was lower than what is expected for the rodent protein (Tofaris et al., 2006). Dopaminergic neurons from the olfactory bulbs and from the substantia nigra presented some a-syn-positive fibrillar inclusions (thioflavin S-positive). Despite the fact that expression of 1–120 resulted in the formation of both non-fibrillar and fibrillar a-syn aggregates, no neuronal loss was observed at 12 months of age, although DA and homovanilic acid (HVA) levels were decreased in 3-month-old animals. Wakamatsu et al. (2008) generated TG mice that over-express either the pathogenic mutant A53T or a truncated variant of this mutant in catecholaminergic neurons comprising residues 1–130 (a-syn A53T, 1–130). The expression of

130 Table 2. Animal models looking at consequences of truncation and phosphorylation on a-syn aggregation and toxicity PTM studied

Model

Strategy

a-syn 1-87

Drosophila

a-syn 1-120

Drosophila

Pan neuronal Promoter Pan neuronal Promoter

Aggregation method of assessment

Toxicity method of assessment

Publication(s)

n.d.

– Number of TH neurons þ Number of TH neurons

Periquet et al. (2007) Periquet et al. (2007)

– Number of TH neurons DA decreased at 3 months – Number of TH neurons DA decreased at 12 months with TH promoter þ number of TH neurons (developmental defect) – PKK2 suppressed asyn-induced cell death – PLK2 suppressed asyn-induced cell death þ Number of TH neurons Ommatidia disruption

Tofaris et al. (2006) Michell et al. (2007) Daher et al. (2009)

þ IHC (a-syn) PK digestion and HMW by WB þ IHC (a-syn) e-microscopy Thio S

Mouse

TH promoter (a-syn KO background)

a-syn 1-119

Mouse

Inducible expression (TH- or nestion-promoter)

– IHC (unpublished)

a-syn A53T 1-130

Mouse

TH promoter

– IHC (a-syn)

a-syn S129-P

Yeast

a-syn þPLK2

n.d.

C. elegans

a-syn þ PLK2

n.d.

Drosophila

a-synþGRK2 pan neuronal or eye targeting promoter

– PK digestion but HMW in WB – IHC (a-syn) PK digestion Solubility but HMW in WB þ IHC (inclusions) PK digestion Solubility þ IHC (a-syn) – IHC (a-syn)

a-syn S129D pan neuronal or eye targeting promoter

Rat

Rat

a-syn S129A pan neuronal or eye targeting a-syn S129D CBA promoter a-syn S129A CBA promoter a-syn S129D CMV promoter

a-syn S129A CMV promoter

– ThioS staining PK digestion e-microscopy þ ThioS staining PK digestion e-microscopy

Wakamatsu et al. (2008a, 2008b) Gitler et al., (2009) Gitler et al. (2009) Chen and Feany (2005) Chen et al. (2009)

þ number TH neurons ommatidia disruption – Number of TH neurons Ommatidia disruption – Number of TH neurons þ Number of TH neurons DA decrease – Number of TH neurons

Gorbatyuk et al. (2008)

Azeredo da Silveira et al. (2008)

þ Number of TH neurons

(Continued )

131 Table 2. (Continued ) PTM studied

a-syn Y125P, Y133-P, Y136-P

Model

Strategy

Rat

a-syn S129D GFP co-expressed CBA promoter a-syn S129A GFP co-expressed CBA promoter a-syn Y125F, Y133F, Y136F pan neuronal or eye targeting promoter a-syn þ shark pan neuronal promoter

Drosophila

Aggregation method of assessment

Toxicity method

of assessment

– IHC (a-syn, ubi)

– Number of TH neurons DA level

– Number of TH neurons DA level þ

– IHC (a-syn, ubi) n.d. but HMW by WB n.d. but reduced HMV by WB

Publication(s)

McFarland et al.

(2009)

Chen et al. (2009)

Number of TH neurons ommatidia disruption – Number of TH neurons Shark reduced a-syn­ induced cell death

In various model over-expressing a-syn, the appearance of a-syn S129-P deposits correlates with the development of ubiquitin immunoreactivity, proteinase K-resistant and thioflavine S-positive inclusions. n.d.: no data; þ: present; -: absent; CBA: CMV beta-actin; CMV: cytomegalovirus; DA: dopamine; Dopac: 3,4-dihydroxyphenylacetic acid; e- microscopy: electron microscopy; GFP: green fluorescent protein; HVA: homovanilic acid; HMW: high molecular weight species; IHC: immunohistochemistry; KO: knockout; PK: proteinase K; TH: tyrosine hydroxylase positive; Thio S: thioflavine S staining; ubi: ubiquitin; WB: western blot.

exogenous a-syn ranged from 1.3- to 1.6-fold the level of the endogenous protein. When the A53T 1–130 variant was over-expressed, the animals dis­ played a developmental decrease in the number of dopaminergic neurons from the substantia nigra, while animals expressing the full-length a-syn A53 did not present any defect. No abnormal proteinac­ eous accumulations were detected in the affected brain regions, despite the fact that the absence of endogenous a-syn was expected to promote aggre­ gation (Wakamatsu et al., 2008). Finally, Daher et al. (2009) described the consequences of Cre­ dependent expression of the C-terminal truncation a-syn 1–119. The authors did not observe any protein deposition by immunohistochemistry or neuronal loss, although dopamine, 3,4-dihydroxy­ phenylacetic acid and HVA were decreased in the striata of 10-month-old mice; the low protein level likely explains this mild phenotype, as the expres­ sion of a-syn 1–119 was 10-fold lower than that of the endogenous protein. The toxicity of the C-terminal truncated forms of a-syn was also assessed using in vitro cell death

assays. The addition of hybrid protofibrils, com­ prising truncated (1–110 or 1–120) and full-length a-syn, significantly increased a-syn-induced toxi­ city in SH-SY5Y neuroblastoma compared to the addition of monomeric truncated or full-length a­ syn separately (Li et al., 2005). These data suggest that truncated a-syn may enhance cell death by promoting the formation of toxic protein aggre­ gates (Liu et al., 2005). Co-over-expression of the C-terminal fragments (1–110 and 1–120) and the full-length protein also enhanced a-syn-induced cell death by increasing the cell vulnerability to oxidative stress (Li et al., 2005; Liu et al., 2005). The emerging results from the various studies discussed above demonstrate that C-terminal trun­ cated a-syn aggregates more than the full-length protein in Drosophila, but the results from TG mice are variable. Indeed, while Tofaris et al. (2006) reported the accumulation of fibril and nonfibrillar 1–120 a-syn inclusions, Wakamatsu et al. (2008) did not report the formation of any deposits of the A53T 1–130 variant, despite the fact that this mutant aggregates more than the full-length protein

132

in vitro. The discrepancies might be due to the lack of endogenous a-syn in the experiment by Tofaris et al. (2006). In vitro studies suggest that the pre­ sence of endogenous a-syn is expected to interfere with the fibrillization of human a-syn in the rat and mouse models. Consistent with this hypothesis, fibrillar aggregates were observed in mouse and fly models that do not express human a-syn (Chen and Feany, 2005; Tofaris et al., 2006). C-terminal trunca­ tions slightly increase neuronal loss compared to the normal a-syn in Drosophila, but not in two out of three TG mouse models. For comparison, pre­ viously published studies in mouse, targeting expres­ sion of full-length WT or PD-associated a-syn mutants (A30P & A53T) into dopaminergic neu­ rons with the TH-promoter, report no neuronal loss (Matsuoka et al., 2001), or a age-associated neurodegeneration at 19 months in the cases of A30P and A53T (Richfield et al., 2002; Thiruchel­ vam et al., 2004). Only expression of a-syn 1–130 provoked a strong phenotype, with impaired devel­ opment of neurons from the substantia nigra pars compacta. However this model does not reproduce the progressive cell loss or the associated neuro­ pathology characteristic of synucleinopathies.

Ubiquitination Ubiquitin-positive inclusions are a neuropathologic hallmark of PD and related disorders Several neuropathologic studies have shown that a large proportion of LBs present in the substantia nigra, the locus coeruleus, the hippocampus and the cortex of brain with PD and related disorders, are positive for a-syn (Baba et al., 1998; Spillantini et al., 1997, 1998) and ubiquitin (Gomez-Tortosa et al., 2000; Hasegawa et al., 2002; Kuzuhara et al., 1988; Lowe et al., 1988; Manetto et al., 1988; Sampathu et al., 2003; Tofaris et al., 2003) (Fig. 6). Double-immunostaining using anti-a-syn and anti-ubiquitin antibodies revealed that the core of LBs is immunoreactive for both proteins and is

surrounded by a rim of a-syn (Gomez-Tortosa et al., 2000; Mezey et al., 1998) (Fig. 6c). Other intra-cytoplasmic inclusions, larger than LB and without halo, called pale bodies, are positive for a-syn but only occasionally for ubiquitin (GomezTortosa et al., 2000; Tofaris et al., 2003). a-Synu­ clein and ubiquitin co-staining is also frequent in Lewy neurites (e.g. in the hippocampus) (GomezTortosa et al., 2000). Moreover, a-syn and ubiqui­ tin show extensive co-localization in the glial cyto­ plasmic inclusions in MSA. Biochemical analysis of purified LBs and/or LBderived preparations, using western blotting and mass spectrometry techniques, confirmed that some of the a-syn within LBs is ubiquitinated (Anderson et al., 2006; Hasegawa et al., 2002; Sam­ pathu et al., 2003; Tofaris et al., 2003). Interest­ ingly, the majority of a-syn species found in LB are mono- or di-ubiquitinated. Some tri-ubiquiti­ nated a-syn species have been detected by western blots, but no polyubiquitin chains were detected on a-syn isolated from LB or other brain tissues (Anderson et al., 2006; Hasegawa et al., 2002; Nonaka et al., 2005; Sampathu et al., 2003). These find­ ings suggest that ubiquitination of a-syn may be involved in regulating some of its pathophysiologic properties and imply that ubiquitin-mediated degradation is not likely to be the major physiolo­ gical mechanism for degrading a-syn. Consistent with this hypothesis, Tofaris et al. (2001) demon­ strated that non-ubiqutinated a-syn can be degraded by the proteasome. Furthermore, studies from several groups have implicated other protein clearance pathways in the degradation and turn­ over of a-syn, including lysosomal and autophagic pathways (Cuervo et al., 2004; Vogiatzi et al., 2008; Webb et al., 2003; Xilouri et al., 2008).

E3 ubiquitin-protein ligases implicated in the ubiquitination of a-syn Three E3 ubiquitin-protein ligases have been iden­ tified to play an important role in the ubiquitination of a-syn in vitro and in vivo: Parkin, ubiquitin

133 a

b

c

d Parkin, SIAH, UCH-L1 Lewy bodies

K6 K10 K12 K21 K23 K32 K34 K43 K45 K58 K60 61

1

In vitro fibrils

K80

K96 K97 K102 95

140

In vitro monomers

+

Cell culture

Fig. 6. Ubiquitin, a hallmark of LB, covalently modifies a-syn. Ubiquitin immunostaining in a nigral (a) and a cortical (b) LB (arrow) from synucleopathies diseased brains. (adapted from Chu et al., 2000). (c) Co-immunofluorescent labelling of a-syn and ubiquitin showing their co-localization in a LB from the substantia nigra of a PD patient: the staining shows ubiquitin and a-syn in the core of this inclusion and the a-syn alone in the periphery. Scale bar = 10 mm (adpated from Mezey et al., 1998). (d) Schematic representation of a-syn showing the lysine which can be ubiquitynated and the major sites of ubiquitination identified in LB (upper part; Anderson et al., 2006) or in cell culture and in vitro studies (lower part; Nonaka et al., 2005, Rott et al., 2008).

134

carboxy-terminal hydrolase L1 (UCH-L1) and seven in absentia homologue (SIAH) (Lee et al., 2008; Liani et al., 2004; Rott et al., 2008). Interest­ ingly, the genes coding for Parkin and UCH-L1 are linked to familial forms of PD and to PD suscept­ ibility, respectively (Liu et al., 2002; Shimura et al., 2000). Both Parkin and SIAH have been detected in LBs in PD brains (Bandopadhyay et al., 2005; Liani et al., 2004)

at K6, K10 and K12 (Nonaka et al., 2005). This observation may be explained by the fact that in a-syn fibrils, the N-terminal region remains acces­ sible for interaction with ubiquitin-protein ligases. The ubiquitination sites linked to PD and related disorders have been identified form LB-purified a-syn. Using trypsin digestion followed by LC­ MS/MS analysis, Anderson et al. (2006) identified residues K12, K21 and K23 as a major sites of ubiquitination in a-syn.

Ubiquitination sites Ubiquitination of a-syn occurs at multiple lysine residues and the sites of ubiqutination depend on the conformational and/or aggregation state of the protein. There are 15 lysine (K) residues in a-syn, the majority of which are distributed within the N-terminal repeat sequences. The remaining resi­ dues are K80, K96, K97 and K103 (Fig. 6). Using single-site mutagenesis of lysine residues (K ! R) and enzyme (lysyl endopeptidase AP1) digestion followed by peptide mapping using mass spectro­ metry, different groups have identified the possi­ ble lysine residues in a-syn that undergo ubiquitination in vitro and in vivo. In vitro ubiqui­ tination of recombinant a-syn revealed that the monomeric and fibrillar forms of a-syn undergo ubiquitination at distinct lysine residues. In vitro, monomeric a-syn undergoes ubiqutination at sev­ eral lysine residues including K10, K21, K23, K32, K34, K43 and K96, with the major ubiquitin-con­ jugated sites represented by K21, K23, K32 and K34 (Nonaka et al., 2005; Rott et al., 2008). Muta­ tion of these residues to argnine (R) results in a >90% reduction of ubiquitinated a-syn (Nonaka et al., 2005). Similar results were obtained when these mutants were expressed in cell lines. Ubiqui­ tination of recombinant a-syn using rabbit reticu­ locytes fraction II or rat brain extracts revealed similar ubiquitination patterns with K21 and K23 being the major ubiquitination sites (Nonaka et al., 2005). Interestingly, ubiquitination of a-syn fibrils prepared from recombinant a-syn, under identical conditions, occurs predominantly

Does ubiquitination of a-syn enhance or prevent its aggregation and toxicity? While the role of ubiquitination in modulating a­ syn aggregation in vivo remains poorly under­ stood, it is not essential for inclusion formation in vivo, as evidenced by the fact that not all a-syn inclusions in TG mouse models are ubiquitinated (Sampathu et al., 2003; van der Putten et al., 2000). The role of ubiquitination in modulating a-syn aggregation and toxicity was addressed by investigating the in vitro ubiquitination of recom­ binant a-syn, in cell culture and TG animal model. In the animal models, attempts to modu­ late the level of a-syn ubiquitnation, and thereby attenuate a-syn-induced neurotoxicity, were based on the regulation of Parkin expression or the over-expression of ubiquitin. In Drosophila (Haywood and Staveley, 2004; Yang et al., 2003), Parkin over-expression protects against a-syn­ induced toxicity without modifying a-syn levels. Recently, Lee and collaborators demonstrated that the over-expression of ubiquitin has no effect per se on overall adult retinal or dopami­ nergic neuronal structure or viability (Lee et al., 2009), but co-expression of ubiquitin and a-syn suppresses a-syn-induced motor impairment (negative geotactic locomotor response) and cell degeneration in Drosophila eyes and in the DM1 cluster of dopaminergic neurons. Furthermore, the authors demonstrated that expression of the K48R, and not K63R ubiquitin mutants, suppresses the protective effect of ubiquitin

135

(Lee et al., 2009), suggesting that the ubiquitin­ mediated neuroprotective effect is potentially dependent on the K48 polyubiquitin linkage, a signature targeting proteins for proteasomal degradation (Lim et al., 2005). Together, these data suggest that a-syn ubiquitination could pro­ tect against a-syn toxicity in Drosophila PD mod­ els, by targeting a-syn for proteasomal degradation and enhancing the function of the ubiquitin proteasome system. In a rat model, over-expressing Parkin protects against a-syn toxicity without affecting a-syn levels (Lo Bianco et al., 2002). Unfortunately, none of the reported studies in fly and rat models characterized the amount of ubiquitinated a-syn or attempted to map the site of modification and/ or characterize the ubiquitination pattern of a-syn. In TG mice over-expressing a-syn, the knockout of the parkin gene does not modify a-syn quantity (Fournier et al., 2009; Stichel et al., 2007; von Coelln et al., 2006) or levels of ubiquitinated a­ syn (von Coelln et al., 2006). However in one of these models, the levels of S129-P a-syn deposits that are ubiquitinated decreased in the absence of Parkin (Fournier et al., 2009). But the same study showed, by in vitro ubiquitination, that neither a­ syn nor S129-P a-syn is a Parkin substrate, sug­ gesting that the fibrils rather than the monomeric protein might be ubiquitinated by Parkin. The decreased ubiquitination of S129-P a-syn inclu­ sions is associated with a delayed appearance of the neurodegenerative phenotype, indicating a possible toxicity of Parkin-mediated ubiquitina­ tion. On the one hand, this report agrees with data describing a lack of synergy between Parkin deficiency and a-syn over-expression (von Coelln et al., 2006) and the previously described protec­ tive effect of Parkin expression against a-syn­ induced toxicity (Haywood and Staveley, 2004; Lo Bianco et al., 2002; Petrucelli et al., 2002; Yang et al., 2003). On the other hand, these results are in agreement with data showing that ubiquiti­ nation of a-syn by SIAH promotes the formation of cytotoxic inclusions (Rott et al., 2008). To con­ clude, the limited studies reported in the literature

indicate that Parkin does not modulate a-syn levels, but it might be involved in its deposition in vivo. Whether the ubiquitinated aggregates are toxic or protective is still controversial and requires further investigations. In cell culture, endogenous SIAH co-localizes with a-syn and is, in part, responsible for its mono and di-ubiquitination in mammalian cell lines and human neuroblastoma, since the suppression of SIAH expression using shRNA completely abolishes a-syn ubiquitination (Lee et al., 2008; Rott et al., 2008). Moreover, the co-expression of a-syn and SIAH enhances a-syn mono and di­ ubiquitination (Lee et al., 2008; Rott et al., 2008). Co-expression of a-syn, an E3 ubiquitin­ protein ligase (Siah-1) and ubiquitin results in the formation of predominantly mono and di-ubi­ quitinated a-syn species (Lee et al., 2008). Siah-1­ or Siah-2-mediated ubiquitination enhances the aggregation of a-syn and formation of a-syn-posi­ tive inclusion in PC12 cells and SH-SY5Y human neuroblastoma (Lee et al., 2008; Rott et al., 2008). In vitro ubiquitination of a-syn by SIAH promotes the formation of higher molecular weight a-syn aggregates as determined by western blot analysis (Rott et al., 2008). This observation suggests that ubiquitination by SIAH may enhance a-syn aggre­ gation in vitro. These findings were confirmed by electron microscopy studies showing that SIAH­ ubiquitinated a-syn forms more aggregates than the non-ubiquitinated form (Rott et al., 2008). Interstingly, the SIAH-ubiquitinated a-syn was reported to enhance the aggregation of non-ubi­ quitinated a-syn (Rott et al., 2008), suggesting that ubiquitinated a-syn may promote aggregation by seeding the non-ubiquitinated forms. The impact of the phosphorylation and the disease-linked mutations on a-syn ubiquitination in vitro is minor (Nonaka et al., 2005; Rott et al., 2008). A30P and 453T mutations reduce the ability of a-syn to bind to SIAH (Lee et al., 2008) but they do not disrupt the efficiency of the ubiquitination (Rott et al., 2008). The formation of ubiquitinated inclusions asso­ ciated with cell death after the inhibition of the

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proteasome system in neuronal cell culture and in vivo has been reported by several groups (McNaught et al., 2002, 2004; Rideout and Stefa­ nis, 2002). Inhibition of the proteasome or impair­ ment of UCH-L1 activity induced neuronal degeneration and an increased intracellular expression of a-syn and ubiquitin (McNaught et al., 2002; Rideout and Stefanis, 2002). After 12 h incubation with proteasome inhibitors, intracellular inclusions immunoreactive for a-syn, ubiquitin and the chaperone Hsp70 were detected in the apoptotic cells (McNaught et al., 2002; Rideout and Stefanis, 2002). Interestingly, McNaught and collaborators highlighted the vul­ nerability of the dopaminergic versus the GABAergic neurons to proteasome inhibition (McNaught et al., 2002), which they suggested could explain the specific degeneration of the nigral dopaminergic neurons in PD. Similar observations were reported in differentiated and undifferentiated PC12 neuroblastoma after the inhibition of the proteasome (Rideout et al., 2001). Lactacystin-exposed cells showed diffuse ubiquitin immunoreactivity, and in some other cells, focal cytoplasmic accumulation of ubiquitin immunoreactivity was detected (Rideout et al., 2001). It is noteworthy that not all the ubiquitin inclusions were positive for a-syn. This observa­ tion suggests that the accumulation of a-syn within the intracellular inclusion depends on the cell type. In a rat model, McNaught showed that the impairment of the proteasome by systemic expo­ sure to proteasome inhibitors induced the devel­ opment of progressive parkinsonism, neuronal degeneration in different brain regions including the substantia nigra and the formation of LB-like inclusions positive for a-syn and ubiquitin (McNaught et al., 2004). Together, the data generated from the proteasome inhibition models show that the formation of the ubiquitin and a-syn-positive inclusions could be associated with neuronal toxicity in vivo and in vitro. However, whether the a-syn in these inclusions is ubiquiti­ nated and which residues are implicated remain to be elucidated.

Conclusions Significant advances have been made towards the identification of post-translational modifications of a-syn (Fig. 7). To date, several post-translational modifications have been identified, some of which appear to be strongly linked to the pathology in PD and related synucleinopathies (e.g. phosphor­ ylation). Although the results from the studies discussed above demonstrate that introducing these modifications into a-syn or mutating the site of modification influences the structure and/ or aggregation properties of the protein, reconcil­ ing the effects of these modifications in the dif­ ferent model systems is complicated by several factors: (1) different mechanisms of toxicity may be involved in modulating a-syn aggregation and toxicity in different model organisms (e.g. Droso­ phila vs. rodents); (2) differences in the methods used to assess aggregation and toxicity and lack of standardized rigorous approaches for the ana­ lysis of soluble and aggregated forms of a-syn, particularly in cellular and animal models, which have made it difficult to make direct comparisons between the various studies; (3) mutations that are generally used to mimic post-translational modifications do not truly reproduce all aspect of these modifications (e.g. use of phosphomi­ mics; see below); (4) the majority of the in vivo studies focused primary on the effect of modifica­ tions on a-syn aggregation and toxicity (Table 2) and very little on elucidating the molecular mechanisms and cellular pathways altered by these modifications and (5) the cross-talk between different modifications is currently diffi­ cult to reproduce and study in vitro or in vivo. Recent studies suggest that a-syn is phosphory­ lated at multiple sites, and phosphorylation at tyrosine residues modulates a-syn aggregation and toxicity induced by S129-P, highlighting the importance of investigating the interplay between the different post-translational modifications. These challenges, combined with the lack of good cellular and animal models that reproduce the pathology and neuronal loss in humans, have

137

Enzymes/ effectors Kinases:

CK, DyrK1A

Ser 87

Fyn, Src, Syk

Y125

Parkin SIAH UCH-L1

Truncation

Ubiquitination

Cross-linking

Lewy body formation

Tissue Transglutaminase (tTG)

Membrane interaction

Aggregation WT.0h WT.96h

Consequences of α -syn PTMs on: Pathological properties of a-syn − property to aggregate in vivo − neuronal toxicity

A-syn function at the synapse − modulation of DAT activity − modulation of vesicle trafficking − interaction with protein of the synaptic scaffolding

Protein-Protein interaction

Sub-cellular localization − nuclear localization − association with membranes − transport to synapses

Subcellular localization WT pS129

Protein degradation − targeting to the proteasome − targeting to the CMA − activation of macroautophagy

Fibrils

Oxidation Nitrosylation

Oligomers

Proteasome 20S Neurosin Calpain Cathepsin D

Protein conformation WT pS87/pS129

Phosphorylation Ser 129 Monomers

CK, GRKs, PLKs, LRRK2,CaMK

Aggregation state

PTM

Fig. 7. Effects of a-syn post-translational modifications on its properties.

limited our ability to translate the knowledge gained from the identification of these modifica­ tions to an improved understanding of the molecular mechanisms underlying a-syn toxicity and developing new therapies for PD and related disorders. Therefore, several key outstanding questions concerning the role of post-transla­ tional modifications in PD remain unanswered (see Box 1).

Looking beyond aggregation The majority of a-syn modifications were initially identified and implicated in disease pathogenesis solely on the basis of isolation and/or co-localiza­ tion of modified a-syn within LBs or inclusions from diseased brains or TG animal models. There­ fore, studies on these modifications have focused primarily on their role in modulating a-syn aggre­ gation and toxicity rather than their effect on the functional and physiologic properties of the pro­ tein, including its stability, subcellular localization,

membrane interactions and clearance mechanisms (Fig. 7). Post-translational modifications of pro­ teins represent important molecular switches for regulating protein–protein and protein–ligand interactions and thus protein function in health and disease. The C-terminal region of a-syn has been implicated in the majority of a-syn interac­ tions with proteins (Fernandez et al., 2004; Gias­ son et al., 2003; Jensen et al., 1999) and metal ions (Brown, 2007; Paik et al., 1999). Therefore, trun­ cation and/or phosphorylation at single or multi­ ple sites is likely to influence a-syn affinity to proteins, metals and other ligands (e.g. dopamine and polyamines) and alter the biochemical and biological processes regulated by its interaction with these molecules. For example, a-syn interacts with the microtubule-associated protein tau and stimulates tau phosphorylation (Jensen et al., 1999) and fibrillogenesis (Frasier et al., 2005) both in vitro and in vivo. The tau binding site was mapped to the C-terminal region (AA 87–140) of a-syn. Phosphorylation at S129, but not Y125, was also reported to reduce the rate of

138

Box 1 1. What percentage of a-syn is modified in vivo and is there a correlation between the level of modified a-syn and disease progression? With the exception of phosphorylation of a-syn at S129, quantitative assessments of the levels of modified a-syn in the soluble and aggregated states of the proteins, relative to total a-syn, are lacking. Studies by Iwatsubo and colleagues suggest that >90% of a-syn within LBs is phosphorylated at S129. 2. Do these modifications occur before and/or after a-syn aggregation and LB formation? For all reported modifications, it remains unclear whether the presence of modified a-syn in LB and other inclusions reflects their active role in the initiation of a-syn aggregation and development of pathology or a cellular response aimed at clearing unmodified forms of a-syn within LB. Interestingly, all disease-associated modifications, with the exception of phosphorylation at S87, occur at flexible regions that remain accessible in the monomeric, oligomeric and fibrillar states of a-syn. Recent findings that phosphorylation of a-syn at serines (S87 and S129) and tyrosines (Y125, Y133 and Y136), covalent cross-linking by tissue transglutaminase and nitration all inhibit a-syn fibril formation in vitro support the notion of these post-translational modifications being a late event rather than a prerequisite for a-syn aggregation. 3. What is the effect of each modification on the structure of monomeric a-syn and its binding to membranes, oligomerization and fibrillogenesis? The answer to this question lies in our ability to introduce site-specific modifications in a-syn and produce the desired protein in sufficient quantities to perform structural and biophysical studies. These site-specific modifications may not be possible at this stage in vivo but are achievable at the protein and single-cell level. Recent advances in chemistry have made it possible to introduce site specifically single or multiple post­ translational modifications into proteins. A detailed structural understanding of how these modifications alter the structural properties and dynamics of monomeric a-syn will provide important insight into their role in triggering or inhibiting a-syn aggregation and/or interactions with other proteins and cellular compartments. 4. What is the effect of each modification on the stability, degradation and functional properties of a-syn? Only an integrative interdisciplinary approach with standardized methods and measures for assessing changes in a-syn properties would bring us closer to addressing the key outstanding mechanistic questions on the role of a-syn post-translational modifications in PD and translate this knowledge into novel therapies. 5. Is there cross-talk between the different post-translational modifications in a-syn? Thus far, all the modifications of a-syn have been investigated separately, and studies aimed at elucidating the interdependence and relationship between the different modifications are lacking. Different forms of a-syn are modified at multiple sites. Phosphorylation at Y125, ubiquitination or C-terminal truncations co-exist with S129-P, although the sequence of modification remains unknown. More importantly, the residues involved in these modifications are in close proximity to each other and result in a dramatic change in the structure and aggregation of monomeric a-syn. Therefore, it is clear that modifications at these residues will likely have a dramatic effect on a-syn interactions with other enzymes and susceptibility to modifications by these enzymes.

139

6. What are the natural enzymes involved in regulating each of these modification? Selective and efficient site-specific modification of a-syn at single or multiple sites in vivo are currently not possible because the identity of the natural enzymes (e.g. kinases, phosphatases, E3 ubiquitin ligases, hydrolyases and proteases) responsible for regulating the dynamics of these modifications remain unknown. The existing tools and methods (e.g. the use of phosphomimics or expression of truncated a-syn variants) do not allow for investigating the effect of post-translational modification with spatial and temporal resolution. Several candidate enzymes have been implicated in the phosphorylation and proteolysis of a-syn and are currently being tested and validated as potential therapeutic targets. The identification and validation of enzymes that regulate specific a-syn post-translational modifications will provide more effective means for modulating the level of these modifications and determining their role in disease pathogenesis in vivo, using genetic manipulations and/or small molecule inhibitors of these enzymes. Furthermore, this knowledge will allow us to identify the cellular pathways regulating these modifications, which may yield more effective therapeutic targets. 7. Can we prevent a-syn aggregation and toxicity in vivo by modulating the type and extent of post­ translational modification at specific residue(s)? To be able to answer this question, we must use approaches and tools that allow site-specific modulation of post-translational modifications in vivo. Although the identification of candidate enzymes involved in regulating these modifications represents a first important step, it is crucial to demonstrate that the effect of modulating the activity of these enzymes is mediated specifically by a-syn, more effective means for modulating the level of these modifications and determining their role in disease pathogenesis in vivo using genetic manipulations and/or small molecule inhibitors of these enzymes. Furthermore, this knowledge will allow us to elucidate the cellular pathways in regulating these modifications, which may yield more tractable therapeutic targets and pathways.

a-syn transport (Saha et al., 2004). Together, these findings suggest that reversible phosphoryla­ tion or truncations within the C-terminal region (Y125 or S129) may be involved in regulating the association/dissociation with tau and other neuro­ nal proteins (e.g. tau, synphilin (Lee et al., 2004), phospholipase D (PLD) (Payton et al., 2004; Pro­ nin et al., 2000), 14-3-3 (Ostrerova et al., 1999), metals (Liu et al., 2005) and lipids. Phosphoryla­ tion within the C-terminal region (S129 or Y125) or the incorporation of phosphorylation mimick­ ing mutations at these residues also reportedly reduces membrane binding and blocks a-syn­ mediated inhibition of PLD2 (Okochi et al., 2000; Payton et al., 2004; Pronin et al., 2000), an enzyme involved in the hydrolysis of phosphati­ dylcholine and vesicular trafficking.

Recent studies by McFarland and colleagues demonstrated that phosphorylation at S129 and Y125 constitute an important switch for regulating a-syn interaction with other proteins. They explored the role of phosphorylation at S129 or Y125 in protein–protein interactions involving a-syn by comparing the protein–protein interac­ tions of phosphorylated and non-phosphorylated C-terminal peptides encompassing these residues (residues 101–140) using pull down assays and mass spectrometry (McFarland et al., 2008). Their studies showed great differences in the set of proteins pulled down by phosphorylated forms of a-syn. The phosphorylated peptides showed preferential interactions with pre-synaptic cytos­ keletal proteins and proteins involved in synaptic vesicle endocytosis, subunits of serine/threonine

140

kinases and phosphatases, whereas the non-phos­ phorylated peptide interacted preferentially with mitochondrial electron transport chain complexes. Some of the proteins reported to interact with a-syn [e.g. 14-3-3 and microtubule-associated pro­ tein 1B (MAP1B)] show a preference to the phos­ phorylated state (S129-P) of a-syn. The NAC region in a-syn plays an important role in mediating a-syn fibrillization (Paleologou et al., 2010; Waxman and Giasson, 2008), mem­ brane binding (Lotharius and Brundin, 2002) and interactions with other proteins, such as the enzyme PLD2 (Payton et al., 2004). Recent stu­ dies from our laboratory show that S87 phosphor­ ylation alters the conformation of membranebound a-syn and decreases its affinity to lipid vesicles, but does not abrogate binding, probably by destabilizing the helical conformation and decreasing the lipid-binding affinity of the protein around the phosphorylation site. Together, these findings underscore the fact that elucidating the role of post-translational modifications in the pathogenesis of a-syn will require a better under­ standing how these modifications alter the phy­ siologic and functional properties of the protein and how potential cross-talk between the differ­ ent modifications influences the function of a-syn in health and disease. Only an integrative inter­ disciplinary approach with standardized methods and measures for assessing changes in a-syn prop­ erties would bring us closer to answering the key outstanding mechanistic questions regarding the role of a-syn post-translational modification in PD and translate this knowledge into novel therapies.

Abbreviations a-syn AD CKI/II DLB ER GFP

a-synuclein Alzheimer’s disease casein kinase I/II dementia with Lewy bodies endoplasmic reticulum green fluorescent protein

GRKs LBD LRRK2 MSA MSA NAC region PD PLKs S129-P TG WT

G protein-coupled receptor kinases Lewy body disease leucine-rich repeat kinase 2 multiple system atrophy multiple system ztrophy non-amyloid component region Parkinson’s disease Polo-like kinases phosphorylated at serine 129 transgenic wild type

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SECTION II

Cellular and system-level pathophysiology of the basal ganglia in PD

A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 8

The role of dopamine in modulating the structure and function of striatal circuits D. James Surmeier
, Weixing Shen, Michelle Day, Tracy Gertler, Savio Chan,

Xianyong Tian and Joshua L. Plotkin

Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

Abstract: Dopamine (DA) is a key regulator of action selection and associative learning. The striatum has long been thought to be a major locus of DA action in this process. Although all striatal cell types express G protein-coupled receptors for DA, the effects of DA on principal medium spiny neurons (MSNs) understandably have received the most attention. In the two principal classes of MSN, DA receptor expression diverges, with striatonigral MSNs robustly expressing D1 receptors and striatopallidal MSNs expressing D2 receptors. In the last couple of years, our understanding of how these receptors and the intracellular signalling cascades that they couple to modulate dendritic physiology and synaptic plasticity has rapidly expanded, fuelled in large measure by the development of new optical and genetic tools. These tools also have enabled a rapid expansion of our understanding of the striatal adaptations in models of Parkinson’s disease. This chapter highlights some of the major advances in these areas. Keywords: Striatum; Dopamine; Synaptic plasticity; Dendritic excitability; Dendritic spines; Parkinson’s Disease

happens have been built upon the notion that reward prediction errors signalled by mesencephalic dopaminergic neurons innervating the striatum provide a means by which experience shapes the strength of corticostriatal synapses of principal medium spiny neurons (MSNs) and, in so doing, action selection (Cohen and Frank, 2009; Schultz, 2007; Yin and Knowlton, 2006). One of the most compelling pieces of evidence for this view comes from the inability of Parkinson’s disease (PD) patients, who have

lost their striatal dopaminergic innervation, to

Introduction The dorsal striatum integrates information about sensory, motivational and motor state conveyed by cortical and thalamic neurons, facilitating the selection of actions that achieve desirable outcomes, like reward, and avoid undesirable ones. Current models of how this
Corresponding author.

Tel.: 312-503-4904; Fax: 312-503-5101 E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83008-0

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translate thought into action (Dujardin and Laurent, 2003). Although there is strong support for the basic tenets of these models, precisely how dopamine (DA) modulates the neural circuitry of the dorsal striatum to achieve this end has been the subject of debate. One of the experimental obstacles that has slowed physiological study is the cellular heterogeneity of the striatum and the seemingly random anatomical distribution of cell types within it. The principal neurons of the striatum are MSNs, constituting roughly 90% of all striatal neurons in most mammals (Kawaguchi, 1997). MSNs can be divided into at least two groups based on their DA receptor expression and axonal projection site: striatopallidal MSNs send their principal axonal arbor to the globus pallidus and express high levels of the D2 DA receptor, whereas striatonigral MSNs send their principal axonal arbor to the substantia nigra and express high levels of the D1 DA receptor (Gerfen et al., 1990). In physiological studies performed either in vitro or in vivo, these two types of MSNs have been virtually impossible to tell apart, clouding the interpretation of plasticity studies exploring the role of DA. The recent development of bac­ terial artificial chromosome (BAC) transgenic mice in which the expression of D1 or D2 receptors is reported by expression of red or green fluores­ cent protein (Gong et al., 2003; Shuen et al., 2008) has eliminated this problem. These studies have revealed that in mice, D1 and D2 MSNs differ in their intrinsic excitability and dendritic morphol­ ogy (Fig. 1) (Day et al., 2008; Gertler et al., 2008). These mice have led to a flurry of discoveries about dopaminergic regulation of intrinsic excit­ ability and striatal synaptic plasticity – providing the primary motivation for this review.

Acute dopaminergic modulation of striatal MSN excitability D1 receptors are positively coupled to adenylyl cyclase (type V) through Golf (Herve et al., 1995).

Elevation in cytosolic cAMP levels leads to the activation of protein kinase A (PKA). PKA has a variety of intracellular targets that affect cellular excitability. For example, PKA can regulate gluta­ mate receptor trafficking via the phosphoprotein DARPP-32, the tyrosine kinase Fyn or the protein phosphatase striatal-enriched tyrosine phosphatase (STEP) (Braithwaite et al., 2006; Hallett et al., 2006; Lee et al., 2002; Scott et al., 2006; Snyder et al., 2000). Although slightly less clear, D1 recep­ tor activation may also directly enhance NMDA receptor currents, via L-type voltage-gated calcium channels (Blank et al., 1997; Cepeda et al., 1993; Liu et al., 2004). In addition, D1 receptor activation has several other consequences on the milieu of conductances being integrated during cellular activ­ ity, such as reducing Naþ channel (likely Nav1.1) conductivity and inhibiting N-type voltage-gated calcium channels (Carr et al., 2003; Kisilevsky et al., 2008; Scheuer and Catterall, 2006; Surmeier and Kitai, 1993). Such actions of D1 receptors are consistent with the classical notion of D1 receptor signalling as ‘excitatory’. D2 receptors couple to Gi/o proteins, leading to inhibition of adenylyl cyclase through Gai subunits (Stoof and Kebabian, 1984). In parallel, released Gbg subunits are capable of reducing Cav2 Ca2þ channel opening and of stimulating phospholipase Cb isoforms, generating diacylglycerol (DAG) andprotein kinase C (PKC) activation as well as inositol trisphosphate liberation and the mobi­ lization of intracellular Ca2þ stores (HernandezLopez et al., 2000; Nishi et al., 1997). D2 receptors also are capable of transactivating tyrosine kinases (Kotecha et al., 2002). Studies of voltage-dependent channels are lar­ gely consistent with the proposition that D2 recep­ tors act to reduce the excitability of striatopallidal neurons and their response to glutamatergic synap­ tic input. Activation of D2 receptors decreases aamino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA) receptor currents in MSNs (Cepeda et al., 1993; Hernandez-Echeagaray et al., 2004) and diminishes pre-synaptic glutamate release, although it is unclear if the latter involves pre- or

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Fig. 1. D1 and D2 MSNs are differentially excitable. (a) Reconstructions of biocytin-filled D1 and D2 MSNs. Striatal neurons from P35–P45 BAC transgenic mice were biocytin filled, imaged and reconstructed in three dimensions. A GABAergic interneuron is included for comparison. (b) Analysis of anatomical differences between reconstructed D1 and D2 MSNs. A three-dimensional Sholl analysis of biocytin filled and reconstructed neurons from P35–P45 BAC transgenic mice. Data are shown as mean (+SEM) number of intersections at 1 mm eccentricities from the soma for 15 D1 and 16 D2 MSNs. D1 MSNs have a more highly branched dendritic tree, as indicated by the increased number of intersections and positive subtracted area (grey shading). (c) Membrane responses to intrasomatic current injection reveal divergence in excitability of D1 and D2 MSNs (d) The higher excitability in the D2 MSN population is illustrated in an F–I plot. (e) Maximum intensity projection image of a D2 MSN using 2PLSM (left) loaded with Alexa Fluor 568 and Fluo-4. Somatic APs were induced and corresponding spine calcium transients were measured at three distances from the soma (line scans indicated by yellow lines) and shown to the right. (f) The decrementation of somatic AP-induced dendritic calcium transients along a dendrite is compared between D1 (n = 11) and D2 (n = 6) MSNs. The data show bAP invasion into MSN dendrites degrades faster in D1 vs. D2 MSNs (Mann–Whitney rank sum test). Figure modified from Day et al. (2008) and Gertler et al. (2008).

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post-synaptically situated D2 receptors (Bamford et al., 2004; Yin and Lovinger, 2006). D2 receptor activation has also been shown to negatively mod­ ulate Cav1.3 Ca2þ channels through a calcineurin­ dependent mechanism (Hernandez-Lopez et al., 2000; Olson et al., 2005), reduce opening of vol­ tage-dependent Naþ channels (presumably by a PKC-mediated enhancement of slow inactivation) (Surmeier and Kitai, 1993) and promote the opening of Kþ channels (Greif et al., 1995). Such actions of D2 receptors are consistent with the classical notion of D2 receptor signalling as ‘inhibitory’. Given the consequences DA has on MSN excit­ ability, post-synaptic response to glutamate and pre-synaptic glutamatergic release, it is not a large conceptual leap to assume that it may play a role in corticostriatal synaptic plasticity. Indeed, the pioneering work of Calabresi and others (1992) utilized rodent tissue slices containing cor­ tex and striatum to demonstrate long-term depres­ sion (LTD) in striatal MSNs and pointed to the importance of DA in governing its induction. We have recently made great progress in elucidating the role of DA in both LTD and long-term poten­ tiation (LTP) induction in striatal MSNs. This work will be a focus of the remainder of this chapter.

LTD at glutamatergic synapses on MSNs The easiest form of synaptic plasticity to see at MSN glutamatergic synapses is LTD (Calabresi et al., 2000). Unlike the situation at many other synapses, striatal LTD induction requires pairing of post-synaptic depolarization with moderate to high-frequency afferent stimulation at physiologi­ cal temperatures (Calabresi et al., 2000; Kreitzer and Malenka, 2005). Typically for the induction to be successful, post-synaptic L-type calcium chan­ nels and Gq-linked mGluR5 receptors need to be co-activated (Kreitzer and Malenka, 2005; Lovin­ ger et al., 1993). Both L-type calcium channels and mGluR5 receptors are found near glutamatergic

synapses on MSN spines, making them capable of responding to local synaptic events (Carter and Sabatini, 2004; Carter et al., 2007; Day et al., 2006; Testa et al., 1994). The interaction between these two membrane proteins in the process of LTD induction undoubtedly involves calcium. Recent work showing that prolonging the opening of L-type channels with an allosteric modulator eliminates the need to stimulate mGluR5 recep­ tors (Adermark and Lovinger, 2007), points to shared regulation of dendritic calcium concentra­ tion. However, there is an asymmetry, as increas­ ing mGluR5 activation by bath application of agonists does not eliminate the need for L-type calcium channel opening (Kreitzer and Malenka, 2005; Ronesi et al., 2004). This might reflect a requirement for calcium-induced calcium release (CICR) from intracellular stores in LTD induc­ tion. In many cell types, CICR depends upon calcium influx through voltage-gated calcium channels, including L-type channels (Nakamura et al., 2000). Activation of mGluR5 and the pro­ duction of inositol-1,4,5-triphosphate (IP3) could serve to prime these dendritic calcium stores, boosting CICR evoked by activity-dependent cal­ cium entry through L-type calcium channels and thus promoting LTD induction (Berridge, 1998; Taufiq Ur et al., 2009; Wang et al., 2000). A key event in the induction of LTD is the post­ synaptic generation of endocannabinoids (ECs). ECs diffuse retrogradely to activate pre-synaptic CB1 receptors and decrease glutamate release probability. Having both pre- and post-synaptic induction criteria confers synaptic specificity on LTD expression (Singla et al., 2007). The molecu­ lar identity of the metabolic pathway leading to EC production in MSNs is still uncertain. There are two abundant striatal ECs: anandamide and 2­ arachidonylglycerol (2-AG). Although previous studies have underscored the neural regulation of anandamide synthesis in the striatum (Giuffrida et al., 1999), collateral support for it has been modest (Ade and Lovinger, 2007). Recent work has provided compelling support for the proposi­ tion that 2-AG and its synthetic enzyme

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diacylglycerol lipase a (DAGLa) are essential (Gao et al., 2010; Lerner et al., 2010; Tanimura et al., 2010). The door is still slightly open for anandamide however. In Lerner et al.’s elegant and focused study, they found that inhibition of DAGLa was effective in preventing LTD induc­ tion only in response to moderate frequency affer­ ent stimulation, not to higher frequency stimulation (~100 Hz). Why this would be is unclear. Both DAGLa and phospholipase D (PLD) are calcium-stimulated enzymes (Breno­ witz et al., 2006). It could be that PLD requires a greater elevation in post-synaptic calcium concen­ tration that would come with higher frequency afferent stimulation. One still unresolved question about the induc­ tion of striatal LTD is whether activation of D2 receptors is necessary. Activation of D2 receptors is a potent stimulus for anandamide production (Giuffrida et al., 1999). However, recent work showing the sufficiency of L-type channel open­ ing in EC-dependent LTD (Adermark and Lovinger, 2007), makes it clear that D2 receptors play a modulatory – not obligatory – role. The real issue is the role of D2 receptors in LTD induction using synaptic stimulation. Attempts to address this question using BAC mice have consistently found that in D2 receptor expressing striatopallidal MSNs, D2 receptor activation seems to be necessary (Kreitzer and Malenka, 2007; Shen et al., 2008; Wang et al., 2006). This could be due to the need to suppress A2a adenosine receptor signalling that could impede efficient EC synthesis and LTD induction (Fuxe et al., 2007a, 2007b; Shen et al., 2008). Indeed, Lerner et al. demonstrate quite convincingly that antagonism of A2a receptors promotes EC-dependent LTD induction in striatopallidal MSNs (Lerner et al., 2010). Is EC-dependent LTD inducible in the other major population of MSNs that do not express D2 receptors – the D1 receptor dominated striato­ nigral MSNs? Kreitzer and Malenka (2007) reported that LTD was not inducible in these MSNs using a minimal local stimulation. This

result was confirmed subsequently (Fig. 2) (Shen et al., 2008). However, using macroelectrode sti­ mulation, EC-dependent LTD is readily inducible in identified D1 MSNs (Wang et al., 2006), consis­ tent with the high probability of MSN LTD induc­ tion seen in previous work (Calabresi et al., 2007). Thus, the stimulation paradigm seems critical to LTD induction in D1 MSNs. Why? The problem with these induction protocols is that the type of axon and cell activated by the electrical stimulus is poorly controlled. With intra-striatal stimulation or with nominal white matter stimulation in cor­ onal brain slices, glutamatergic afferent fibres, dopaminergic fibres and fibres intrinsic to the striatum are all activated, producing a mixture of neuromodulators that makes the interpretation of results less than straightforward. In Kreitzer and Malenka’s case, minimal local stimulation of both dopaminergic and glutamatergic fibres appears to be critical to the LTD induction failure, as block­ ing D1 receptors unmasked a robust EC-depen­ dent LTD in D1 MSNs (Shen et al., 2008), establishing a clear parallel to the A2a receptor phenomenon described by Lerner et al. (Lerner et al., 2010). This kind of complication also appears to be responsible for the apparent D2 receptor dependence of LTD induction in D1 MSNs using macroelectrodes that more effectively activate cholinergic interneuron axons (Wang et al., 2006). The neuromodulator mixture created by non­ specific electrical stimulation could also be a factor in slice studies implicating nitric oxide (NO) sig­ nalling in LTD induction (Calabresi et al., 1999). The principal sources of NO in the striatum are NO synthase expressing interneurons and endothelial cells. Experiments by Sergeeva et al. (2007) implicate both neuronal and endothelial sources of NO in LTD. Calcium entry and stimu­ lation by NO in both cell types presumably occur in response to elevation in extracellular glutamate. In nitric oxide synthase (NOS) interneurons, N-methyl-D-aspartate (NMDA) receptors are necessary for NOS activation (Ondracek et al., 2008). However, this creates a problem in that LTD

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Fig. 2. STDP in D1 and D2 MSNs. (a) Model of a typical MSN dendritic spine, showing glutamatergic and dopaminergic inputs. (b) (Left) Positive spike timing (theta burst patterns of pre- and post-synaptic stimulation, pre-synaptic stimulation at –5 ms) produces LTP and negative spike timing (pre-synaptic stimulation at þ10 ms) produces LTD in D2 MSNs. (Right) Positive spike timing produces LTP, whereas negative timing does not induce plastic changes in D1 MSNs. When D1 receptors are blocked by SCH23390, however, negative timing induced LTD is unmasked. (c) Model showing the behavioural consequences of differential corticostriatal STDP on D2 and D1 MSNs. Figure modified from Shen et al. (2008).

induction in the dorsal striatum of adult rodents is not NMDA receptor dependent. Recent work by Sergeeva’s group has shown that there is a devel­ opmental dependence to the signalling mechan­ isms responsible for NO production and LTD, with engagement of NMDA receptor-stimulated NO production being necessary for EC-dependent

LTD induction only in juvenile rodents (Chep­ kova et al., 2009). This suggests that endothelial cells play a more pivotal role in the adult dorsal striatum. Another interesting aspect of the NO story is where it is acting. MSNs express very high levels of NO-stimulated soluble guanylyl cyclase and protein kinase G (Ariano, 1983). But

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these cells appear not to be the target of NO in LTD. Rather, it appears that the site of NO action is downstream of CB1 receptor activation, in the pre-synaptic terminal (Sergeeva et al., 2007). How this relates to Lovinger’s evidence implicating pre­ synaptic gene expression in the expression of LTD is unclear. The lack of specificity in activating inputs to MSNs during the induction of plasticity also raises questions about the type of glutamatergic synapse being affected by EC-dependent LTD. Studies using nominal white matter or cortical stimulation in a coronal brain slices typically assume that the glutamatergic fibres being stimulated are of corti­ cal origin, but very few of these fibres are left intact in this preparation (Kawaguchi et al., 1989). The thalamic glutamatergic innervation of MSNs is similar in magnitude to that of the cere­ bral cortex, perhaps constituting as much as 40% of the total glutamatergic input to MSNs, termi­ nating on both shafts and spines (Smith et al., 2009; Wilson, 2004). As a consequence, it is not really known whether EC-dependent LTD is pre­ sent at corticostriatal or thalamostriatal synapses or both. The localization of CB1 receptors on corticostriatal terminals, but not thalamostriatal terminals (Uchigashima et al., 2007), is consistent with the hypothesis that LTD is a corticostriatal phenomenon, but more definitive studies are needed. Cutting brain slices in planes that pre­ serve cortical and/or thalamic connectivity is one way to sort this out (Ding et al., 2008; Kawaguchi et al., 1989; Smeal et al., 2007). But these approaches have limitations given the highly con­ vergent nature of the glutamatergic input to MSNs (Wilson, 2004). Optogenetic approaches offer a powerful alternative strategy (Zhang et al., 2006) that would allow glutamatergic inputs from var­ ious cortical and thalamic regions to be dissected.

LTP at glutamatergic synapses on MSNs Less is known about the mechanisms controlling induction and expression of LTP at glutamatergic

synapses. Most of the work describing LTP at glu­ tamatergic synapses has been done with sharp elec­ trodes (either in vivo or in vitro), not with patchclamp electrodes in brain slices that afford greater experimental control and definition of the cellular and molecular determinants of induction. How­ ever, there have been a number of studies using these approaches in the last few years that have made progress in characterizing LTP mechanisms. Previous studies have argued that LTP induced in MSNs by pairing high-frequency stimulation of glutamatergic inputs, and post-synaptic depolariza­ tion depends upon co-activation of D1 DA and NMDA receptors (Calabresi et al., 2007). The involvement of NMDA receptors in LTP induction is not controversial. What is controversial is the involvement of D1 receptors. Robust expression of these receptors is only found in striatonigral MSNs, roughly half of the MSN population, making it difficult to understand how LTP induction could be universally dependent on them unless some rather complicated, indirect mechanism was involved. Again, the advent of BAC transgenic mice has provided a tool to sort this issue out. Using perforated patch recordings to preserve the intracellular milieu controlling the induction of synaptic plasticity, our group found that the induc­ tion of LTP at glutamatergic synapses was depen­ dent on D1 DA receptors only in striatonigral MSNs, not in D2 receptor expressing striatopallidal MSNs (Fig. 2) (Flajolet et al., 2008; Shen et al., 2008). In D2 MSNs, LTP induction required activa­ tion of A2a adenosine receptors. These receptors are robustly expressed in striatopallidal MSNs and have a very similar intracellular signalling linkage to that of D1 receptors; that is, they positively cou­ ple to adenylyl cyclase and PKA. Acting through PKA, D1 and A2a receptor activation leads to the phosphorylation of DARPP-32 and a variety of other signalling molecules, including MAPKs, linked to synaptic plasticity (Sweatt, 2004). The nature of the co-operativity between NMDA receptors and D1/A2a receptor signalling in the induction of LTP remains to be resolved. This interaction governs the timing dependence of

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spike timing-dependent plasticity (STDP) (Pawlak and Kerr, 2008; Shen et al., 2008). For example, when D2 receptors in striatopallidal MSNs were blocked and A2a receptors were stimulated, pair­ ing a short burst of post-synaptic spikes with an excitatory post synaptic potential (EPSP) 10 ms later led to LTP induction, whereas with normal G protein-coupled receptor stimulation this protocol invariably produced LTD. In contrast, in striatoni­ gral MSNs, pairing post-synaptic spiking with a trailing pre-synaptic volley only produced LTD in the absence of D1 receptor stimulation, suggesting that PKA signalling could abrogate LTD induction. Reversing the order of stimulation gave LTP only when D1 receptors were stimulated and yielded LTD otherwise, arguing that PKA signalling not only could shut down LTD induction, but was also necessary for LTP induction. Conceptually similar results have been reported in other cell types (Seol et al., 2007; Tzounopoulos et al., 2007), leading to the notion that LTD and LTP induction are gov­ erned by ‘opponent processes’ that interact at synaptic sites to determine the sign of synaptic plas­ ticity. Altered activation of these processes could be responsible for ‘anti-Hebbian’ plasticity reported in the striatum (Fino et al., 2005). How these oppo­ nent processes interact with one another and the cellular mechanisms underlying changes in synaptic strength remains to be determined. Given the evi­ dence that PKA signalling can potentiate NMDA receptor currents (Blank et al., 1997; Colwell and Levine, 1995), it is tempting to think that A2a and D1 receptors promote LTP induction in this way. Molecules like regulator of calmodulin signalling (RCS), whose affinity for calmodulin and negative regulation of calcium signalling is dramatically ele­ vated by PKA phosphorylation, could also contri­ bute to the opponent interaction (Xia and Storm, 2005). The proposition that there is an LTD ‘can­ celling’ signal arising from D1 or A2a receptors but which requires some measure of co-operativity from NMDA receptor signalling (and CaMKII) would appear to be an economical solution to the plasticity problem, as it makes little sense to allow both processes to proceed independently. Another

potential mediator of this interaction is STEP (Braithwaite et al., 2006). Activation of STEP pro­ motes the endocytosis of both NMDA and AMPA receptors and is inactivated by PKA phosphoryla­ tion (Tashev et al., 2009; Zhang et al., 2008). Cal­ cium activation of STEP also shortens ERK1/2 and Fyn kinase signalling, establishing a connection to striatal LTP (Dunah et al., 2004; Flajolet et al., 2008; Nguyen et al., 2002; Paul et al., 2003; Pelkey et al., 2002). The nature of this interaction also has implica­ tions for the distal reward problem (Sutton and Barto, 1981). The change in DA release produced by the consequences of action selection occurs later in time than the pre- and post-synaptic activity that produced the action. In theoretical treatments of this issue, there are two strategies for dealing with this temporal delay or distal reward. One way is to have temporally co-incident pre- and post-synaptic activity create an eligibility trace (perhaps expressed as elevated CAMKII or calcineurin) that subsequently can be acted on by an outcomedependent signal, in this case DA. However, if DA receptor signalling changes the impact of patterned synaptic stimulation on intracellular signalling cas­ cades controlling the induction of plasticity, it is difficult to see how this could work. An alternative approach is to have repeating/reverberating activity or have the outcome event trigger a fictive replay of the action selection (Drew et al., 2006; Genovesio et al., 2006; Tsujimoto and Sawaguchi, 2004). As the corticostriatal pathway is the first leg of a multisynaptic loop between the cortex, basal ganglia, thalamus and again cortex (Alexander and Crutcher, 1990), it is not hard to imagine how such an approach may work.

Dendritic excitability and synaptic plasticity Although most of the induction protocols that have been used to study striatal plasticity are decidedly unphysiological, involving sustained, strong depo­ larization and/or high-frequency synaptic stimula­ tion that induces dendritic depolarization, they do

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make the necessity of post-synaptic depolarization clear. In a physiological setting, what types of depo­ larization are likely to gate induction? One possi­ bility is that spikes generated in the axon initial segment (AIS) propagate into dendritic regions where synapses are formed. Recent work has shown that STDP is present in MSNs (Fino et al., 2005; Pawlak and Kerr, 2008; Shen et al., 2008). But there are reasons to believe that this type of plasti­ city is relevant for only a subset of the synapses formed on MSNs. MSN dendrites are several hundred microns long, thin and modestly branched. Their initial 20–30 mm are largely devoid of spines and glutamatergic synapses. Glutamatergic synapse and spine density peak near 50 mm from the soma and then modestly decline with distance (Wilson, 2004). Because of their geometry and ion channel expression, AIS generated spikes rapidly decline in amplitude as they invade MSN dendrites (as judged by their ability to open voltage-dependent calcium channels), producing only a modest depolarization 80–100 mm from the soma. This is less than half the way to the dendritic tips (Day et al., 2008), arguing that a large portion of the synaptic surface area is not normally accessible to somatic feedback about the outcome of aggregate synaptic activity. Highfrequency, repetitive somatic spiking improves dendritic invasion, but distal (>100 mm) synapses remain relatively inaccessible. In the more distal dendritic regions, what con­ trols plasticity? The situation in MSNs might be very similar to that found in deep layer pyramidal neurons where somatically generated bAPs do not invade the apical dendritic tuft (Golding et al., 2002). In this region, convergent synaptic stimula­ tion is capable of producing a local calcium spike or plateau potential that produces a strong enough depolarization to open L-type calcium channels, to unblock NMDA receptors and promote plasticity. In vivo, convergent synaptic inputs to MSNs can trigger plateau potentials called up-states (Wilson and Kawaguchi, 1996). Although transitions from the resting down-state to the up-state have all the hallmarks of an active, regenerative process (e.g. stereotyped transition kinetics, a narrow range of

up-state potentials), transitions are very difficult to manipulate with a sharp electrode impaling the somatic region (Wilson and Kawaguchi, 1996). This suggests that the site of up-state generation is in distal dendritic regions that cannot be easily manipulated. If this were the case, distal dendrites should have ionic conductances that could support a plateau. Calcium imaging using two-photon laser scanning microscopy (2PLSM) has shown that there is robust expression of both low-threshold Cav3 and Cav1 channels in MSN dendrites (Carter and Sabatini, 2004; Carter et al., 2007; Day et al., 2008), a result that has been confirmed using cell­ type-specific gene profiling (Day et al., 2006) (unpublished observations). The rich investment of MSN dendrites with strongly rectifying Kir2 Kþ channels also creates a favourable biophysical condition for plateau potential generation. The question is how the plateaus or up-states are normally generated. Based on the sparse con­ nectivity between individual cortical axons and MSNs (Kincaid et al., 1998; Wilson, 2004), model­ ling studies have concluded that several hundred pyramidal neurons need to be near simultaneously active for a sufficient amount of current to be injected into dendrites for an up-state to be gen­ erated (Stern et al., 1997; Wilson, 2004). These studies have assumed that MSN dendrites are pas­ sive. However, if dendrites are not passive but active, then the convergence requirements could be dramatically different. Although glutamate uncaging experiments at proximal spines have not revealed regenerative processes (Carter et al., 2007), the situation could be different at more distal locations. If this is the case, spatial convergence of glutamatergic inputs onto a distal dendrite could induce a local plateau potential capable of pulling the rest of the cell into the up­ state, fundamentally altering the impact of synap­ tic input on other dendrites. This is a way in which spatially convergent excitatory input to one den­ drite could gate synaptic input to another. The lack of temporal correlation between up-state transitions and EPSP-driven spike generation is consistent with a scenario like this one (Stern

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et al., 1998). If this were how MSNs operated, it would fundamentally change our models of stria­ tal information processing. In vivo studies of striatal synaptic plasticity have provided an important counterpoint to the perspec­ tives based on reduced in vitro preparations. The pioneering work of (Charpier and Deniau, 1997) demonstrated that with more intact input, LTP was readily inducible in MSNs, contrary to the prevail­ ing model. More recently, Stoetzner et al. have shown that the sign of synaptic plasticity in MSNs is influenced by anaesthetic and presumably the degree of cortical synchronization in corticostriatal projections (Stoetzner et al., 2010). In particular, they show that in barbiturate anaesthetized rats, 5 Hz stimulation of motor cortex evokes LTP in the striatum, but that in awake animals the same stimulation induced LTD. A challenge facing the field is how to bridge these observations. Because glutamatergic connections are sparse, it is virtually impossible to reliably stimulate a collection of synapses onto a particular MSN dendrite with an electrode in a brain slice. Optogenetic techniques might provide a feasible alternative strategy. Another strategy would be to employ two-photon laser uncaging (2PLU) of glutamate at visualized synaptic sites (Carter and Sabatini, 2004). These tools are becoming more widely available and should allow the regenerative capacity of MSN dendrites to be tested soon. If it turns out to be the case that up-states are locally generated in den­ drites, then it also becomes feasible to characterize their role in the induction of synaptic plasticity. Up­ states could be sufficient, as in the apical tuft of pyramidal neurons, or they could simply be neces­ sary by promoting back-propagation of spikes into the distal dendrites (Kerr and Plenz, 2002).

Homeostatic plasticity in PD models Sorting out how DA regulates synaptic plasticity in striatal MSNs has obvious implications for disease states that are triggered by alterations in the function of dopaminergic neurons. Second in prominence

among DA-dependent disorders only to drug abuse, PD is a common neurodegenerative disorder whose motor symptoms are attributable largely to the loss of dopaminergic neurons innervating the dorsal striatum. In the prevailing model, the excit­ ability of the two major populations of MSNs shifts in opposite directions following DA depleting lesions, creating an ‘imbalance’ in the regulation of the motor thalamus favouring suppression of move­ ment (Albin et al., 1989; Wichmann and DeLong, 1996). In particular, D2 receptor expressing striato­ pallidal MSNs spike more, whereas D1 receptor expressing striatonigral MSNs spike less in the PD state. The mechanisms underlying this shift were not known at the time the model was formulated, but have widely been assumed to reflect changes in intrinsic excitability that accompanied loss of inhibi­ tory D2 receptor signalling and excitatory D1 recep­ tor signalling. Indeed, studies by our group and others have found electrophysiological support for this view (Mallet et al., 2006; Surmeier et al., 2007). What about synaptic remodelling? Several stu­ dies have suggested that in the absence of DA, synaptic plasticity is lost, essentially ‘freezing’ the striatal circuit in its pre-depleted state (Calabresi et al., 2007; Kreitzer and Malenka, 2007). How­ ever, recent studies of defined MSN populations have shown that although DA is necessary for plasticity to be bidirectional and Hebbian, it is not necessary for the induction of plasticity per se (Shen et al., 2008). Following DA depletion, pair­ ing pre-synaptic and post-synaptic activity – regardless of which came first – induced LTP in D2 MSNs and LTD in D1 MSNs. This result adds a new dimension to the prevailing model by show­ ing that activity-dependent changes in synaptic strength parallel those of intrinsic excitability fol­ lowing DA depletion. Work in vivo examining the responsiveness of anti-dromically identified MSNs to cortical stimulation following unilateral lesions of the striatal dopaminergic innervation is consis­ tent with this broader model (Mallet et al., 2006). But this poses a problem. Neurons are homeo­ static; sustained perturbations in synaptic or intrinsic properties that make neurons spike more or less

159

than their set-point engage homeostatic mechan­ isms that attempt to bring activity back to the desired level (Marder and Goaillard, 2006; Turri­ giano, 1999). One of the most common mechanisms of homeostatic plasticity is to alter synaptic strength or to scale synapses. In striatopallidal MSNs, the elevation in activity following DA depletion triggers a dramatic down-regulation of glutamatergic synapses formed on spines (Fig. 3) (Day et al., 2006). This can be viewed as a form of homeostatic plasticity. Like scaling seen in other cell types, the

synaptic modification depends upon calcium entry through voltage-dependent L-type calcium channels that presumably report activity levels. In an attempt to better characterize the homeo­ static mechanisms controlling synapse density in MSNs, striatum from transgenic mice expressing a D2 receptor reporter construct was co-cultured with wild-type cerebral cortex. In these co-cultures, MSN dendrites develop nearly normal spine density with pre-synaptic glutamatergic terminals (Fig. 4) (Segal et al., 2003; Tian et al., 2010).

DA depleted BAC D1

40 μm

40 μm

DA depleted BAC D2

5 μm

5 μm

control D1

control D2

DA depleted

DA depleted 10 pA

10 pA

1s

1s

Fig. 3. Dopamine depletion causes a reduction in spine density in D2 MSNs but not D1 MSNs. Alexa 594 loaded D2 (left) and D1 (right) MSNs 5 days after dopamine depletion (reserpine). High-power images of spines indicated by red boxes are shown below. After dopamine depletion spine density is significantly decreased in D2 MSNs, but appears normal in D1 MSNs. mEPSC traces taken from control and dopamine depleted MSNs (bottom) show that following dopamine depletion mEPSC frequency is decreased in D2 MSNs but unaltered in D1 MSNs, correlating with the observed change in spine density. Figure modified from Day et al. (2006).

160

+K+

10

10 pA

+K+

8

2 0

control

4

+nimodipine

6

control

spines/10 µm

12

+K++nimodipine

1s

8

*

* +K+

6

+nimodipine

(d)

4 2 0

control

(c) control

***

***

mEPSC frequency (Hz)

(b) 14

+K++nimodipine

+K+

control

(a)

Fig. 4. L-type Ca2þ channels are necessary for spine and synapse elimination. (a) Images of D2 MSNs in corticostriatal co-cultures treated with 35 mM KCl and ionotropic receptor blockers for 24 h, in absence or presence of 10 mM nimodipine. Bar, upper panels 10 mm; lower panels, 5 mm. (b) Quantification of spine density showing that nimodipine blocked the membrane depolarizationinduced spine loss (control, median = 11.9, n = 15; þKþ, median = 5.6, n = 18; þKþþnimodipine, median = 11.9, n = 13). (c) Examples of mEPSCs recording from the D2 MSNs treated as in (a). (d) Box plot showing membrane depolarization resulted in reduction of mEPSC frequency (control, median = 2.17, n = 19; þKþ, median = 1.29, n = 14), which was blocked by nimodipine (þKþþnimodipine, median = 2.92, n = 18).
p < 0.05,


p < 0.001, Mann–Whitney rank sum test. Figure taken from Tian et al. (2010).

Sustained (>3 h) depolarization induced a pruning of glutamatergic synapses and spines in striatopallidal MSNs. This pruning was antagonized by dihydropyridines, implicating L-type calcium channels as with DA depletion (Fig. 4) (Day et al., 2006; Neely et al., 2007; Segal et al., 2003). However, unlike the situation in vivo, L-type channels with a Cav1.3 pore-forming subunit were not necessary, but rather ones with a Cav1.2 subunit. It could be that this reflects some abnormality in the cultured

MSNs. But it seems more likely that this difference is a reflection of local and global mechanisms underlying spine pruning. In vivo, low-threshold Cav1.3 channels are located near glutamatergic synapses where they are capable of being activated by synaptic depolarization. Their activation could be impor­ tant to effective propagation of synaptic depolarization to the soma. Thus, eliminating or antagonizing Cav1.3 channels should attenuate the synaptic consequences of DA depletion, mitigating

161

the homeostatic drive. High-threshold Cav1.2 channels appear to be largely somatic where they report spiking (or strong depolarization). In our experiments, by bath application of elevated potas­ sium, the normal dendritic synaptic mechanisms were bypassed and somatic Cav1.2 channels directly activated. These channels have been implicated in other forms of homeostatic synaptic plasticity induced by global alterations in excitability or synaptic activity (Turrigiano, 1999). In MSNs, calcium entry through Cav1.2 L-type calcium channels triggered a signalling cascade that led to a transcriptionally dependent spine pruning. The first step in this cascade was activation of the calcium-dependent protein phosphatase calci­ neurin. Calcineurin dephosphorylates myocyte enhancer factor 2 (MEF2) (Flavell et al., 2006), increasing its transcriptional activity. As in other neurons (Flavell et al., 2006; Shalizi et al., 2006), MEF2 up-regulation increased the expression of two genes linked to synaptic remodelling – Nur77 and Arc (Fig. 5). These experiments establish a translational framework within which adaptations in striatal synapses that are linked to the symptoms of PD can be explored. There are other recently described network adaptations relevant to homeostatic plasticity in PD models. For example, although feed-forward inhibition through fast spiking GABAergic interneurons does not appear to be directly altered, low-threshold GABAergic interneurons do elevate their input to at least a subset of MSNs in PD models (Dehorter et al., 2009; Mallet et al., 2005). Recurrent collateral inhibition between MSNs, which is normally strongest between D2 MSNs, is almost abolished following DA depletion (Taverna et al., 2008). These adaptations in conjunction with enhanced striatopallidal MSN excitability are likely to contribute to the transmission of beta band activity from the cortex through the striatum to the globus pallidus (Murer et al., 2002). A major gap in the existing literature is a descrip­ tion of the intrinsic changes in MSN excitability following prolonged DA depletion. All the work with identified cell types has relied on short-term

(~<1 week) DA depletions (e.g. Day et al., 2006; Kreitzer and Malenka, 2007), but there clearly are slower adaptations that take 3–4 weeks to stabilize. Given the robust differences in the anatomy and intrinsic physiology of striatonigral and striatopalli­ dal MSNs that exist in the normal striatum (Gertler et al., 2008), it is easy to conjecture that these rest­ ing differences are due to differential regulation of basal excitability by DA. If that were true, losing DA could trigger homeostatic processes that make MSNs much more alike.

Concluding remarks In the last few years, our understanding of the mechanisms controlling synaptic plasticity in the corticostriatal circuits has significantly deepened. Although DA is the central player in the induction of plasticity at corticostriatal synapses on principal MSNs, other acetylcholine, adenosine and NO have joined the drama. However, much remains to be done. How the relatively sparse but func­ tionally important interneuron populations contri­ bute to plasticity remains to be clearly defined, although there are a number of recent advances in this area (Gittis et al., 2010; Higley et al., 2009; Martella et al., 2009). The development of trans­ genic mice expressing Cre in select neuronal populations (and the growing stable of mice with floxed genes) should propel this effort forward and allow a molecular dissection of these mechan­ isms in coming years. The growing application of optical approaches, like 2PLSM and 2PLU, also promises to yield insights into synaptic integration and plasticity not achievable with any other approach. Coupling these new tools with optogenetic strategies for activating microcircuits relevant to action selection should prove to be a watershed for basal ganglia and motor systems research. These approaches should allow us to gain a better grasp of basal ganglia pathophy­ siology in disease states – like PD, Huntington’s disease and drug abuse – and in so doing develop a new generation of therapeutics.

(a)

GFP/Arc

B

(c)

Ca2+ Cav1.3

Cav3 plateau potentials?

NMDA

X D2R spine loss

MEF2A/2D RNAi

Scrambled shRNA

(b)

GFP/

Arc

GFP/ Arc

Ca2+

Cav1.2

Arc, Nurr77

Ca2+ PP2B

MEF2p CDK5 MEF2

RCSp PKA RCS

Fig. 5. Membrane depolarization induces MEF2-dependent Arc expression. (a) A D2 MSN in a corticostriatal co-culture treated with 35 mM KCl and ionotropic receptor blockers for 2 h and stained with anti-GFP and anti-Arc antibodies. High-magnification images (right panels) show Arc expression in dendrites. (b) Images of D2 MSNs in corticostriatal co-cultures transfected with indicated shRNAs and depolarized for 2 h. Transfected cells are shown in yellow squares, an untransfected cell is shown in a blue square. (c) Model showing the cellular signaling that mediates the spine loss in D2 MSNs. Scale bars: low-magnification images, 10 mm; high-magnification images 5 mm. Figure taken from Tian et al. (2010).

163

Acknowledgements This work was supported by NS34696 to DJS.

Abbreviations 2-AG 2PLSM 2PLU AIS BAC DA DAG DAGLa EC GPCR IP3 LTD LTP MEF2 MSN NO PKA PKC PLD RCS STDP STEP

2-arachidonylglycerol 2 photon laser scanning microscopy 2 photon laser uncaging axon initial segment bacterial artificial chromosome dopamine diacylglycerol diacylglycerol lipase a endocannabinoid G protein-coupled receptor inositol 1,4,5-trisphosphate long-term depression long-term potentiation myocyte enhancer factor 2 medium spiny neurons nitric oxide protein kinase A protein kinase C phospholipase D regulator of calmodulin signalling spike timing-dependent plasticity striatal-enriched tyrosine phosphatase

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A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright � 2010 Elsevier B.V. All rights reserved.

CHAPTER 9

Assemblies of glutamate receptor subunits with post-synaptic density proteins and their alterations in Parkinson’s disease Fabrizio Gardoni†, Veronica Ghiglieri‡, Monica di Luca† and Paolo Calabresi
,‡,§ †

Department of Pharmacological Sciences, University of Milano, Milano, Italy

‡ Fondazione Santa Lucia IRCCS, Rome, Italy

§ Clinica Neurologica, Università degli Studi di Perugia, Ospedale S. Maria della Misericordia, Perugia, Italy

Abstract: N-methyl-D-aspartate (NMDA) receptors have been implicated as a mediator of neuronal injury associated with many neurological disorders including ischemia, epilepsy, brain trauma, dementia and neurodegenerative disorders such as Parkinson’s disease (PD) and Alzheimer’s disease. To this, non­ selective NMDA receptor antagonists have been tried and have been shown to be effective in many experimental animal models of disease, and some of these compounds have moved into clinical trials. However, the initial enthusiasm for this approach has waned, because the therapeutic index for most NMDA antagonists is quite poor, with significant adverse effects at clinically effective doses, thus limiting their utility. More recently, the concept that the exact pathways downstream NMDA receptor activation could represent a key variable element among neurological disorders has been put forward. In particular, variations in NMDA receptor subunit composition could be important in different disorders, both in the pathophysiological mechanisms of cell death and in the application of specific symptomatic therapies. As to PD, NMDA receptor complex has been shown to be altered in experimental models of parkinsonism and in PD in humans. Further, it has become increasingly evident that the NMDA receptor complex is intimately involved in the regulation of corticostriatal long-term potentiation, which is altered in experimental parkinsonism. The following sections will examine the modifications of specific NMDA receptor subunits as well as post-synaptic associated signalling complex including kinases and scaffolding proteins in experimental parkinsonism. These findings may allow the identification of specific molecular targets whose pharmacological or genetic manipulation might lead to innovative therapies for PD.




Corresponding author.

Tel.: þ39-075 5784230; Fax: þ39-075 5784229;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83009-2

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Keywords: Striatum; Synaptic plasticity; NMDA receptor; Post-synaptic density; Experimental parkinsonism

Molecular and functional interactions between glutamate receptors and dopamine (DA) receptors regulate an incredible variety of functions in the brain and, when abnormal, they contribute to and underlie numerous central nervous system (CNS) diseases. Cross-talk between DA and glutamate signalling is relevant in a variety of different functions as motor control, cognition and memory, neurodegenerative disorders, schizophrenia and addiction. On this view, a huge number of studies have been performed in the last decade in the attempt of understanding the molecular and functional mechanisms coordinating functions of glutamate and DA receptors. There is a general agreement that an integrated interplay between DA and glutamate is essential to drive correct motor behaviours. In the striatum, dopaminergic terminals from the substantia nigra pars compacta converge with glutamatergic signals from the cortex on dendritic spines of striatal medium spiny projecting GABAergic neurons. Therefore, the striatum is thought to be a primary substrate for numerous forms of learning and memory and for controlling behavioural output. This critical role of striatum in basal ganglia relies also on the close interactions between medium spiny neurons and several subtypes of interneur­ ons, including three subtypes of GABAergic neu­ rons and large aspiny cholinergic interneurons that receive a powerful excitatory input from cor­ tex and exert a modulatory action on striatal synaptic transmission through pre- and post­ synaptic mechanisms and thus also affecting the function of glutamatergic system (Brown and Arbuthnott, 1983; Centonze et al., 2001; Cepeda et al., 1993; Garcia-Munoz et al., 1991; Kerkerian et al., 1987; Mitchell and Doggett, 1980; Rowlands and Roberts, 1980) Thus, the integrative action exerted by striatal projection neurons on the con­ verging information arising from the cortex, nigral DA neurons and interneurons shapes the activity

of neurons throughout the entire basal ganglia circuitry. It is well known that Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a mas­ sive degeneration of the dopaminergic neurons of the substantia nigra pars compacta. The progressive neurodegeneration of nigrostriatal terminals leads to a depletion of DA in the striatum altering the above-described basal ganglia functioning and is subsequently responsible for most of the PD motor symptoms such as akinesia, rigidity and tre­ mor. The degeneration of the nigrostriatal pathway in PD leads also to significant morphological and functional modifications in the striatal neuronal circuitry such as over-activity of the corticostriatal glutamatergic pathway (Centonze et al., 2005). At molecular level, the sub-cellular organization and the functional interactions of glutamate receptors within the striatum appear to be crucial in the patho­ genesis of PD (Picconi et al., 2004) as well as in the development of L-DOPA-induced dyskinesia (Gardoni and Di Luca, 2006; Hallett et al., 2006). In particular, alterations of N-methyl-D-aspartate (NMDA)-type glutamate receptors localization in striatum have been described in DA-denervated rats (Picconi et al., 2004), as well as in L-DOPA­ treated dyskinetic rats and monkeys (Gardoni and Di Luca, 2006; Hallett et al., 2006). Accordingly, it has been suggested that the normalization of corticostriatal glutamatergic transmission in the PD brain could potentially prevent functional alterations at the dendritic spines of striatal project­ ing neurons (Gardoni et al., 2009a).

Alterations of the NMDA receptor complex in experimental PD The NMDA-type glutamate receptors are abun­ dant, ubiquitously distributed throughout the CNS, fundamental to excitatory glutamatergic

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transmission and critical for normal brain func­ tion. NMDA receptors are oligomeric complexes formed by the co-assembly of members of three receptor subunit families: NR1, NR2 subfamily and NR3. NMDA receptors are enriched in the post-synaptic density (PSD) of the glutamatergic synapse, an electron dense and highly differen­ tiated structure associated with the post-synaptic membrane. The PSD consists of numerous scaffolding cytoskeletal and signalling proteins, some of which are in close contact with the cyto­ plasmic domain of glutamate ionotropic receptors in the post-synaptic membrane. This accumulation of NMDA receptors at the post-synaptic sites ensures a rapid response to neurotransmitter release and provides a molecular mechanism for linking the transmembrane ion flux to the signal­ ling machinery responsible for specific second messenger pathways. In the last decade, the increasing knowledge of the structure and func­ tion of the excitatory glutamatergic PSD (Gardoni et al., 2009a) has led to the identification of key protein families, such as PSD-MAGUKs (membrane-associated guanylate kinases), that play a fundamental role in governing NMDA receptor localization at synapse and, consequently, glutamate receptor function (Gardoni et al., 2009a). Thus, understanding the molecular mechanisms regulating the correct assembly of the NMDA receptors at synapses is a challenge in our comprehension of the strength of glutamater­ gic synapse in physiological and pathological con­ ditions. In other words, the localization of NMDA receptors in the PSD has a key role in the modula­ tion of the response of the post-synaptic neuron to different stimuli, both in activity-dependent synap­ tic plasticity and in cell death (Gardoni, 2008). CNS diseases are often characterized, at least in the early stages of the disease, by alterations in synaptic function/plasticity at the excitatory gluta­ matergic synapse without the concomitant occur­ rence of massive neuronal cell death. These alterations represent a key initial pathogenic event especially in some neurodegenerative disor­ ders such as Alzheimer’s disease (AD) and PD. Of

relevance, modifications of synaptic plasticity par­ alleled by alterations of NMDA receptor complex, molecular composition and function have been recently described in several CNS disorders such as AD, PD, Huntington’s disease (HD) and epi­ lepsy. Concerning to PD, it has become increas­ ingly evident that in striatal spiny neurons, NMDA receptor complex is intimately involved in the reg­ ulation of corticostriatal long-term potentiation (LTP) (Calabresi et al., 1996) and is altered in experimental parkinsonism (Dunah et al., 2000; Ingham et al., 1998; Ulas and Cotman, 1996). Early studies evaluated NMDA receptor abun­ dance, composition and phosphorylation in the rat lesioned with 6-hydroxydopamine (6-OHDA) as a model of parkinsonism. Dunah and co-workers (2000) found in the lesioned striatum a decrease of the abundance of NR1 and NR2B subunits of NMDA receptor in striatal membranes compared to the unlesioned striatum; conversely, the abun­ dance of NR2A was unchanged (Dunah et al., 2000; Ingham et al., 1998). Further studies in the 6-OHDA model showed similar results and corre­ lated the alteration of NMDA receptor composition at synapses with the reduction of corticostriatal synaptic plasticity (Gardoni et al., 2006; Picconi et al., 2003, 2004). In particular, NR2B subunit was specifically reduced in synaptic fraction purified from 6-OHDA rats when compared with sham­ lesioned rats in the absence of parallel alterations of NR1 and NR2A (see Fig. 1) (Gardoni et al., 2006). Intriguingly, molecular modifications of NMDA receptor at synaptic sites have been further con­ firmed in the primate model of PD (Hallett et al., 2005). In fact, it was showed that in the striatum of macaques lesioned with 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine (MPTP) DA depletion induces substantial alterations in the abundance of striatal NMDA receptor proteins, namely a reduction in the abundance of NR1 and NR2B but not NR2A subunit in the synaptic membrane fractions. In the PSD, it has been demonstrated that NMDA-type receptors are bound to scaffolding and signalling proteins, which regulate NMDA receptor clustering at synaptic sites and,

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NR1 NR2A NR2B Synaptic NMDA receptors

MAGUK

Synaptic NMDA receptors

Extrasynaptic NMDA receptors

Physiological

Parkinson’s disease

Fig. 1. Diagrammatic representation of the structural rearrangement of glutamatergic synapse in parkinsonian animals. Sub-cellular redistribution of NMDA receptors containing the NR2B subunit from synaptic to extra-synaptic sites represents the key element in the modifications of the glutamatergic synapse in 6-OHDA-lesioned animals compared with controls. These events are paralleled, and most probably triggered, by modifications of NMDA receptor NR2B subunit association with members of the MAGUKs protein family.

consequently, the strength of synaptic transmission (Gardoni et al., 2009a). In particular, C-tails of the NR2A and NR2B subunits of NMDA receptor bind to PDZ domain of members of the PSD-MAGUK family. Maintenance of the PDZ interaction is a critical element in keeping NMDA receptors at the synapse and disrupting this interaction may be the first step in the removal of the receptor (Gardoni et al., 2009b). Interestingly, alterations of NMDA receptor interaction with PSD-MAGUK proteins have been recently put forward as a common event in several neurological disorders such as epilepsy, HD and ischemia (Gardoni et al., 2009b). On this line, in the denervated striatum of parkinsonian animals the alteration of NMDA receptor subunit localization at synaptic sites is accompanied by a decreased recruitment of PSD-95 to NR2A–NR2B subunits; these events are paralleled by an increased activation of the pool of alphaCaMKII associated to the NMDA receptor complex (Picconi et al., 2004). Further, other studies reported that experimental parkinson­ ism in rats appears to be associated with decreased synaptic membrane localization and increased vesi­ cular localization of PSD-95 and SAP97 members of the PSD-MAGUK family (Lee et al., 2008; Nash

et al., 2005) that could account for dysregulation of NMDA receptors at synapses.

Alterations of AMPA receptors in experimental PD Functions of glutamate are also mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropio­ nic acid (AMPA) receptors, tetrameric proteins composed of subunits GluR1-4 that cluster at the PSD. Upon binding with glutamate, synaptic AMPA receptors induce membrane depolariza­ tion and consequently remove magnesium (Mg2þ) block from NMDA, reducing the thresh­ old to induce long-term increases of the synaptic responses. AMPA receptor-dependent depolari­ zation also opens L-type calcium (Ca2þ) channels and lead to activation of CRE elements that, bind­ ing to specific promoter regions, are responsible for gene transcription. It has been put forward that the levels of AMPA receptors at synaptic sites are very dynamic. Con­ sequently, the understanding of the cellular machinery that controls AMPA receptor traffick­ ing will be critical for unravelling the molecular and cellular basis of the function of the excitatory

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synapse and its involvement in many neurological diseases (Shepherd and Huganir, 2007). However, even if it is very difficult to ascertain if dysfunction of AMPA receptor is involved in the initial or in the late steps of a disorder, there are many evi­ dence that alterations of AMPA receptor traffick­ ing may be one of the first manifestations of synaptic dysfunction that underlies neurodegene­ ration. On the other hand, although a direct evi­ dence on the role of AMPA receptors in PD has been recently put forward (Lee et al., 2008), there is still no general consensus on the mechanism underlying dysregulation of AMPA receptor sub­ cellular distribution in PD or their pathological changes in subunit composition. Early studies reported that levels of GluR1 sub­ unit were not changed in the neostriatum of par­ kinsonian rats (Bernard et al., 1996; Betarbet et al., 2000) while GluR1 immunoreactivity was seen to be markedly increased in the caudate and putamen of MPTP-lesioned monkeys (Betarbet et al., 2000). More recent works have provided evidence that GluR1 immunoreactivity is decreased in medium spiny neurons (Lai et al., 2003) and in striatal membrane fractions of 6-OHDA-lesioned rats (Ba et al., 2006); on the contrary, no alteration of GluR1 levels in a triton-insoluble synaptic fraction was seen in striatum of 6-OHDA-lesioned rats (Picconi et al., 2004). Although controversial, these data indicate that altered AMPA receptor-mediated transmis­ sion in the basal ganglia network could play a critical contribution to the motor symptoms of PD.

Pathological synaptic plasticity in the striatum: implications for PD Two forms of synaptic plasticity long-term depres­ sion (LTD) and LTP that are thought to underlie cognitive performance have been described at cor­ ticostriatal synapse both in vitro (Calabresi et al., 1992a, 1992b; Charpier and Deniau, 1997; Lovin­ ger et al., 1993; Partridge et al., 2000; Walsh, 1993; Walsh and Dunia, 1993) and in vivo (Charpier and

Deniau, 1997; Mahon et al., 2004; Reynolds and Wickens, 2000). Both LTP and LTD are induced by using an high-frequency stimulation (HFS) pro­ tocol of the corticostriatal fibers (Calabresi et al., 1992a, 1992b; Lovinger et al., 1993) being the type of the long-lasting change critically dependent on the level of membrane depolarization and on the ionotropic glutamate receptor subtype acti­ vated during the HFS. A third form of striatal synaptic plasticity, distinct from LTD and defined synaptic depotentiation, results from the reversal of an established LTP by the application of a lowfrequency stimulation (LFS) of corticostriatal fibers (1–5 Hz) (O’Dell and Kandel, 1994; Picconi et al., 2003). At the molecular level, it has become increasingly evident that the NMDA receptor complex, which is altered in experimental parkin­ sonism (see below), is a dynamic structure involved in the regulation of corticostriatal longterm synaptic changes (Calabresi et al., 1996; Dunah et al., 2000; Hallett et al., 2005; Ingham et al., 1998; Menegoz et al., 1995; Ulas and Cotman, 1996). Of relevance, also DA, function­ ing at D1- and D2-like receptors crucially influ­ ences both the induction and the reversal of neuroplasticity at corticostriatal synapses making the concurrent involvement of glutamatergic and dopaminergic pathways a characteristic of striatal synaptic plasticity. Thus, activation of DA receptors seems to represent a crucial factor in the induction of neuroplasticity at corticostriatal synapse which is lost after pharmacological manip­ ulation or genetic disruption of the DA-mediated signalling pathway. Accordingly, corticostriatal plasticity has been shown to be impaired in striatal spiny neurons of 6-OHDA-lesioned animals (Centonze et al., 1999). Striatal spiny neurons show spontaneous membrane depolarization in coincidence with phasic release of glutamate from corticostriatal glutamatergic terminals (‘up state’). Conversely, they are silent at rest, when membrane potential is hyperpolarized (‘down state’) (Calabresi et al., 2007; Plenz and Kitai, 1998). In the ‘up state’, the membrane depo­ larization relieves the voltage-dependent Mg2þ

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block of NMDA receptor channel. This oscillatory behaviour of membrane potential accounts for the involvement of NMDA receptors in the induction of LTP. Conversely, the NMDA receptor does not play a prominent role in LTD. Moreover, the defective synaptic plasticity induction in the PD rats, which parallels the development of motor abnormalities, is accompanied by an increase in alphaCaMKII autophosphorylation along with a higher recruitment of activated alphaCaMKII to the regulatory NR2 NMDA receptor subunits (Picconi et al., 2004). This evidence suggests that CaMKII may play a critical role in this process, functioning as a signal integrator downstream of DA and glutamate receptors in the PSD.

Alterations of the glutamatergic synapse in L-DOPA-induced dyskinesia While the current DA replacement therapy offers remarkable symptomatic benefits in many PD patients, these treatments are often accompanied by severe side effects, motor fluctuations and wearing-off phenomena. In the unilateral 6-OHDA model of PD in rats, chronic treatment with L-DOPA is able to restore synaptic plasticity (Picconi et al., 2003). However, a consistent number of treated animals experiences a progressive shortening of the motor response to each drug dose, similar to L-DOPA-induced wearing-off fluctuations in PD patients (Lee et al., 2003). Moreover, rats develop abnormal involuntary movements (AIMs) of the limb con­ tralateral to the lesion, the trunk and the orofacial region, resembling human dyskinesias (Lundblad et al., 2002). Strikingly, electrophysiological recordings from dyskinetic rats demonstrated a selective impairment of striatal synaptic depoten­ tiation. Interestingly, however, experimental PD rats or mice with virtually the same degree of nigrostriatal denervation may or may not develop AIMs following chronic therapeutic doses of L-DOPA (Cenci et al., 1998; Lundblad et al., 2004; Picconi et al., 2003). Animals that do not

develop involuntary movements maintain the phy­ siological reversal of synaptic strength after LFS protocol (Picconi et al., 2003). After chronic L-DOPA therapy, the glutamater­ gic signalling from the cortex to the striatum undergoes further adaptive changes; abnormal composition and function of NMDA receptor complex have been suggested to be correlated also with the development of L-DOPA-induced dyskinesia (Gardoni et al., 2006). In particular, it has been recently demonstrated that L-DOPA­ treated dyskinetic rats are characterized by signif­ icantly higher levels of NR2A subunit while expression of NR2B subunit is reduced in the post-synaptic compartment by redistribution in extra-synaptic membranes. These events are accompanied by modifications of NMDA receptor NR2B subunit association with PSD-95, SAP-97 and SAP-102. Notably, it has been demonstrated that these molecular alterations are strictly corre­ lated to abnormal synaptic plasticity and motor behaviour in L-DOPA-treated dyskinetic rats (Gardoni and Di Luca, 2006; Gardoni et al., 2006; Picconi et al., 2003). Treatment of non-dyskinetic animals with a synthetic peptide (TAT2B) able to affect synaptic localization of NR2B, and its bind­ ing to MAGUK proteins, causes a worsening of motor symptoms with appearance of dyskinetic behaviours (Gardoni et al., 2006). These data further support the idea that molecular distur­ bances of the glutamatergic synapse, initially caused by DA denervation, create a pathological substrate that may have a causal role in the devel­ opment of L-DOPA-induced dyskinesia (Gardoni et al., 2006). Conversely, according to Nash and co-workers (2005), L-DOPA-induced dyskinesias are asso­ ciated with increased total levels of PSD-95 and SAP97, reflecting an increase at the synaptic membrane, whereas vesicular levels of both pro­ teins are decreased. Even if there are some dis­ crepancies between the above-mentioned studies, probably related to the different lesioning para­ digms and L-DOPA treatment used, they all con­ firm a central role for PSD-MAGUK proteins in

175

modulating NMDA receptor function in experi­ mental parkinsonism as well as in L-DOPA­ induced dyskinesia.

NMDA receptor antagonist in experimental PD and in dyskinesia Several studies in the last decade have exam­ ined the beneficial effects of NMDA receptor antagonists in animal models of PD (Losch­ mann et al., 2004; Nash et al., 2000) and in blocking the development of L-DOPA-induced dyskinesias (Hadj Tahar et al., 2004; Wessell et al., 2004). Overall, there is a general agree­ ment that NMDA receptor blockade attenuates parkinsonian motor symptoms and improves dopaminergic therapy. However, these agents are not well tolerated in primates due to a high number of unwanted side effects. Research has then focused on selective NMDA receptor antagonists in order to achieve anti-parkinso­ nian effects with a reduction in adverse effects. Subtype-specific antagonists might allow block­ ade of a specific subunit of the NMDA com­ plex. This would facilitate cell preservation during excitotoxic processes while not causing complete inhibition of the receptor and there­ fore allowing physiological neurotransmission. The NR2B-selective antagonist ifenprodil and its derivatives seem well suited for this purpose. These agents are effective in cell culture models of excitotoxicity and in anti-ischemic therapy. Ifenprodil has anti-parkinsonian actions in reserpine-treated rats, 6-OHDA-lesioned rats and MPTP-lesioned non-human primates (Nash and Brotchie, 2002; Nash et al., 1999, 2000). However, this agent has affinity for other recep­ tor types such as adrenergic, serotoninergic and sigma receptors. CP-101,606, a derivative of ifenprodil, acts as selective NR2B antagonist over the NR2A, reducing parkinsonian symp­ toms in both haloperidol-treated rats and MPTP-lesioned non-human primates (Nash et al., 2004; Steece-Collier et al., 2000).

In addition, it has been shown that selective NMDA receptor blockers can reduce L-DOPA­ induced dyskinesia in both experimental parkinsonism and PD patients (Del Dotto et al., 2001; Verhagen Metman et al., 1998) with­ out affecting the beneficial effects on parkinso­ nian symptoms. Selective antagonists targeting NMDA receptors composed by the NR1/NR2B subunits are able to prevent L-DOPA-induced dyskinesia in primate models of PD (Hadj Tahar et al., 2004; Morissette et al., 2006). How­ ever, apparently contradictory results have been provided by recent studies on the effects of NR2B-selective NMDA receptor antagonists on L-DOPA-induced dyskinesia in experimental par­ kinsonism (Rylander et al., 2009). Two studies described conflicting results on the effects of CP­ 101,606 on L-DOPA-induced dyskinesias in two different models of experimental parkinsonism (Nash et al., 2000; Wessell et al., 2004). However, all studies agreed that NR2B is a key element both in the experimental parkinsonism and in the devel­ opment of L-DOPA-induced dyskinesias. On the other hand there have been limited studies of NR2A-selective agents. The competitive NR2A­ selective antagonist MDL 100,453 not only increased motor activity in MPTP-lesioned non­ human primates but also increased dyskinesia caused by L-DOPA (Blanchet et al., 1999). Additionally, this agent has proved ineffective in restoring L-DOPA-associated alterations in the 6-OHDA rat (Blanchet et al., 1999).

AMPA receptor modulation in PD therapy Much effort has been recently put into developing of pharmacological agents able to modulate AMPA receptor function in order to get more insights into the mechanisms underlying the pathophysiology of neurodegenerative diseases, including PD (Johnson et al., 2009). Interestingly, if NMDA receptor antagonists have shown promise in reversing motor symptoms and delaying L-DOPA-induced dyskinesias in

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preclinical PD models, drugs targeting AMPA receptors exert more complex effects. In fact, while antagonists of these receptors may be useful in the treatment of L-DOPA-induced dyskinesias (Chase et al., 2000), AMPA receptor potentiators, also known as ampakines, are allosteric modula­ tors that slow desensitization or deactivation of AMPAR ion channels [reviewed in Arai and Kessler (2007)] and represent a promising thera­ peutic approach to contrast neurodegeneration (Johnson et al., 2009). The most interesting aspect of these positive allosteric modulators is linked to their capability to increase the expression of neurotrophic factors. In fact, by enhancing AMPA receptor activation, ampakines stimulate the downstream pathways that regulate brain-derived neurotrophic factor (BDNF) expression. In particular, strong mem­ brane depolarization induces activation of L-type Ca2þ channels leading to activation of CRE ele­ ments that, binding to BDNF promoter region, positively regulate the protein transcription. Among different functions, BDNF is also a key factor in the regulation of neuronal activity and synaptic plasticity and, through its binding with the high-affinity tyrosine kinase-linked receptor, TrkB, it modulates the activity of several receptor systems and molecular pathways of the PSD (Wu et al., 1996; Yoshii and Constantine-Paton, 2007). Many studies have been conducted in hip­ pocampal and cortical preparations to demon­ strate that BDNF, through activation of TrkB receptors, finely modulates the activity of NMDA receptor by inducing phosphorylation of NR1 sub­ unit (Lin et al., 1999; Suen et al., 1997) and NR2B subunit and its binding with protein phosphatases (Lin et al., 1999). Relevant to striatal neurons is the evidence that BDNF regulates maturation and expression of dopamine and cAMP-regulated phosphoprotein 32 (DARPP-32) (Bogush et al., 2007; Ivkovic et al., 1997), which have a key role in the maintenance of long-term plastic changes (Calabresi et al., 2000). It is therefore conceivable that alterations of BDNF signalling may play an important role in PD and dyskinesias.

On this view, recent studies conducted by O’Neill’s group using 6-OHDA and MPTP rodent models of PD suggest that selective potentiators of AMPA receptors may be useful for protection against nigral degeneration (Murray et al., 2003; O’Neill et al., 2004a, 2004b, 2005). Interestingly, these protective effects occur when the nigrostria­ tal lesion is established, suggesting that these com­ pounds exert a neurotrophic effect in animal models of PD-like neurodegeneration. In support of this, some of these compounds cause increases in expression of BDNF in the substantia nigra pars compacta suggesting that enhancing AMPA receptor activation may slow the normal loss of these neurons that occurs with age and perhaps prevent levels of nigral degeneration that cause PD symptoms. In line with these evidences, a recent paper by Jourdy and colleagues demonstrated that by increasing BDNF expression and activating BDNF-dependent signalling pathways, ampakines exert neuroprotective effect against MPP(þ)­ induced toxicity (Jourdi et al., 2009). These results provide a strong support for testing positive AMPA modulators as a new possible therapy for neurodegenerative disorders, including PD.

Functional and molecular cross-talk between D1 and NMDA receptors: role in physiological synaptic transmission, in experimental PD and in L-DOPA-induced dyskinesia DA and glutamate receptors’ functional interac­ tion in the striatum, as well as in other brain structures, has been shown to regulate locomo­ tion, positive reinforcement, attention and work­ ing memory (Cepeda and Levine, 1998). As stated above, dopaminergic terminals from the substantia nigra pars compacta converge with glutamatergic signals from the cortex on dendritic spines of stria­ tal medium spiny projecting GABAergic neurons. Several studies, using different experimental approaches, have shown that in striatal spiny neu­ rons the D1 receptors are located within dendritic

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spines, both in the spine neck and, probably to a lesser extent, in the PSD (Fiorentini et al., 2003); in this way, PSD-associated D1 receptors co-localize in striatal spiny neurons with NMDA receptors. D1 receptor-mediated potentiation of NMDA responses in the striatum has been described in the early 1990s (Cepeda et al., 1992, 1993) and then confirmed in other brain areas (Chen et al., 2004; Wang and O’Donnell, 2001). Notably, D1 receptor-mediated increase of NMDA responses can lead to significant functional consequences. In fact, potentiation of NMDA receptor-mediated responses can enhance glutamate activity up to predisposing to excitotoxicity. On the other hand, as stated also above, activation of D1 recep­ tors in the striatum is required for the induction of LTP (Calabresi et al., 2000; Kerr and Wickens, 2001), suggesting further that activation of D1 receptors is needed for the correct integration of cortical glutamatergic signals to the striatum. Different mechanisms have been proposed to be involved in the functional cross-talk existing in the striatum between D1 and NMDA receptors, ranging from second messenger-mediated phos­ phorylation of NMDA receptor subunits, and con­ sequent regulation of receptor trafficking at synaptic sites, to direct interaction leading to the formation of heteromeric D1/NMDA receptor complexes (Cenci and Lundblad, 2006). Early studies reported that D1 receptor co­ immunoprecipitates with NMDA receptor subu­ nits from isolated PSD, suggesting that these two receptor types are co-clustered in the post-synaptic compartment (Fiorentini et al., 2003). Further, of key relevance, a direct interaction has been clearly demonstrated between the C-terminal tails of D1 receptor and the NR1 and NR2A sub­ units of NMDA receptor (Lee et al., 2002). These studies also addressed the functional role of the direct D1/NMDA receptor molecular interaction. In particular, D1 interaction with the NR2A subunit is involved in the inhibition of NMDA receptor-gated currents obtained through a decrease in the number of cell surface receptors

(Lee et al., 2002). Moreover, D1/NR1 interaction has been clearly correlated with the attenuation of NMDA receptor-mediated excitotoxicity through a PI-3 kinase-dependent pathway. Recent studies showed that, in striatal neurons, D1 receptor activation leads to rapid trafficking of NMDA receptor subunits, with increased NR1 and NR2B subunits in dendrites and enhanced co-clustering and surface expression of these sub­ units at synaptic sites (Hallett et al., 2006). Inter­ estingly, D1 receptor-mediated NMDA receptor trafficking is blocked by tyrosine kinase inhibi­ tors, while blockers of tyrosine phosphatases also induce NMDA subunit trafficking, but the effect is non-selective and alters both NR2A­ and NR2B-containing receptors. Other signalling cascades have been shown to regulate D1 recep­ tor-dependent enhancement of NMDA responses in the striatum. The most important involves pro­ tein kinase A (PKA) and DARPP-32-regulated phosphorylation of NMDA receptor NR1 subu­ nits (Snyder et al., 1998). Further, NMDA recep­ tor potentiation by phospholipase C-coupled D1­ like receptors has been shown to occur via PKC activation (Chergui and Lacey, 1999). Although much effort has been put into under­ standing the D1/NMDA relationships, a fine reg­ ulation of the interplay between glutamate and DA systems cannot be achieved without the inter­ action of D1 receptor with AMPA receptors. In particular, Wolf and co-workers have demon­ strated that D1 DA receptor stimulation enhances phosphorylation of GluR1 at the PKA site, increases surface expression of AMPA receptors and facilitates their synaptic insertion in several brain areas (Gao et al., 2006; Sun et al., 2005). Notably, in the last few years it has been demon­ strated that alterations of NMDA receptor cluster­ ing with DA receptors can play a central role in the pathogenesis of PD as well as in L-DOPA-induced dyskinesias (Fiorentini et al., 2006). Interestingly, D1/NMDA receptor clusters containing NR2B subunits were decreased in the PSD of 6-OHDA­ lesioned striatum (Fiorentini et al., 2006). Pro­ longed L-DOPA treatment normalizes synaptic

178

D1/NMDA receptor complexes in non-dyskinetic rats, but remarkably reduces them in the dyski­ netic group without changing their interaction. Notably, the levels of D1/NMDA receptors com­ plexes are unchanged in total membrane proteins, suggesting that the decrease of these species in the PSD is likely to reflect an altered receptor traffick­ ing (Fiorentini et al., 2006). Modification of these pathways may become an additional therapeutic target for PD and L-DOPA­ induced dyskinesias in which abnormal function of striatal glutamate receptors contributes to the symptoms.

CNS DA HD HFS LTD LTP MAGUKs MPTP NMDA PD PSD

Conclusions In the last decade, the increasing knowledge of the structure and function of the excitatory glutama­ tergic PSD has led to the identification of key protein families, such as PSD-MAGUKs, that play a fundamental role in governing NMDA receptor localization at synapse and, consequently, NMDA receptor function. It has been shown that alterations of NMDA receptor complexes’ locali­ zation in the PSD could represent an important event in different central system disorders (Gardoni et al., 2009a). As to PD, because altera­ tions in NMDA receptor localization at synapse contribute to the clinical features of the experimen­ tal parkinsonism and may underlie the develop­ ment of dyskinesias, therapies targeted to modulate protein–protein interactions between NMDA receptor subunits and PSD-associated scaf­ folding elements able to regulate striatal NMDA receptor trafficking/localization of specific subunits may be useful in the treatment of the disease.

Abbreviations 6-OHDA AD AIMs

AMPA

6-hydroxydopamine Alzheimer’s disease abnormal involuntary movements

alpha-amino-3-hydroxy-5­ methyl-4-isoxazolepropionic acid central nervous system dopamine Huntington’s disease high-frequency stimulation long-term depression long-term potentiation membrane-associated guanylate kinases 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine N-methyl-D-aspartate Parkinson’s disease post-synaptic density

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181 Morissette, M., Dridi, M., Calon, F., Hadj Tahar, A., Meltzer, L. T., Bedard, P. J., et al. (2006). Prevention of dyskinesia by an NMDA receptor antagonist in MPTP monkeys: Effect on ade­ nosine A2A receptors. Synapse, 60, 239–250. Murray, T. K., Whalley, K., Robinson, C. S., Ward, M. A., Hicks, C. A., Lodge, D., et al. (2003). LY503430, a novel alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor potentiator with functional, neuroprotective and neurotrophic effects in rodent models of Parkinson’s disease. Journal of Pharmacology and Experimental Therapeutics, 306, 752–762. Nash, J. E., & Brotchie, J. M. (2002). Characterisation of striatal NMDA receptors involved in the generation of parkinsonian symptoms: Intrastriatal microinjection studies in the 6-OHDA-lesioned rat. Movement Disorders, 17, 455–466. Nash, J. E., Fox, S. H., Henry, B., Hill, M. P., Peggs, D., McGuire, S., et al. (2000). Antiparkinsonian actions of ifen­ prodil in the MPTP-lesioned marmoset model of Parkinson’s disease. Experimental Neurology, 165, 136–142. Nash, J. E., Hill, M. P., & Brotchie, J. M. (1999). Antiparkin­ sonian actions of blockade of NR2B-containing NMDA receptors in the reserpine-treated rat. Experimental Neurol­ ogy, 155, 42–48. Nash, J. E., Johnston, T. H., Collingridge, G. L., Garner, C. C., & Brotchie, J. M. (2005). Subcellular redistribution of the synapse-associated proteins PSD-95 and SAP97 in animal models of Parkinson’s disease and L-DOPA-induced dyski­ nesia. The FASEB Journal, 19, 583–585. Nash, J. E., Ravenscroft, P., McGuire, S., Crossman, A. R., Menniti, F. S., & Brotchie, J. M. (2004). The NR2B-selective NMDA receptor antagonist CP-101,606 exacerbates L-DOPA-induced dyskinesia and provides mild potentiation of anti-parkinsonian effects of L-DOPA in the MPTP­ lesioned marmoset model of Parkinson’s disease. Experi­ mental Neurology, 188, 471–479. O’Dell, T. J., & Kandel, E. R. (1994). Low-frequency stimulation erases LTP through an NMDA receptor-mediated activation of protein phosphatases. Learning & Memory, 1, 129–139. O’Neill, M. J., Bleakman, D., Zimmerman, D. M., & Nisenbaum, E. S. (2004a). AMPA receptor potentiators for the treatment of CNS disorders. CNS & Neurological Disorders – Drug Targets, 3, 181–194. O’Neill, M. J., Murray, T. K., Clay, M. P., Lindstrom, T., Yang, C. R., & Nisenbaum, E. S. (2005). LY503430: Pharma­ cology, pharmacokinetics, and effects in rodent models of Parkinson’s disease. CNS & Neurological Disorders, 11, 77–96. O’Neill, M. J., Murray, T. K., Whalley, K., Ward, M. A., Hicks, C. A., Woodhouse, S., et al. (2004b). Neurotrophic actions of the novel AMPA receptor potentiator, LY404187, in rodent models of Parkinson’s disease. European Journal of Pharmacology, 486, 163–174. Partridge, J. G., Tang, K. C., & Lovinger, D. M. (2000). Regional and postnatal heterogeneity of activity-dependent

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A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 10

Pathophysiological roles for purines: adenosine, caffeine and urate Micaela Morelli†,
, Anna R Carta†, Anil Kachroo‡ and Michael A. Schwarzschild‡ † Department of Toxicology, University of Cagliari, Cagliari, Italy MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Boston, MA, USA

‡

Abstract: The motor symptoms of Parkinson’s disease (PD) are primarily due to the degeneration of the dopaminergic neurons in the nigrostriatal pathway. However, several other brain areas and neurotransmitters other than dopamine such as noradrenaline, 5-hydroxytryptamine and acetylcholine are affected in the disease. Moreover, adenosine because of the extensive interaction of its receptors with the dopaminergic system has been implicated in the pathophysiology of the disease. Based on the involvement of these non-dopaminergic neurotransmitters in PD and the sometimes severe adverse effects that limit the mainstay use of dopamine-based anti-parkinsonian treatments, recent assessments have called for a broadening of therapeutic options beyond the traditional dopaminergic drug arsenal. In this review we describe the interactions between dopamine and adenosine receptors that underpin the pre-clinical and clinical rationale for pursuing adenosine A2A receptor antagonists as symptomatic and potentially neuroprotective treatment of PD. The review will pay particular attention to recent results regarding specific A2A receptor–receptor interactions and recent findings identifying urate, the end product of purine metabolism, as a novel prognostic biomarker and candidate neuroprotectant in PD. Keywords: A2A receptors; neuroprotection; neurodegeneration; Parkinson’s disease

Adenosine receptors: localization and functional interactions with dopamine receptors

only the A1 and A2A are thought to play an impor­ tant role at physiological concentrations of adeno­ sine in the CNS (Fredholm et al., 2005). The A1 receptors has a widespread distribution in the brain whereas, of particular relevance for Parkin­ son’s disease (PD), the A2A receptor is highly enriched in dopamine innervated areas. High densities of adenosine A2A receptors are present in both the ventral and the dorsal striatum of rodents and primates, including humans. These

Adenosine acts in the brain via four cloned and pharmacologically characterized G-proteins’ coupled receptors: A1, A2A, A2B and A3, however,
Corresponding author. Tel.: 0039-0706758663; Fax: 0039-0706758665 E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83010-9

183

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receptors co-localize in the striatum with the dopa­ mine D2 receptor in the dendritic spines of enke­ phalin-rich striatopallidal gamma-aminobutyric acid (GABA) neurons and on glutamatergic term­ inals (Rosin et al., 1998; Schiffmann et al., 1991). This anatomical framework provides an important structural basis to our understanding of previously discovered A2A/D2 functional interactions. In addition, A2A receptors are highly expressed in the globus pallidus (GP), mainly in the neuropil, where their stimulation enhances striatopallidal GABA outflow, and their blockade reduces it (Ochi et al., 2000; Rosin et al., 1998; Shindou et al., 2003). In 6-hydroxydopamine (6-OHDA)­ lesioned rats, intra-pallidal infusion of A2A recep­ tor antagonists, while not eliciting any motor response per se, does potentiate motor activity induced by L-DOPA (L-3,4-dihydroxyphenylala­ nine) or dopaminergic agonists. This suggests that blockade of pallidal A2A receptors, by redu­ cing extracellular GABA, may stabilize GP activ­ ity and in turn subthalamic nucleus (STN) activity (Simola et al., 2006). Therefore, both structures may contribute to the therapeutic action of A2A receptor antagonists. Adenosine A2A receptors exert an excitatory influence on striatopallidal neurons, in part through their antagonistic effect on dopamine D2 receptor activation (Fig. 1). The basis of this antagonistic action of adenosine A2A receptors is their ability to decrease the binding affinity of D2 receptors for dopamine as demonstrated in rat striatal membrane, in human striatal tissue and in different cell lines (Canals et al., 2003; DiazCabiale et al., 2001; Ferré et al., 1991; Hillion et al., 2002). In agreement with these studies, sti­ mulation of adenosine A2A receptors counteracts the D2 receptor-mediated inhibition of cyclic ade­ nosine monophosphate (cAMP) formation and D2 receptor-induced intracellular Ca2þ responses (Kull et al., 1999; Olah and Stiles, 2000; Salim et al., 2000). Of great importance, A2A receptors exert a strong influence on dopamine- and cAMP­ regulated phosphoprotein-32 (DARPP-32), a DARPP, which is expressed at high levels in the

GABAergic efferent neurons and is deeply involved in dopamine-mediated signalling (Linds­ kog et al., 2002) (Fig. 1). The regulation of dopaminergic signal transduc­ tion by A2A receptors is also illustrated by the regulation of cAMP response element-binding (CREB) activity by A2A receptor stimulation, which increases cAMP formation and in turn phosphoryla­ tion of CREB. Selective D2 receptor agonists dose dependently counteracted these effects (Kull et al., 1999). Furthermore, a variety of in vivo studies sup­ port the reciprocal antagonistic influence of A2A and D2 receptors in induction of immediate early gene expression (e.g. c-fos, zif/268, NGFI-B and jun-B) (Morelli et al., 1995; Svenningsson et al., 1999; Tronci et al., 2006). Interestingly, the gene expression of striatal A2A receptors and the A2A/D2 receptor inter­ action are increased in the dopamine-denervated striatum (Ferré and Fuxe, 1992) (Fig. 1). Despite these mentioned findings, it has been shown that stimulation, as well as blockade, of adenosine A2A receptors induces behavioural and biochemical responses in mice lacking dopamine D2 receptors, suggesting that adenosine A2A receptor actions can occur independently of dopamine signalling (Aoyama et al., 2000). Receptor–receptor interaction: heterodimeric complexes as basis for new anti-parkinsonian therapies An important finding, with respect to A2A recep­ tors, is the formation of functional receptor complexes (receptor mosaics) with other G protein-coupled receptors (GPCRs). A2A receptors, like many other GPCRs, form both homo and heterodimers (Agnati et al., 2003). This discovery has further increased our understanding of the biol­ ogy of the A2A receptor with particular emphasis on molecular interactions with receptors for other neurotransmitters such as dopamine D2, D3, canna­ binoid CB1 and metabotropic glutamate mGlu5 receptors (Fig. 1). Heterodimerization may have functional and pharmacological consequences;

185 A2A

D2

GS Golf

CB1

mGlu5

Gq

Gi/o

AC

PLC

PP-2 Ca2+ DARPP-32-P (Thr75)

cAMP

DARPP-32-P (Thr 34)

PKA

PKC

CaMK II / IV

PP-1

MAPK/ERK

CREB-P

Gene expression

(c-fos, ENK, neurotensin, zif 268)

Fig. 1. Functional interactions between dopamine D2, adenosine A2A, cannabinoid CB1 and glutamate mGlu5 receptors in striatopallidal neurons. Adenosine A2A receptors interact antagonistically with D2 and CB1 receptors at the intramembrane level and at the adenylyl cyclase level; metabotropic glutamate mGlu5 and adenosine A2A receptors act synergistically to counteract the D2 dopamine receptor signalling in striatopallidal neurons. Synergistic interactions exist between A2A and mGlu5 receptors at the level of c-fos expression, MAP kinases and phosphorylation of DARPP-32 protein; for further explanation see text. broken arrows, inhibitory effect; ‘þ’, stimulation; ‘–’, inhibition; AC, adenylyl cyclase; Ca2þ, calcium ions; CaMK II/IV, calcium/calmodulin­ dependent protein kinase type II/IV; cAMP, cyclic AMP; CREB, cAMP response element-binding protein; Kþ, potassium channel; DARPP-32, dopamine- and cAMP-regulated phosphoprotein; DARPP-32-P (Thr75) and DARPP-32-P (Thr34), DARPP­ 32-phopshorylated at threonine residues 75 and 34, respectively; Gi, Go, inhibitory G proteins; Gq, Gs, Golf, stimulatory G proteins; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PP-1, protein phosphatase-1; PP-2, protein phosphatase-2.

therefore, the presence of heterodimeric complexes has constituted a considerable step forward in the neurobiology of adenosine, suggesting new ways of modulating neuronal activity by targeting the A2A receptor (Ferré et al., 2009; Fuxe et al., 2003). Co-immunoprecipitation studies with fluorescence resonance energy transfer (FRET) and BRET (Bioluminescence Resonance Energy Transfer) analyses have demonstrated the existence of constitutive A2A/D2 heteromers as well as A2A homodimers within the plasma membrane,

indicating less than 10 nm separating the receptors (Canals et al., 2003; Hillion et al., 2002). Functional implications of the A2A/D2 intramem­ brane receptor–receptor interaction through heteromerization include a decrease in receptor affinity for dopamine agonists acting on D2 recep­ tors, as well as a reduction of D2 receptor G protein coupling and signalling. Thus, the essence of this A2A/D2 receptor heteromerization may be to con­ vert the D2 receptor into a state of strongly reduced functional activity. The discovery of heteromeric

186

A2A/D2 complexes has added to the substantial evidence for antagonistic molecular, cellular, elec­ trophysiological and behavioural interactions between A2A and D2 receptors. These form the basis for anti-parkinsonian strategies that simulta­ neously block adenosine A2A and stimulate dopa­ mine D2 receptors (Morelli et al., 2007, 2009). Moreover, evidence for functional A2A/D3 het­ eromers has recently been obtained in co-trans­ fected A2A/D3 cells using FRET. A2A activation reduces the affinity of D3 agonist binding sites as well as D3 signalling (Torvinen et al., 2005). It should be noted, however, that while this interac­ tion could provide interesting clues for mediation of ventral striatal (limbic) functions, it might not be relevant for dorsal striatum functions where the D3 receptor, which is expressed only after dopa­ mine denervation, and the A2A receptor are seg­ regated in different neuronal populations (of striatonigral and striatopallidal neurons, respec­ tively) (Bordet et al., 2000). Although functional properties of multiple receptor heteromers remain to be determined, the interaction of A2A receptors with receptors other than those for dopamine is also relevant (Fig. 1). Co-immunoprecipitation evidence shows that A2A and mGlu5 receptors form heteromeric complexes, and combined stimulation of both of these receptor types synergistically reduced the affinity of the D2 receptor agonist binding sites in striatal membranes (Fuxe et al., 2003). These observations were supported by the high degree of A2A and mGlu5 co-localization in primary cultures of striatal neurons and in striatal glutama­ tergic nerve terminals (Rodrigues et al., 2005). Co-activation of A2A and mGlu5 receptors causes a synergistic interaction at the level of c-fos expression and on extracellular signal-regulated kinases (ERK) as well DARPP-32 phosphoryla­ tion, indicating a possible role of this heteromeric complex in striatal plasticity (Ferré et al., 2002; Nishi et al., 2003) (Fig. 1). Combined A2A and mGlu5 receptor activation may also produce synergistic cellular effects on striatal output neu­ rons in vivo, as demonstrated by a greater than

additive increase in GABA release from ventral striatopallidal neurons after local perfusion with both A2A and mGlu5 agonists (Diaz-Cabiale et al., 2002). Similarly, the discovery of functional A2A/ mGlu5 receptor interactions and heteromeric A2A/mGlu5 complexes led to recent findings of a synergistic anti-parkinsonian potential of combin­ ing A2A and mGlu5 antagonists (Coccurello et al., 2004; Kachroo et al., 2005). Another interesting interaction that may have important implications for the design of new drugs useful in the treatment of PD is that of adenosine or dopamine receptors with cannabinoid CB1 recep­ tors whose presence has been described in basal ganglia, most specifically in GABAergic striatal neurons (Egertova and Elphick, 2000) (Fig. 1). CB1 receptors co-localize with D2 and A2A recep­ tors predominantly in the soma and dendrites of the striatopallidal GABA neurons and in corticostriatal glutamate terminals. CB1–D2 heteromers as well as CB1–A2A heteromeric complexes have been described in HEK-293 cell lines (Ferré et al., 2009; Marcellino et al., 2008). Interestingly post-synaptic CB1 receptor signalling was found to be dependent on A2A receptor activation. Accordingly, blockade of A2A receptors counteracted the motor depres­ sant effects and extracellular field potentials, in corticostriatal neurons, produced by cannabinoid CB1 agonist (Carriba et al., 2007; Tebano et al., 2009). At the same time, CB1 receptors mediate psychomotor activation by A2A receptors antago­ nists (Lerner et al., 2010). Antagonistic CB1/D2 interactions have been described at the behavioural level as well. The CB1 receptor agonist CP 55940 at a dose that did not change basal locomotion is able to block quin­ pirole-induced increases in locomotor activity. In addition, not only the CB1 receptor antagonist rimonabant but also the specific A2A antagonist MSX-3 blocked the inhibitory effect of CB1 recep­ tor agonists on D2-like receptor agonist-induced hyperlocomotion (Marcellino et al., 2008). These results find support in the existence of antagonistic CB1/D2 receptor–receptor interactions within CB1/D2 heteromers in which A2A receptors

187

might also participate (Marcellino et al., 2008) (Fig. 1). Based on this biochemical evidence show­ ing how CB1 receptors interact with both A2A and D2 receptors, it has been proposed that these receptors are putative targets for PD. Role of adenosine receptors in neuroprotection Arresting disease progression at the level of its underlying neuronal degeneration remains a criti­ cal unmet goal of neurotherapeutics for PD. More than a decade of research has suggested that manipulating adenosine neurotransmission might offer a valuable strategy to achieve neuroprotec­ tion in PD. The first studies investigating adenosine and neuroprotection were conducted in models of ischemic and excitotoxic brain injury [reviewed in Fredholm et al. (2005)]. Under these conditions, increased extracellular adenosine in response to brain injury has been shown to act as a neuropro­ tectant (Dux et al., 1990; Evans et al., 1987). How­ ever, a pro-neurotoxic role of adenosine has also been demonstrated, suggesting that blockade of adenosine receptors may confer neuroprotection across a range of neurodegenerative disorders (Chen et al., 1999; de Mendonca et al., 2000; Jones et al., 1998; Melani et al., 2003; Pinna et al., 2010; Popoli et al., 2002). This apparent paradox reflects the complexity of adenosine transmission, with several receptor sub-types selectively localized in brain areas and uniquely coupled to G proteins and signalling pathways, as described above. Moreover, the important role played by adenosine in the immune response in the central nervous system (CNS) should be con­ sidered as a component of degenerative and pro­ tective processes. Hence, the same adenosine receptor sub-type expressed in different cell types such as neurons and glia may mediate opposing effects in response to different neuro­ toxic insults (Fig. 2). In PD, the initial suggestion that manipulating adenosine neurotransmission might be beneficial

in terms of affecting disease onset or progression came from the epidemiological evidence that con­ sumption of caffeine, a non-specific A1/A2A recep­ tor antagonist, is associated with a reduced risk of developing PD (Ascherio et al., 2001; Schwarzs­ child et al., 2003b). Convergent laboratory studies investigating the effect of caffeine and more spe­ cific adenosine receptor antagonists suggested that blockade of the A2A receptor sub-type prevents nigrostriatal degeneration in several models of PD (Schwarzschild et al., 2006). Interestingly, epide­ miological studies have identified a second purine as another robust inverse risk factor for PD. Blood levels of urate (the end product of adenosine metabolism in humans) are also strongly linked to a reduced risk of PD and, more recently, of its progression, as described below.

Neuroprotection and A1 adenosine receptors Preclinical evidence for a role of A1 receptors in neuroprotection in PD is sparse; therefore this section will focus mostly on a general role of A1 receptors on glutamate release and toxic cytokine control. A1 adenosine receptors are widely distributed throughout the CNS, being expressed both in neu­ ronal and in glial cells (Daré et al., 2007; Ochiishi et al., 1999; Svenningsson et al., 1997). Neuronal A1 adenosine receptors exist in pre-synaptic term­ inals as well as in post-synaptic membranes. How­ ever, the most striking effect of their stimulation is the inhibition of neurotransmitter release, mediated by a reduction of pre-synaptic calcium influx (Borycz et al., 2007; Brown et al., 1990; Masino et al., 2002; Ochiishi et al., 1999). Although similar effects have been reported for different neurotransmitters, including glutamate, dopamine, acetylcholine and GABA, the inhibition of glutamate release holds the main interest for neurodegeneration. Based on this mechanism, A1 receptor agonists have been mainly pursued for their potential to protect under conditions characterized by a massive release of glutamate,

188

Astroglia Pre-synaptic neuron

– –

+

–

Post-synaptic neuron

Glu release

A2A- R Ca2+

GDNF-R

–/+ ? NMDA-R TNF-α IL-Iβ IL-6 iNOS COX-2

–

Glutamate GLT-1

Microglia

A2A R antagonist NO/COX-2

Fig. 2. Schematic representation of possible cellular mechanisms affected by A2A receptor blockade in a neuroprotective model of PD. In pre-synaptic neurons, A2A antagonism inhibits glutamate efflux either directly or indirectly through an inhibition of GDNF receptors (see text for more details on this mechanism). A decrease in glutamate release results in reduced glial response and as a consequence release of toxic factors. In microglia, direct A2A receptor blockade inhibits NO and COX-2 production. In astroglia, direct A2A receptor blockade inhibits glutamate release directly or indirectly through an inhibition of GLT-1.

as occurs in ischemic stroke. Few studies have investigated the effects of A1 receptor agonism in models of PD. Lau and Mouradian (1993) have shown that an adenosine A1 agonist was able to prevent the decrease in striatal dopamine levels induced by a single injection of the neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). Accordingly, the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxathine (CPX) may have slightly exacerbated striatal MPTP toxicity (Chen et al., 2001). Although the mechanism has not been fully clarified, an inhibition of excessive release of glutamate, which may contribute to MPTP toxicity, might be involved in the protective effect displayed by A1 receptor agonists in the

striatum. In line with this interpretation, blockade of N-methyl-D-aspartic acid (NMDA) glutamate receptors achieved a similar protective effect against an acute MPTP insult (Chan et al., 1993). Other clues to A1-mediated neuroprotection in PD models came from evidence that caffeine pre-treat­ ment protected against methamphetamineinduced nigrostriatal toxicity via an upregulation of A1 receptors (Delle Donne and Sonsalla, 1994). Accordingly, Alfinito and colleagues have shown that administration of an A1 antagonist exacer­ bated dopamine neuron degeneration induced by the mitochondrial inhibitor malonate, although nigral but not striatal A1 receptors were selectively involved in the effect (Alfinito et al., 2003).

189

Glial A1 receptors (Haskó et al., 2008) may play an important role in dopamine neuron demise in PD. The involvement of this receptor in neuroinflammatory responses has been well documented, although direct evidence towards its involvement in neuroinflammation associated with nigrostriatal damage is lacking. In the pre­ sence of ischemia and brain injury, extracellular adenosine stimulates glial A1 receptors, resulting in inhibition of astrocyte proliferation and exces­ sive reactive astrogliosis, as well as increased pro­ duction of trophic factors, such as nerve growth factor (NGF), S100beta protein and transforming growth factor beta, which in turn may help to protect neurons from injury. Interestingly, the anti-inflammatory cytokine IL-6 enhances the expression of A1 receptors in astrocytes, suggest­ ing that a self-modulating loop involving A1 recep­ tors is triggered in presence of a neuronal damage (Ciccarelli et al., 2001; Schubert et al., 1997; van Calker and Biber, 2005). Although the topic is still poorly investigated, based on this evidence it is tempting to speculate that a modulation of the astroglial response through the A1 receptor might be beneficial in PD nigrostriatal degeneration.

Neuroprotection and adenosine A2A receptors In contrast to the A1 receptor, the A2A receptor has been consistently implicated as a mediator or modulator of dopaminergic neuron degeneration across a range of laboratory models of PD. An early suggestion of the neuroprotective potential of A2A receptor blockade in PD came with the demonstration that caffeine can attenuate the loss of striatal dopamine induced by acute MPTP administration in mice (Chen et al., 2001). Caffeine has also been shown to be neuroprotec­ tive in other models of PD such as the unilaterally 6-OHDA-lesioned rat (Joghataie et al., 2004) and more recently in a dual pesticide, chronic expo­ sure model (Kachroo et al., 2010), in which repeated systemic administration of paraquat

plus maneb leads to degeneration of nigral dopa­ minergic neurons. In addition, two major demethylation metabolites of caffeine, namely theophylline and paraxanthine, both adenosine receptor antagonists themselves, have also been shown to protect against MPTP-induced neuro­ toxicity in mice (Xu et al., 2010). These consistent findings of neuroprotection by caffeine and its metabolites in multiple models of PD have strengthened the hypothesis that a true protective effect of caffeine is the basis for its inverse asso­ ciation with PD risk in epidemiological studies (see below). A similar protective effect was observed upon administration of the selective A2A receptor antagonist KW-6002, but not with the A1 receptor antagonist CPX (Chen et al., 2001; Pierri et al., 2005; Schwarzschild et al., 2006). In line with these results, genetic deletion of A2A receptors pre­ vented the loss of striatal dopamine induced by acute MPTP (Chen et al., 2001). Thereafter, a protective effect by A2A antagonists was reported in a different toxin model of PD, in which 6-OHDA is infused within the rat striatum (Ikeda et al., 2002). In these studies, neuroprotec­ tion was assessed as attenuation of the striatal dopamine depletion or of the loss of tyrosine hydroxylase (TH)-positive cells in the substantia nigra pars compacta (Table 1). Recently, the A2A receptor antagonists SCH-58261 and ANR 94 were shown to prevent the death of nigral dopa­ minergic neurons induced by sub-chronic MPTP administration in mice (Carta et al., 2009; Pinna et al., 2010). Therefore, blockade of the A2A receptor seems to confer a functional protection of striatal dopamine transmission, as well as to prevent the loss of nigral dopaminergic neurons induced by neurotoxin exposure. Despite considerable evidence suggesting the neuroprotective potential of A2A receptor block­ ade in PD, the underlying mechanism is still a matter of debate. In neurons, A2A adenosine receptors have been identified both pre- and post-synaptically, where they control neurotrans­ mitter release and neuronal stimulation,

190 Table 1. Neuroprotective outcome of A2AR manipulation in different rodent PD models

PD model 6-OHDA in Str MPTP acute

MPTP subchronic

A2AR manipulation

Functional protection in Str

SNc neurons survival

Attenuation of glial response

A2A antag.

þ

þ

NA

A2A antag.

þ

þ

þ

totA2AKO

þ

NA

NA

fbnA2AKO

–

NA

NA

A2A antag.

þ

þ

þ

fbnA2AKO

þ

þ

þ

References Ikeda et al. (2002) Chen et al. (2001) Ikeda et al. (2002) Pierri et al. (2005) Yu et al. (2008) Carta et al. (2009)

Parkinson’s disease, PD; antag., antagonists; 6-OHDA, 6-hydroxydopamine; MPTP, 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine.

Summary of recent data highlighting the neuroprotective outcome observed in both the striatum and the substantia nigra by pharmacologically and

genetically targeting the A2A receptor in different PD models.

respectively (Rebola et al., 2005; Rosin et al., 1998; Schiffman et al., 1991; Svenningsson et al., 1999) (Fig. 2). Moreover, cells involved in the neuroinflammatory response such as astroglia, microglia and bone marrow-derived cells all express the A2A receptor (Fiebich et al., 1996; Saura et al., 2005). Since many factors may con­ tribute to neuronal demise in PD, including neu­ ronal pathological processes and chronic neuroinflammation, several mechanisms have been investigated to explain the neuroprotective effect of A2A receptor blockade. Interestingly, recent reports have highlighted that multiple mechanisms, involving either neuronal or glial receptors, may be recruited in different PD mod­ els to achieve neuroprotection by A2A receptor blockade (Carta et al., 2009; Yu et al., 2008).

Neuronal A2A receptors and neuroprotection An alternative strategy employed to complement pharmacological approaches in demonstrating the neuroprotective efficacy of A2A receptor blockade has been offered by targeted gene mutations

producing mice lacking this receptor. In general, A2A receptor knockout mice display attenuated brain damage in models of ischemia or excito­ toxin-induced brain injury as had been previously observed using traditional pharmacological antagonists (Chen et al., 1999; Monopoli et al., 1998; Pedata et al., 2005; Phillis, 1995). More recently, studies using a conditional knockout (Cre/loxP) system to generate mice with a selec­ tive post-natal depletion of forebrain neuronal A2A receptors have contributed to the under­ standing of cellular mechanisms of A2A receptormediated neuroprotection in PD. In a sub-chronic MPTP model of PD, loss of these A2A receptors fully prevented neurotoxin-induced degeneration of nigral dopaminergic neurons, endorsing a pri­ mary role of neuronal A2A receptors in the neu­ roprotective effects of A2A antagonists in this model (Carta et al., 2009). However, neuronal A2A receptors seemed to play a minor role in striatal dopamine loss induced by a more acute MPTP intoxication, since Yu et al. (2008) reported that neuronal A2A receptors inactivation did not protect striatal terminals from a one-day, highdose MPTP exposure (Yu et al., 2008).

191

Interestingly, in this study mice globally lacking A2A receptors were partially protected against striatal MPTP toxicity. This suggests that blockade solely of neuronal A2A receptors might not be sufficient to prevent neurotoxicity induced by acute MPTP in the striatum, or alternatively that cells other than forebrain neurons, likely glial cells, may play a role in A2A-mediated protection in the striatum (Yu et al., 2008) (Fig. 2). Studies of toxin-induced striatal neuron death modelling the core neurodegenerative features of Huntington disease rather than PD have also given opposing results regarding the role of A2A receptors. In corticostriatal slices A2A receptor antagonism reduced the irreversible functional alterations caused by rotenone in striatal neurons, supporting a beneficial role of neuronal A2A receptor blockade against neurotoxic insults (Bel­ castro et al., 2009). By contrast, Huang et al. reported that in a model of acute striatal neuron damage produced by local infusion of the mito­ chondrial toxin 3-nitropropionic acid (3-NP), selective deletion of A2A receptors on forebrain neurons was unable to protect against neurotoxi­ city (Huang et al., 2006). In this model global A2A receptor inactivation actually exacerbated 3-NP­ induced neurotoxicity. Moreover, the cell-type­ specific inactivation of A2A receptors located in bone marrow-derived cells also exacerbated stria­ tal damage (Huang et al., 2006). Altogether, these data, while supporting a neuroprotective outcome of neuronal A2A receptor blockade in PD, high­ light the complexities of the roles played by A2A receptors, pointing to distinct actions of cell-type­ specific receptors in different neurodegenerative conditions. In neurons, A2A receptors are enriched in the striatum, but also have been described in other basal ganglia nuclei, such as GP, and at lower levels in the substantia nigra (Brooks et al., 2008; Cunha et al., 1994; Johansson et al., 1997; Rosin et al., 1998; Schiffmann et al., 1991). At the pre-synaptic level striatal A2A receptors are mostly located on glutamatergic terminals where they modulate the efflux of glutamate

(Marchi et al., 2002; Melani et al., 2003; Popoli et al., 2002; Tebano et al., 2004) (Fig. 2). It has been extensively demonstrated that antagonism of the A2A receptor protects against ischemic damage and toxin-induced excitotoxicity in the hippocampus or striatum (Jones et al., 1998; Melani et al., 2006; Popoli et al., 2002). Excessive excitotoxic glutamate efflux from the STN to sub­ stantia nigral neurons is a component of PD neu­ ropathology, likely contributing to nigral neuron death. Therefore a reduction of glutamate efflux in the substantia nigra, and perhaps in the stria­ tum, might provide a mechanism for protection against dopaminergic degeneration in PD models (Wallace et al., 2007). It is important to point out that the effect of A2A receptor antagonists on striatal glutamate levels is dependent on the dose and experimental condi­ tions (Tebano et al., 2004). In intact striatum, the A2A antagonist SCH-58261 decreased evoked glu­ tamate efflux at low doses only, whereas it lost this effect at higher doses (Pintor et al., 2001). More­ over local infusion of an A2A antagonist directly into the 6-OHDA-lesioned striatum increased glu­ tamate outflow (Corsi et al., 2003). Tebano and colleagues have shown that in corticostriatal slices A2A antagonists inhibit glutamate outflow, whereas in striatal neurons they amplify excito­ toxic mechanisms due to direct NMDA receptor stimulation (Tebano et al., 2004). Therefore, the potential neuroprotective effect mediated by pre­ synaptic A2A receptor blockade might emerge specifically at the low-dose range, whereas other mechanisms, likely mediated by post-synaptic A2A receptors, might mask this effect at higher doses. The higher binding affinity displayed by pre- ver­ sus post-synaptic A2A receptors supports this con­ cept (Cunha et al., 1996). An interesting mechanism of adenosine– glutamate interaction that has emerged recently involves cross-talk between adenosine and glialderived neurotrophic factor (GDNF) receptors, co-localized on striatal glutamatergic nerve end­ ings (Gomes et al., 2009). In rat striatal synapto­ somes, GDNF enhanced glutamate release by

192

13% from corticostriatal terminals, an effect potentiated by A2A agonist CGS21268 and pre­ vented by the A2A receptor antagonist SCH58261 (Gomes et al., 2009). Therefore, it is suggested that A2A receptor blockade would impair GDNF-stimulated increase of corticostria­ tal glutamate release, providing a beneficial effect on neurodegeneration (Gomes et al., 2009).

Glial A2A receptors and neuroprotection Neuroinflammation, characterized by reactive astrocytes and activated microglia, is a hallmark of PD and may play a pathogenic role in dopamine neuron degeneration. Classical studies have described activated microglia in the substantia nigra of PD patients, whereas most recent studies have reported a more widespread distribution of activated microglia in the brain, both in early and in late stages of the disease, involving pons, basal ganglia, striatum and frontal and temporal cortex (Gerhard et al., 2006; Mc Geer and McGeer, 2008). Persistent microglial activation leads to ele­ vated levels of glial-derived cytokines and chronic neuroinflammation, which exert neurotoxic effects on highly vulnerable dopaminergic neurons. On the basis of this evidence it appears of great impor­ tance that, in addition to neurons, adenosine A2A receptors are located on both microglia (Fiebich et al., 1996; Saura et al., 2005) and astrocytes (Lee et al., 2003) (Fig. 2). Recent reports in vivo have shown that A2A receptor antagonists were able to prevent the astroglial and microglial activation induced by acute or sub-chronic administration of MPTP in mice (Carta et al., 2009; Pierri et al., 2005). These results are in line with that obtained in an in vivo model of ischemia, where the specific A2A receptor antagonist SCH58261 inhibited phos­ phorylation of P38-MAPK (mitogen-activated pro­ tein kinase), supporting an inhibitory effect of A2A receptor antagonism on the response in microglial cells (Melani et al., 2006). Therefore, an effect on glial cells has been considered as a potential mechanism of neuroprotection by A2A receptor

antagonists. However, the effect on glial response observed in different models of neurodegeneration might either result from a direct effect through stimulation of A2A receptors on these cells, or might be secondary to an effect mediated by A2A receptors on neurons. The molecular effects of glial A2A receptor sti­ mulation have been investigated by a number of in vitro studies, leading to somewhat contradictory results and suggesting that receptors located on microglia and astroglial cells might play opposing roles in neurodegeneration. Supporting a beneficial effect of glial A2A receptor blockade, A2A receptor agonist CGS21680 potentiated lipopolysaccharide (LPS)-induced nitric oxide (NO) release and NO synthase-II expression by microglial cells in a con­ centration-dependent manner, whereas an A2A antagonist suppressed this effect (Saura et al., 2005). Accordingly, A2A receptors mediated the induction of cyclooxygenase-2 and NO synthase in microglia (Fiebich et al., 1996, 1998). However, A2A receptor agonists inhibited cyto­ kines production by activated microglia (van der Putten et al., 2009). In cultured astroglia, both adenosine kinase inhibitors and the A2A receptor agonist CGS21280 counteracted LPS-induced pro­ duction of NO (Brodie et al., 1998; Lee et al., 2005). In line with a positive role of A2A receptor blockade, the A2A receptor agonist CGS21680 increased the astrocytic release of glutamate via an A2A receptor/PKA signalling pathway and via inhibition of glutamate transporter glutamate transporter-1 (GLT-1), resulting in increased synaptic concentrations of this neurotransmitter and likely in deleterious effects on neurons survi­ val. Most importantly, this effect was inhibited by the A2A receptor antagonist DMPX (Nishizaki, 2004). Furthermore, adenosine and the A2A receptor agonist 50 -(N-cyclopropyl)-carboxami­ doadenosine (CPCA) increased astrocyte prolif­ eration after brain injury in rat cortex, whereas adenosine antagonists where shown to counteract astrocytic proliferation in both in vitro and in vivo models of brain injury (Brambilla et al., 2003; Hindley et al., 1994; Pugliese et al., 2009).

193

Therefore, whereas substantial evidence suggests that manipulating glial A2A receptors might be beneficial in neurodegenerative conditions, their selective stimulation or blockade might be bene­ ficial depending on neurodegenerative conditions and time of intervention (Fig. 2). Moreover, reports on glial A2A receptors manipulation in in vivo PD models are currently lacking. Lately, a new alternative mechanism of neuro­ protection has been suggested, involving A2A receptors located on oligodendrocytes. In a stroke model A2A receptor antagonist SCH58261 reduced striatal Olig 2 transcription factor induced by ischemia (Melani et al., 2009).

Epidemiology and clinical trials of adenosine antagonists Caffeine epidemiology Case control studies: coffee Considerable epidemiological and laboratory data have suggested that A2A receptor blockade by caffeine, a non-selective adenosine receptor antagonist, may protect against the underlying neurodegeneration of PD. Drinking caffeinated beverages (coffee and to a lesser extent tea) has emerged as the dietary factor most consistently linked to an altered risk of PD, with greater con­ sumption associated with a reduced risk (Ascherio et al., 2001; Benedetti et al., 2000; Checkoway et al., 2002; Fall et al., 1999; Hellenbrand et al., 1996; Hu et al., 2007; Ragonese et al., 2003; Ross et al., 2000; Tan et al., 2003). In the early 1990s case–control studies suggested a reduced risk of developing PD associated with drinking coffee but the reduction either was not statistically significant (Jimenez-Jimenez et al., 1992; Morano et al., 1994) or was significant but partially attributable to the confounding association of smoking in cof­ fee drinkers (Grandinetti et al., 1994) and thus difficult to interpret. Following on from this, lar­ ger, case–control studies, using better-matched

cohorts, demonstrated that even after adjusting for tobacco smoking and other potential con­ founding factors, the significant inverse relation­ ship between prior coffee drinking exposure and PD remained (Benedetti et al., 2000; Fall et al., 1999; Hellenbrand et al., 1996). These latter stu­ dies also specifically investigated dose–response relationships, with increasing coffee consumption (measured in cups per day) associated with decreasing likelihood of having developed PD. In these retrospective analyses PD patients were 4–8 times less likely than control subjects to have reported being heavy coffee drinkers in the past. While case–control studies have some advantages, weaknesses of these designs for investigating diet­ ary aetiology of chronic diseases are well known (Willett, 1998) and include, for example, difficulty selecting appropriate control subjects and recall bias. The introduction of large cohort prospective investigations (see below) has overcome many of these limitations through follow-up evaluations to determine disease incidence in subjects from a single population in which the exposure in ques­ tion (i.e. coffee or caffeine consumption) had been reported years earlier.

Case–control studies: tea Relatively few studies have considered the effect of tea drinking and its association with PD risk. This could be attributed to its low consumption in North America and Europe (Ascherio et al., 2001; Chan et al., 1998). These studies usually (Ayuso-Peralta et al., 1997; Chan et al., 1998; Checkoway et al., 2002; Hellenbrand et al., 1996; Ho et al., 1989; Tan et al., 2003, 2007), although not always (Preux et al., 2000), indicated a reduced risk of developing PD amongst frequent tea drinkers. Interestingly, epidemiological studies from China have observed that the prevalence of PD is much lower than in the Caucasian population (Li et al., 1985; Zhang and Roman, 1993). Barranco et al., 2009 reviewed observational studies that evaluated tea con­ sumption and the risk of PD (11 case–control

194

and 1 cohort) between 1981 and 2003. The studies represented documented cases from North Amer­ ica, Europe and Asia. Amongst the case–control studies, the pooled OR was 0.8 (95% CI 0.71–0.90) suggesting that tea consumption is inversely asso­ ciated with the risk of PD. It is unclear whether the active ingredient(s) mediating this observed pro­ tective effect is caffeine or some biologically active substance(s) present in tea but not coffee.

observed in two separate cohort studies of Finnish men and women free from PD at baseline (Hu et al., 2007; Saaksjarvi et al., 2008). Heavier coffee consumption was associated with a reduced risk of PD even after adjustment for confounding factors. The Finnish population is of particular interest since it exhibits one of the world’s highest rates of coffee consumption (Fredholm et al., 1999).

Gender differences (oestrogen interactions) Cohort studies Caffeinated beverages More convincing epidemiological evidence that caffeine and coffee consumption are linked to a reduced risk of developing PD has been obtained from the study of prospectively followed large populations (Ascherio et al., 2001; Ross et al., 2000). Three decades after ~8000 Japanese-Amer­ ican men were enrolled in the Honolulu Heart Program (and provided details of their dietary caffeine consumption), over 100 had gone on to develop PD. Higher initial coffee intake was dose dependently associated with a reduced incidence of PD, with a fivefold lower risk amongst those who drank over 24 oz per day (Ross et al., 2000). Confirmation of these findings was provided by two prospective studies of larger, multiethnic populations, namely the Health Professionals Fol­ low-up Study (HPFS) of 50 000 men followed for a period of 10 years and the Nurses Health Study (NHS) of 90 000 women followed over 16 years (Ascherio et al., 2001). Amongst the men, increased coffee, tea and non-coffee caffeine con­ sumption and total caffeine consumption were all significantly, dose dependently and negatively cor­ related with the incidence of subsequent PD. These associations were independent of smoking and other potential confounding factors. By con­ trast, the rates of consuming decaffeinated coffee were unrelated to the risk of PD, implicating caf­ feine as the component in coffee that is inversely associated with PD risk. Similar findings were

Interestingly, a stark gender difference in how caffeine relates to PD risk emerged from compar­ ison of female and male cohorts within a large protective study of health care workers (Ascherio et al., 2001). Initially, analysis of women in the NHS study revealed no clear relationship between PD and caffeine or coffee intake. This gender difference was consistent with the observations of PD incidence rates in Olmstead County, MN, which were strongly inversely related to prior cof­ fee drinking in men (with a ~17-fold reduction in risk amongst drinkers vs. non-drinkers; p < 0.01) but did not vary with coffee exposure in women (with a relative risk of 1.0 ; Benedetti et al., 2000). Ascherio and colleagues gained insight into stra­ tification of the women by oestrogen exposure his­ tory. In two separate prospective studies (Ascherio et al., 2003, 2004) they showed that amongst women who did not use post-menopausal oestro­ gens, caffeine was in fact associated with a reduc­ tion in the risk of subsequent PD (just as in men). Conversely, for women who had used oestrogen replacement caffeine use did not carry a lower risk of PD, suggesting a hormonal basis for the gender difference in caffeine’s association with PD. Interestingly, in contrast to the result from stu­ dies by Ascherio and colleagues, the two prospec­ tive cohort studies of Finnish populations (Hu et al., 2007; Saaksjarvi et al., 2008) reported no gender differences in the (inverse) relationship between coffee consumption and PD risk. How­ ever, as Saaksjarvi and colleagues note in their study the effect of post-menopausal hormone use

195

could not be examined due to small number of users, which was reported to be ~5% of women and markedly less than in the US cohorts. For example, in the NHS, a third of the women were currently taking post-menopausal oestrogens and 54% reported ever taking them. Thus, the varia­ bility in an overall association between caffeine and PD risk among women between cohorts may be explained by wide variation in their use of post­ menopausal oestrogen, which appears to compli­ cate the relationship between caffeine and PD. Recent laboratory experiments have explored the biology that may underlie gender differences in the caffeine–PD link observed in populations with higher rates of oestrogen use. Xu et al. (2006) found that the ability of caffeine to attenuate MPTP toxicity in a mouse model of PD was greater in male versus female mice and in ovariectomized versus sham-operated female mice. They also demon­ strated that chronic oestrogen replacement under­ mined caffeine’s protective effect both in male and in ovariectomized female mice, providing direct evidence of a hormonal influence on caffeine’s neu­ roprotective properties in lab animals. The study also implicated a biological basis for the gender difference in the association between caffeine con­ sumption and PD risk. In general, these and other laboratory studies (as reviewed above) support – but do not prove – the hypothesis that the consis­ tent epidemiological association between caffeine and a reduced risk of PD is causal.

over 5 years from 268 participating PD patients (87% of the original CALM-PD cohort). No asso­ ciation was found between caffeine intake (from coffee, tea and soda sources) and rate of PD pro­ gression even after adjustment for treatment group, gender, tobacco and alcohol use. Outcome measures assessed progression by change in the Unified Parkinson Disease Rating Scale (UPDRS) or by alteration in the striatal uptake of iodine-123-labeled 2-b-carboxymethoxy-3-b-(4­ iodophenyl) tropane ([123I] b-CIT) in the subset of patients that underwent neuroimaging. The study also allowed for assessment of caffeine consump­ tion and dyskinesia development. A trend albeit non-significant towards lower risk of dyskinesias with increasing caffeine intake was observed. The lack of an association between caffeine consump­ tion and rate of subsequent progression was also observed in a NET-PD (NIH Exploratory Trials in Parkinson’s Disease) trial cohort (Simon et al., 2008) monitoring changes in the UPDRS score and likelihood of disease progression to the point of requiring symptomatic therapy as measurable out­ comes. These studies do not lend support to a pro­ tective benefit of caffeine by PD patients. However, they leave open the possibility that disease progres­ sion among individuals who develop PD despite ingesting caffeine at higher levels may be less influ­ enced by any true protective effect it may have.

Clinical trials of A2A antagonists for PD PD progression Convergent epidemiological and laboratory data support the possibility that dietary caffeine reduces the risk of developing PD. Less well known is the relationship between caffeine intake and the rate of progression of the disease. A pre­ liminary investigation (Schwarzschild et al., 2003a) reported a secondary analysis of the ‘Com­ parison of the agonist pramipexole versus L-DOPA on the motor complications of PD’ (CALM-PD) trial database, which included data

Trials for symptomatic anti-parkinsonian indications Non-selective adenosine antagonists: caffeine and theophylline Although caffeine is a non-specific adenosine receptor antagonist that blocks with similar potency at the A1, A2A and A2B sub-types (Fred­ holm et al., 1999), it appears to have its main psychomotor stimulant actions primarily through blockade of CNS A2A receptors (Xu et al., 2004;

196

Yu et al., 2008). Thus the first trials of A2A antag­ onism in PD may have been a pair of doubleblind, placebo-controlled studies of caffeine effects on parkinsonian symptoms of patients tak­ ing L-DOPA or a dopamine agonist in the 1970s (Kartzinel et al., 1976; Shoulson and Chase, 1975). Both studies found no benefit of caffeine. How­ ever, their small size (<10 subjects), clinical het­ erogeneity (some subjects did not have PD) and extremely high dosing 1–1.5 g of caffeine daily precludes meaningful conclusions about the symp­ tomatic utility of caffeine for PD from these stu­ dies. A more rigorously designed randomized, placebo-controlled trial of caffeine at more phar­ macologically informative doses (100 or 200 mg bid) is currently underway and projects enroll­ ment of some 50 subjects with idiopathic PD and excessive daytime somnolence (McGill University Health Center; clinical trials.gov identifier # NCT00459420). Although its primary outcome focuses on daytime sleepiness, a secondary analy­ sis of parkinsonian motor symptoms is planned using the standard UPDRS. Theophylline, a demethylation metabolite of caffeine as well as an anti-asthmatic agent in com­ mon use, is also a non-specific adenosine antago­ nist. Although small open-label trials of theophylline in PD seemed to suggest anti-parkin­ sonian benefit without exacerbation of dykinesias (Kostic et al., 1999; Mally and Stone, 1994), a subsequent double-blind, placebo-controlled trial of theophylline in PD did not clearly demonstrate relief from symptoms (Kulisevsky et al., 2002). However, the power and design of all these trials like the earlier caffeine studies in PD were not sufficient to rule out clinically useful symptomatic effects, as have been suggested by long-standing preclinical studies of motoric enhancement in PD models (Stromberg and Waldeck, 1973).

Selective adenosine A2A antagonists A clear indication of the promise of adenosine A2A receptor antagonism for PD is the depth of

industry investment in programs to develop a vari­ ety of xanthine and non-xanthine structure-based adenosine receptor antagonists as anti-parkinso­ nian agents. Amongst at least 10 publicly announced programs are some 5 that have entered human studies, with 4 being now actively pursued through phase II and III clinical trials: istradefyl­ line (aka KW-6002) from Kyowa-Kirin (Kyowa Hakko Kirin Co., 01.15.2009 announcement), Pre­ ladenant (aka SCH 420814) from Schering Plough (now Merck) (Schering Plough Co., 11.24.2008 announcement), BIIB014 (aka V2006) from Bio­ genIdec (licensed from Vernalis) (Papapetropou­ los et al., 2010) and Syn-115 from Synosia (licensed from Roche) (Black et al., 2010a, 2010b). The early frontrunner amongst these clinical development programs was istradefylline, which had progressed through encouraging phase II trial results (Bara-Jimenez et al., 2003; Fernandez et al., 2010; Hauser et al., 2003, 2008; Kase et al., 2003; Lewitt et al., 2008; Stacy et al., 2008) to as yet unpublished, reportedly mixed results of phase III studies (Kyowa Hakko Kirin Co., 06.03.2007 announcement) before submission of a New Drug Application (NDA) to US Food and Drug Admin­ istration (FDA). However, the FDA in response issued a ‘Non-Approvable letter’ in 2008 based on concerns of the adequacy of overall efficacy (Kyowa Hakko Kirin Co., 02.08.2008 announce­ ment). Since then clinical development of istrade­ fylline has continued, but the company has pulled back from an international program to focus on trials and a potential indication in Japan. Based on available data for the published phase II trials, istradefylline employed (at once-daily doses from 20 to 80 mg) as adjunctive therapy in relatively advanced subjects, produced a modest but significant reduction in ‘off’ time (i.e. in motor dysfunction). Of note, these ‘positive’ outcome measures were based on reports of the patients themselves, whereas no significant treatmentinduced improvement was observed based on the clinician-scored UPDRS. Although preclinical evi­ dence supporting the original study designs had suggested adjunctive A2A antagonism might

197

assuage PD symptoms without exacerbating dys­ kinesias [reviewed in Xu et al. (2004)], istradefyl­ line treatment in this relatively advanced PD population actually increased dyskinesias in most studies. However, when dyskinesias were strati­ fied into ‘troublesome’ and ‘non-troublesome’, only the latter were significantly increased on the drug. Recently, istradefylline was also tested as monotherapy for its potential symptomatic benefit in early PD (Fernandez et al., 2010). Despite a trend towards improvement on istradefylline, again the difference between placebo and A2A antagonist-treated groups did not reach signifi­ cance for the change in UPDRS score, the primary outcome in this study. Retrospective assessment of why this early A2A antagonist development program fell short in its initial efforts to gain an indication for PD treat­ ment has focused on trial design elements, includ­ ing dose selection (for both istradefylline and concomitant L-DOPA) and disease stage of the targeted PD subpopulation (i.e. possibly too advanced in adjunctive trials, though possibly too early in the monotherapy trial). In addition, none of the trial reports addressed the potential confound of concomitant A2A antagonism by caffeine use, which apparently was neither excluded nor monitored. At doses relevant to typical human consumption, caffeine and more specific A2A antagonists (including istradefylline) bind to striatal A2A receptors in vivo to a similar extent (El Yacoubi et al., 2001; Moresco et al., 2005). Given also the well-known psychomotor stimulant properties of caffeine and its continued consumption in PD with a mean intake of approximately 200 mg/day among early in the disease (Schwarzschild et al., 2003a; Simon et al., 2008), its use is important to consider in A2A antagonist trials. As caffeine use tends to decrease over the course of the disease, controlling for its use in the analysis of early PD/monotherapy trials may be particularly infor­ mative. If specific adenosine A2A receptor antagonists were found to be more effective

amongst those consuming less caffeine (i.e. less of a general adenosine antagonist), it would sup­ port the distinct possibility of a shared anti-par­ kinsonian effect through a common mechanism. It would then remain to be determined whether specific A2A receptor antagonism offers an advantage of greater efficacy or tolerability (given possibly adverse effects of blocking other adenosine receptors) to offset the lower cost and greater availability of caffeine.

Trials for disease modification in PD Convergent epidemiological and laboratory stu­ dies have suggested that adenosine A2A antagon­ ism may confer disease-modifying benefits beyond the anti-parkinsonian motor effects now being actively pursued in clinical trials as above (Schwarzschild et al., 2006; see Fig. 3). Although caffeine itself was listed amongst the most attrac­ tive candidate neuroprotectants under considera­ tion for clinical investigation (Ravina et al., 2003), if a specific A2A antagonist were to receive an indication for symptomatic relief, it may be more likely than caffeine to first undergo testing in a ‘neuroprotection’ trial given the high costs of con­ ducting the necessarily large and long-term trials required. The prospects of favourable disease modifica­ tion by A2A receptor blockade may extend beyond its neuroprotective potential as A2A receptors have also been implicated in the maladaptive neuroplasticity that underlies the development of L-DOPA-induced dyskinesia (LID) in PD (Morelli et al., 2009). Accordingly, early treatment with an A2A antagonist versus placebo as an adjunct to newly initiated L-DOPA may also be considered as a novel trial design to assess for both an early synergis­ tic benefit (possibly with greater sensitivity than a monotherapy design) and a prophylactic effect on LID during a second long-term phase of observation with subjects maintained in their blinded treatment arms.

198

Purine metabolism pathway

Adenosine

Hypothesized neuroprotective mechanisms

Caffeine

Adenosine deaminase

Inosine

A2A receptor

Nucleoside

phosphorylase

Hypoxanthine Dopamine neuron death

Xanthine oxidase

Xanthine

Neuroprotective effects in cellular models of PD

Xanthine oxidase

Urate Urate

oxidase

Mutations

end product of the metabolism of purines like adenosine. It also possesses antioxidant properties comparable to those of ascorbate (Ames et al., 1981) and accounts for most of the antioxidant capacity in human plasma (Yeum et al., 2004), supporting the hypothesis that our ancestors gained antioxidant benefits from UOx mutations and a resulting elevation of urate concentrations (Proctor, 1970). While higher levels of urate may have conferred an evolutionary advantage through bolstered defences against oxidative damage (Ames et al., 1981) today they are also the core molecular culprit in gout and uric acid kidney stones.

Oxidative damage

Allantoin Fig. 3. Therapeutic targets along the purine metabolic pathway. Adenosine A2A antagonists (including caffeine) and urate have emerged as realistic candidate neuroprotectants. In humans the enzymatic metabolism of purines such as adenosine ends with urate due to multiple mutations within the urate oxidase gene during primate evolution (see text). The schematic suggests a possible homeostatic mechanism linking an adenosinergic neurodegenerative influence with an offsetting neuroprotective influence of urate.

Urate as a novel target for neuroprotection Biology Evolutionary significance During primate evolution a series of mutations occurred in the urate oxidase gene (UOx) likely accounting for the relatively high levels of urate (the physiologically dissociated form of uric acid) in apes and humans compared to other mammals (Oda et al., 2002; see Fig. 3). Urate is not only the

In addition to its antioxidant actions, urate has also been shown to possess other potentially pro­ tective properties in vitro. It has been shown to scavenge peroxynitrite as well as oxygen free radi­ cals in vitro (Franzoni et al., 2006; Whiteman et al., 2002) and displays potent iron-chelating activity independent of its direct antioxidant action (Davies et al., 1986). Direct evidence for a neuro­ protective effect of urate has come initially from cellular and animal models of multiple sclerosis (Hooper et al., 1997, 1998; Scott et al., 2002), stroke (Romanos et al., 2007; Yu et al., 1998) and spinal cord injury (Du et al., 2007; Scott et al., 2002). Urate also confers protection in cellular models of PD. In PC12 cells, urate blocked apoptosis and oxidant production induced by dopamine (Jones et al., 2000) or the pesticide rotenone in combina­ tion with homocysteine (Duan et al., 2002) and reduced cell death induced by MPPþ or Fe2þ (Haberman et al., 2007). Urate also attenuated toxin-induced loss of primary neurons in culture. A recent study of dopaminergic neurons in primary midbrain culture of rat ventral mesencephalon found that their physiological function and survival were significantly enhanced by urate (Guerriero et al., 2009) at concentrations (  30–50 mM)

199

corresponding to those in human cerebrospinal fluid (CSF) associated with a reduced rate of clinical decline in PD (Ascherio et al., 2009).

Epidemiology When first considered in case–control studies, lower urate levels were found in serum (Andrea­ dou et al., 2009; Annanmaki et al., 2007; Bogda­ nov et al., 2008; Johansen et al., 2009; Larumbe et al., 2001), possibly in cerebrospinal fluid (Tohgi et al., 1993), and in post-mortem nigrostriatal tis­ sue samples (Church and Ward, 1994) of PD patients compared to those of controls. These stu­ dies suggested that low CNS and peripheral levels of urate are associated with PD. A series of epidemiological investigations of prospectively followed cohorts has more incisively linked higher blood urate with a reduced risk of developing PD (Chen et al., 2009; Davis et al., 1996; De Lau et al., 2005; Weisskopf et al., 2007). For example, in the largest of these cohorts, 18 000 men were followed for more than 8 years in the HPFS. Weisskopf and colleagues found that those in the top quartile of plasma urate concentration had a two to threefold lower risk of PD than subjects in the bottom quartile (p < 0.02 for trend across all quartiles). Amongst the subset of cases for whom blood was collected at least 4 years before the diagnosis of PD, an even greater reduc­ tion of PD risk was observed – with those in the highest urate quartile having a fivefold lower risk of PD compared to the lowest quartile (p < 0.01 for trend). This further analysis suggests that the low uricemia among individuals with PD precedes the onset of neurological symptoms and is thus unlikely to be a consequence of changes in diet, behaviour or medical treatment early in the course of the disease. This inverse association was independent of age, smoking, caffeine con­ sumption and other aspects of lifestyle that have been related to PD or uricemia. Similarly, urate-elevating diet was also asso­ ciated with a lower risk of PD (Gao et al., 2008).

In the HPFS cohort, the authors found that a higher dietary uricemic index (reflecting dietary patterns linked to higher plasma urate) predicted a reduced risk of developing PD (p < 0.001 for trend). The association between the index and the PD risk remained strong and significant in models further adjusted for age, smoking and caf­ feine intake. Their findings suggest that dietary interventions that raise blood urate concentrations might reduce the risk of PD. In a related set of epidemiological studies of large prospectively followed cohorts, a diagnosis of gout (a form of arthritis due to urate crystal­ lization in joints and associated with hyperurice­ mia) was linked to a lower risk of later being diagnosed with PD (Alonso et al., 2007; De Vera et al., 2008). Together these epidemiological data establish urate exposure – assessed by laboratory, dietary or pathological indicators – as a robust inverse risk factor for PD.

Clinical studies of urate and PD progression The emergence of robust epidemiological data linking higher urate levels amongst healthy popu­ lations to a reduced risk of developing PD prompted a corollary hypothesis: Amongst people already diagnosed with PD, do higher urate levels predict a slower rate of clinical decline? To test the hypothesis, incidentally measured urate levels in two large completed ‘neuroprotection’ trials were related to rates of clinical decline over years. Although neither the PRECEPT (Parkinson Study Group, 2007) nor the DATATOP (Parkin­ son Study Group, 1993) trial had demonstrated efficacy of candidate neuroprotectants, each had collected data for routine safety lab tests – includ­ ing serum urate – at enrollment of some 800 recently diagnosed ‘de novo’ PD patients, who were then followed closely for 2 years. For both trials the primary outcome was time to disability warranting the initiation of L-DOPA or dopami­ nergic agonist therapy, with secondary outcomes including rate of UPDRS change and, in the case

200

of the PRECEPT trial, rate of loss of dopamine transporter (DAT) ligand uptake in the striatum. In the PRECEPT cohort, subjects with higher (but still normal) levels of serum urate at baseline were significantly less likely to develop disability warranting dopaminergic therapy and also retained significantly more striatal DAT binding capacity during the study (Schwarzschild et al., 2008). For example, subjects in the top quintile of serum urate (~7–8 mg/dl with normal value reference ranges typically 3–8 mg/dl) reached the end point at only half the rate of subjects in the lowest quintile (hazard ratio, 0.51; 95% confi­ dence interval, 0.37–0.72; p for trend <0.001). In the DATATOP cohort higher baseline urate con­ centrations in CSF as well as in serum were simi­ larly associated with a slower rate of reaching the primary disability end point (Ascherio et al., 2009). In both cohorts higher urate levels were also predictive of a favourable rate of clinical decline measured by the change in UPDRS score. Although several descriptive clinical fea­ tures of PD have been identified as probable pre­ dictors of the rate of clinical decline in PD (Post et al., 2007), urate may be the first molecular factor clearly linked to clinical progression of idio­ pathic PD. In addition to its emerging utility as a prognostic biomarker of PD in research studies, the demon­ strated antioxidant and neuroprotective proper­ ties of urate raise the possibility of its potential for direct therapeutic benefit. Convergence of these biological, epidemiological and clinical find­ ings has prompted rapid translation to clinical application (Parkinson Study Group; clinical trials.gov identifier # NCT00833690). A multi-cen­ tre, randomized, placebo-controlled trial of ino­ sine (a precursor of urate as well as the deamination product of adenosine in purine meta­ bolism; see Fig. 3) in early PD is currently under­ way to assess its safety and ability to elevate serum and CSF urate levels and its potential for further development as a novel strategy to impede pro­ gression of the disease.

Acknowledgements Supported by NIH grants K24NS060991 and R01NS054978, DoD W81XWH-04-1-0881 and the RJG Foundation. Abbreviations 3-NP 6-OHDA BRET cAMP CNS CPX CREB DARPP-32 DAT ERK FDA FRET GABA GDNF GLT-1 HPFS L-DOPA

LID LPS MAPK MPTP NDA NGF

3-nitropropionic acid 6-hydroxydopamine Bioluminescence Resonance Energy Transfer cyclic adenosine monophosphate central nervous system 8-cyclopentyl-1,3­ dipropylxathine cAMP response elementbinding dopamine- and cAMP­ regulated phosphoprotein-32 dopamine transporter Extracellular signal-regulated kinases US Food and Drug Administration fluorescence resonance energy transfer gamma-aminobutyric acid glial-derived neurotrophic factor glutamate transporter-1 Health Professionals Follow-up Study L-3,4-dihydroxyphenylalanine L-DOPA-induced dyskinesia lipopolysaccharide mitogen-activated protein kinase 1-methyl-4-phenyl-1,2,5,6­ tetrahydropyridine New Drug Application nerve growth factor

201

NHS NO PD STN TH UPDRS

Nurses Health Study nitric oxide Parkinson’s disease subthalamic nucleus tyrosine hydroxylase Unified Parkinson’s Disease Rating Scale

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A. Bjorklund and M.A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 11

Maladaptive striatal plasticity in L-DOPA-induced dyskinesia M. Angela Cenci†,
and Christine Konradi‡ †

Basal Ganglia Pathophysiology Unit, Department of Experimental Medical Science, Lund University, Lund, Sweden ‡ Center for Molecular Neuroscience and Kennedy Center for Research on Human Development, Departments of Pharmacology and Psychiatry, Vanderbilt University, Nashville, TN, USA

Abstract: Dopamine (DA) replacement therapy with L-DOPA remains the most effective treatment for Parkinson’s disease, but causes dyskinesia (abnormal involuntary movements) in the vast majority of the patients. The basic mechanisms of L-DOPA-induced dyskinesia (LID) have become the object of intense research focusing on neurochemical and molecular adaptations in the striatum. Here we review this vast literature and highlight trends that converge into a unifying pathophysiological interpretation. We propose that the core molecular alteration of striatal neurons in LID consists in an inability to turn down supersensitive signaling responses downstream of DA D1 receptors (where supersensitivity is primarily caused by DA denervation). The sustained activation of intracellular signaling pathways induced by each dose of L-DOPA leads to abnormal cellular plasticity and high bioenergetic expenditure. The over-exploitation of signaling pathways and energy reserves during treatment impairs the ability of striatal neurons to dynamically gate cortically driven motor commands. LID thus exemplifies a disorder where ‘too much’ molecular plasticity leads to plasticity failure in the striatum. Keywords: Striatonigral; Complications

Striatopallidal;

Medium

neuron;

ERK;

MAPK;

Transcription;

this disease are due to the demise of nigrostriatal dopamine (DA) neurons (Fearnley and Lees, 1991; Morrish et al., 1996). Accordingly, these symptoms are alleviated by the DA precursor, L-DOPA. Fol­ lowing peripheral administration, L-DOPA crosses the blood–brain barrier and is rapidly decarboxy­ lated to DA in the brain. Importantly, the capacity for L-DOPA uptake and its conversion to DA is not compromised by the loss of nigrostriatal DA

Introduction Although it is presently recognized that several neuronal systems degenerate in Parkinson’s disease (PD), the characteristic motor symptoms that define
Corresponding author. Tel.: (+)46-46-2221431; Fax: (+)46-46-2224546;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83011-0

spiny

209

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neurons, because other cell systems in the brain are endowed with the enzyme that converts L-DOPA to DA [aromatic amino acid decarboxylase; reviewed in Cenci and Lundblad (2006)]. However, with the loss of nigrostriatal DA neurons the pre-synaptic control of DA release and clearance becomes greatly impaired [reviewed in Cenci and Lundblad (2006)], leading to large fluctuations in extracellular levels of DA concomitant with the L-DOPA dosing cycles. These variations are closely associated with the development of abnormal involuntary move­ ments, AIMs (dyskinesia) (Chase, 1998), a very common complication of the pharmacotherapy of PD (Fabbrini et al., 2007). It has been estimated that 10% of PD patients per year develop dyskine­ sia during the first 7 years of L-DOPA pharma­ cotherapy (Grandas et al., 1999), but the reported prevalence of dyskinesia varies greatly among PD patient cohorts (Manson and Schrag, 2006). L-DOPA-induced dyskinesia (LID) most typically consists of choreiform movements that affect the limbs, the head and the trunk when plasma and brain levels of L-DOPA are high (‘peak-dose dyski­ nesia’) (Fabbrini et al., 2007). The appearance of these movements correlates with a large increase in extracellular DA levels in the striatum (de la Fuente-Fernandez et al., 2004; Pavese et al., 2006). This particular brain structure mediates the dyski­ netic effects of PD treatment, as indicated by the results of local drug infusion experiments (Buck et al., 2009; Carta et al., 2006) and cell transplanta­ tion procedures [see Lane et al. (2010), this volume]. Striatal neurons express high levels of DA receptors and undergo profound molecular and structural adaptations following DA depletion [see Surmeier et al. (2010), this volume]. During the past 10 years, evidence has accumulated to indicate that DA replacement therapy, whether pharmacological or cell-based, cannot reset the striatum back to its normal physiological state but introduces novel functional/dysfunctional states that strictly correlate with the profile of treatment-induced motor effects. This knowledge has been gained through studies performed in

rodents and non-human primates in which the nigrostriatal DA pathway was severed using specific neurotoxins, thus producing a model of PD in laboratory animals. The most commonly used neurotoxins are 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine (MPTP) in non-human pri­ mates and 6-hydroxydopamine (6-OHDA) in rats and mice. After DA depletion, animals develop parkinsonian-like motor deficits that cor­ relate with the extent of striatal DA denervation and that can be ameliorated by dopaminergic drugs [reviewed in Fox and Brotchie (2010) and Dunnett (2010), this volume; see also Cenci et al. (2002)]. When treated with L-DOPA, both rodents and non-human primates can develop AIMs of the limbs, head and trunk with dystonic and hyper­ kinetic features (reviewed in Cenci and Ohlin (2009); Cenci et al. (2002) and Jenner (2003)]. Also in these animal models, the expression of LID correlates temporally and quantitatively with an increase in striatal extracellular DA levels (Lindgren et al., 2010), similarly to the situation reported in dyskinetic PD patients. An active area of research is addressing the basic mechanisms of LID and the factors account­ ing for individual differences in the susceptibility to this movement disorder. There is large consen­ sus that dyskinesia is caused by two interacting factors: striatal DA denervation and pulsatile treatment with L-DOPA. These two factors in combination lead to (1) dysregulated DA transmis­ sion, (2) secondary changes in non-dopaminergic transmitter systems, (3) abnormal intracellular sig­ naling and synaptic plasticity in striatal neurons and (4) altered activity patterns in the basal gang­ lia output pathways. We have reviewed the full extent of this pathophysiological cascade in recent articles (Cenci, 2007, 2009; Cenci and Lindgren, 2007). The present chapter will zoom in on the third layer of alterations, namely, molecular changes in striatal neurons. We shall first summar­ ize basic mechanisms of DA receptor-dependent signaling in the intact and DA-denervated stria­ tum. We shall next review intracellular signaling responses and patterns of gene expression that

211

have been found to correlate with LID in animal models. Finally, we shall integrate the main find­ ings into a unifying interpretation of this move­ ment disorder.

DA receptor signaling in the neurons of the intact striatum: modulation of cyclic AMP/PKA pathways and non-canonical signaling Before examining the molecular adaptations asso­ ciated with LID, it is helpful to provide an overview of the signal transduction mechanisms and intracel­ lular events that follow the interaction of DA with its receptors in the intact brain. Molecular cloning has revealed five DA receptors as the products of dif­ ferent genes (Grandy and Civelli, 1992; Lachowicz and Sibley, 1997). All DA receptors are coupled to G proteins, and pharmacological studies have helped to group these receptors into two main classes: on one hand, D1-like DA receptors (D1, D5) and, on the other hand, D2-like receptors (D2, D3, D4), which are oppositely linked to adenylyl cyclase enzyme(s) and to the production/reduction of cyclic AMP (cAMP) (Sibley and Monsma, 1992). D1-like receptors increase cAMP levels and activate protein kinase A (PKA), whereas D2-like receptors decrease cAMP levels as well as PKA activity (Robinson and Caron, 1997; Sibley et al., 1998; Val­ lar and Meldolesi, 1989). D1 and D2 receptors are abundantly expressed in the striatum, whereas the other members of the D1 and D2 subfamilies predominate in mesocorticolimbic structures [reviewed in Sibley and Monsma (1992) and Strange (1993)]. In the striatum, D1 and D2 receptors are largely segregated into the two main classes of effer­ ent neurons (medium-sized spiny neurons, MSN) that give rise to the ‘direct’ and ‘indirect’ striatofugal pathways, respectively (Gerfen, 1992) [see also Sur­ meier et al. (2010), this volume]. Thus, these two neuronal populations are differently affected by DA, with PKA activity induced in one subset of neurons and repressed in the other (Fig. 1). Acti­ vated PKA phosphorylates surrounding proteins that are critical to neuronal plasticity and gene

expression (Schulman, 1995). In particular, activa­ tion of PKA by D1 receptors has facilitatory effects on N-methyl-D-aspartate (NMDA) receptors, amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors and L-type Ca2þ channels (Cepeda and Levine, 1998, 2006; Cepeda et al., 1993; Chao et al., 2002; Dudman et al., 2003; Gray et al., 1998; Konradi et al., 1996). Calcium entry through NMDA receptors or L-type Ca2þ channels can in turn activate calcium-dependent kinases, such as CaM kinase II and IV (Ghosh et al., 1994; Ginty, 1997). PKA as well as CaM kinases can phosphorylate the transcription factor cAMP response element-binding protein (CREB) on Serine 133, inducing the expression of immedi­ ate early genes (c-fos), and opioid precursor genes, such as prodynorphin (preproenkephalin-B) or pre­ proenkephalin (preproenkephalin-A) (Cole et al., 1995; Dudman et al., 2003; Ghosh et al., 1994; Rajadhyaksha et al., 1999) (Fig. 1). Because of their cellular location and effect on PKA, D1 recep­ tor agonists induce the expression of immediate early genes and prodynorphin in ‘direct pathway’ MSN. In ‘indirect pathway’ neurons, immediate early genes and opioid precursor genes (preproen­ kephalin-A) are induced by D2 receptor antagonists (Konradi et al., 1996; Leveque et al., 2000; Robert­ son et al., 1992). It can thus be assumed that normal signaling downstream of D1 and D2-like receptors is essential to maintain the distinctive gene expression patterns and physiological responses of ‘direct path­ way’ and ‘indirect pathway’ MSN. Inactivation of DA receptors is an active pro­ cess. After interaction with their ligand, DA receptors get phosphorylated by G-protein­ coupled receptor kinases (GRKs), which is fol­ lowed by their binding to arrestin (Claing et al., 2002). Arrestin binding blocks further G-protein­ mediated signaling and leads to receptor interna­ lization. Like other G-protein-coupled receptors (GPCRs), DA receptors are subjected to endocy­ tosis (Ariano et al., 1997; Kim et al., 2008; Paspa­ las et al., 2006; Vargas and Von Zastrow, 2004; Xiao et al., 2009). An insufficient internalization of DA receptors has recently emerged as an

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Nigrostriatal nerve terminal

Corticostriatal nerve terminal

Dopamine

Glutamate Ionotropic

glutamate

receptor

Ca2+ channel

Ca2+ channel D1

mGluR 5

β Gαs/olf

D2 γ

?

Ca2+

Gαi Phosphorylation by GRK

Ras

β γ

AC5 D2 receptor β-ARR/Akt/PP2A

Raf cyclic AMP MEK1/2

GSK-3β

Phosphatidylinositol­ 4,5-bisphosphate DAG

PKA

Erk1/2

PLC

IP3

DARPP-32Thr34 IP3R PP-1 Histone modification

p-Elk-1

Ca2+ release

p-CREB

Gene transcription Fig. 1. Canonical and non-canonical signaling cascades downstream of D1 and D2 DA receptors. Being coupled to stimulatory (Gs/olf) and inhibitory (Gi/o) GTP-binding proteins, D1 and D2 receptors have opposite effects on the adenylyl cyclase/cAMP/PKA/ DARPP-32 cascade (‘canonical pathway’), which regulates the levels of phosphorylation of multiple cellular and nuclear targets. In particular, the cAMP/PKA/DARPP-32 pathway modulates the activity of MAPK-dependent signaling pathways downstream of glutamate receptors. It has been recently recognized that cAMP-independent pathways are also recruited following D2 receptor stimulation (non-canonical pathways; cf. Section ‘DA receptor signaling in the neurons of the intact striatum: modulation of cAMP/PKA pathways and non-canonical signaling’). Full names of the molecules shown in this drawing are given in the list of abbreviations.

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important mechanism in LID and will be further discussed below (see Section ‘Altered intracellular trafficking of DA and glutamate receptors’). Beside their role in receptor internalization, arrestins are involved in a non-canonical (cAMP/ PKA-independent) signaling pathway downstream of D2 receptors. Activation of D2 receptors leads to the formation of a protein complex composed of b-arrestin, protein kinase B (Akt) and protein phosphatase 2A (PP2A) (Beaulieu et al., 2005). The formation of this complex results in the depho­ sphorylation/inactivation of Akt by PP2A and sub­ sequent stimulation of glycogen synthase kinase 3 (GSK3)-mediated signaling (Beaulieu et al., 2005) (Fig. 1). GSK3 is a regulator of many cellular func­ tions, including cell architecture, motility and survi­ val (Jope and Johnson, 2004). Thus, even though DA stimulation of D2 receptors decreases the levels of PKA activity in ‘indirect pathway’ MSN, it can promote DA-dependent adaptations through the activation of this non-canonical signaling path­ way, as has been shown to occur following admin­ istration of amphetamine and apomorphine (Beaulieu et al., 2007). Inhibition of D2 receptors with anti-psychotic drugs, a situation that is akin to the loss of DA in PD, prevents D2/beta arrestin 2­ mediated signaling (Masri et al., 2008). These results warrant future investigations on the role of b-arrestin/Akt/PP2A in the pathophysiology of both parkinsonism and LID. In fact, recent data from a non-human primate model of LID lend support to the hypothesis that striatal Akt/GSK3 signaling may be involved in the development of the movement disorder (Morissette et al., 2010). An additional pathway deserving a closer look is one linking D2 receptors to a mobilization of intra­ cellular calcium stores. The coupling of D2 recep­ tors to Gi/o proteins causes both an inhibition of adenylyl cyclase (through the Gai subunit) and a release of Gbg subunits, which are capable of sti­ mulating phospholipase Cb isoforms, leading to production of diacylglycerol (DAG) and inositol trisphosphate (IP3), followed by mobilization of intracellular Ca2þ stores (Fig. 1) (HernandezLopez et al., 2000). A possible role of this pathway

in the cellular adaptations associated with PD and LID has not yet been explored. DA receptor-mediated changes in gene expres­ sion necessitate the re-packaging of histones along the DNA. Tightly packed histones prevent tran­ scription factors from binding to the DNA. Mod­ ifications on the DNA (such as methylation of CpG islands) and post-translational modifications of histone proteins affect the histone–DNA inter­ action and the ability of transcription factors to transactivate a promoter. These epigenetic mechanisms have received much attention in the last couple of years. Histones are subject to a wide variety of post-translational modifications includ­ ing lysine acetylation, lysine and arginine methy­ lation, serine and threonine phosphorylation, lysine ubiquitination and sumoylation (Vaquero et al., 2003). A host of protein-modifying enzymes such as histone acetyltransferases, histone deace­ tylases, histone methyltransferases or kinases help to organize the histone structure and contribute to cell-specific gene expression patterns. DA recep­ tor-mediated signal transduction pathways can selectively activate or repress these modifying enzymes and thus trigger chromatin remodelling (Kumar et al., 2005; Schroeder et al., 2008).

DA receptor signaling in the DA-denervated striatum As striatal DA levels decline in PD (Bernheimer et al., 1973; Hornykiewicz, 1975), allostatic changes take place in the nigrostriatal system. Adaptive mechanisms are employed to maintain stability and functionality including a reduced rate of DA inactivation and increased DA synthesis and turn­ over in residual DA neurons (Zigmond et al., 1990), as well as changes in post-synaptic DA receptor sensitivity (Mishra et al., 1974; Staunton et al., 1981). These adaptive mechanisms can compensate for a large decline in DA concentrations, account­ ing for the fact that manifest parkinsonian motor symptoms only occur when the loss of nigral DA neurons exceeds 50% (Fearnley and Lees, 1991).

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Post-synaptic adaptations affecting DA receptors and their downstream signaling mechanisms are particularly relevant to this chapter, because dener­ vation-induced supersensitivity of striatal DA receptors has long been regarded as an important determinant of LID (Klawans et al., 1977; Marconi et al., 1994; Nutt, 1990). This supersensitivity does not necessarily stem from an increased receptor number, because no consistent changes in ligandbinding activities at D1, D2 or D3 receptors have been found in dyskinetic PD patients (Hurley et al., 1996; Rinne et al., 1991; Turjanski et al., 1997), and findings from animal models have been contradic­ tory, particularly with respect to D1 (cf. Gerfen et al., 1990; Konradi et al., 2004) and D3 receptors (cf. Bezard et al., 2003; Hurley et al., 1996; Mela et al., 2010; Quik et al., 2000). Although increased receptor expression at the plasma membrane may contribute to D1 supersensitivity (Guigoni et al., 2007), denervation-induced supersensitivity of DA receptors is generally attributed to altered signal transduction mechanisms in the dopaminoceptive neuron (Gerfen, 2003; Pifl et al., 1992; Prieto et al., 2009; Zhen et al., 2002). The following altera­ tions have been documented to occur in the DAdenervated striatum: (1) aberrant activation of noncanonical signaling cascades downstream of D1 receptors; (2) exuberant activation of canonical sig­ naling pathways following D1 and D2 receptor sti­ mulation and (3) reduced expression of negative signaling modulators. The most relevant example of non-canonical sig­ naling pathway activation was provided by Gerfen and collaborators, reporting that treatment with D1 receptor agonists induces mitogen-activated pro­ tein kinases (MAPK) in striatonigral neurons in the DA-denervated but not the intact striatum (Gerfen et al., 1990). Because this signaling altera­ tion is particularly relevant to LID, it will be exten­ sively discussed in the following paragraphs. Hyperactive canonical signaling seems to depend on an increased coupling of both D1 and D2 recep­ tors to their G proteins. The G-protein-coupling efficiency of striatal DA receptors has been studied in both rat and monkey models of PD by measuring DA agonist-induced guanosine 50 -O-(gamma[35S]

thio)triphosphate ([35S]GTP-gammaS) binding. These studies have shown that DA denervating lesions result in increased agonist-induced Gprotein-binding activity at both D2- and D1-type receptors (Aubert et al., 2005; Cai et al., 2002; Geurts et al., 1999) and that the G-protein-coupling activity of D1, but not D2 receptors, is further enhanced in LID (Aubert et al., 2005). The increased guanosine triphosphate (GTP)-binding activity of D1 receptors is paralleled by an upregula­ tion of Ga-olf proteins (Corvol et al., 2004; Herve et al., 1993) and by a pronounced overactivity of the D1 receptor-adenylyl cyclase signal transduction pathway (Mishra et al., 1974; Pifl et al., 1992). The duration and extent of DA receptor activa­ tion is limited not only by internalization processes (discussed at Section ‘Altered intracellular traffick­ ing of DA and glutamate receptors’) but also by a range of negative signaling modulators, whose expression and efficiency seem to be altered in the DA-denervated striatum (Geurts et al., 2003; Harrison and LaHoste, 2006; Zhen et al., 2002). Relevant to this chapter are the changes found in a family of proteins named regulators of G-protein signaling (RGS), which promote GTP hydrolysis by the alpha subunit of heterotrimeric G proteins, thereby inactivating the G protein and rapidly switching off GPCR signaling pathways (De Vries et al., 2000). A selective decrease in RGS4 and RGS9 mRNA levels occurs in the rat striatum following DA denervation (Geurts et al., 2003), and this change might predispose to overactive DA receptor signaling, hence to LID. In keeping with this hypothesis, viral vector-mediated overexpression of RGS9-2 in the striatum reduces the severity of L-DOPA-induced AIMs in both rat and monkey models of PD (Gold et al., 2007).

Molecular alterations associated with LID What happens when DA formed from exogenous L­ DOPA acts on supersensitive receptors in the par­ kinsonian striatum? A rapidly growing literature indicates that the molecular responses to DA differ greatly between animals exhibiting dyskinesia and

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those showing a normal pattern of motor activation. Prominent molecular differences between the two response categories have been found at all levels of investigation, as will be explained below.

Altered intracellular trafficking of DA and glutamate receptors Both DA denervation and L-DOPA treatment can alter GPCR desensitization and internalization, and important differences have been observed between dyskinetic and non-dyskinetic subjects in both rodent and non-human primate models of PD. In the striatum of non-human primates treated with MPTP, D1 receptor density at the plasma mem­ brane is increased and becomes further increased in LID (Guigoni et al., 2007), while D2 receptor distribution is only modestly affected. In the rat model of LID, an exaggerated expression of D1 receptors at the cell membrane correlates with the severity of the treatment-induced AIMs (Berthet et al., 2009). Importantly, a recent study has shown that the internalization of D1 receptors in striatal neurons can be enhanced by lentiviral over-expres­ sion of GRK6 and that this intervention alleviates LID in both rat and non-human primate models of PD (Ahmed et al., 2010). These observations indi­ cate that therapeutic strategies increasing D1 recep­ tor internalization may have a useful role in the future treatment of LID. In addition to the D1 receptor, ionotropic gluta­ mate receptors show altered subcellular localiza­ tion in LID. This phenomenon has been studied extensively with respect to the NMDA receptor complex in both monkey and rat models of LID. In drug-naïve MPTP-treated macaques, NR1 and NR2B subunits were found to be significantly decreased in synaptosomal membranes, while the abundance of NR2A was unaltered (Hallett et al., 2005). Dyskinesiogenic L-DOPA treatment nor­ malized the abundance of NR1 and NR2B and raised NR2A levels significantly above unlesioned control values. These results led to the suggestion that a relative enhancement in the synaptic abun­ dance of NR2A is implicated in LID (Hallett et al.,

2005). Studies in 6-OHDA-lesioned rats have emphasized the role of NR2B, showing that the receptor-trafficking alteration most critically asso­ ciated with LID consists in a re-distribution of NR2B subunits between synaptic and extrasynaptic membranes (Fiorentini et al., 2006; Gardoni et al., 2006). Protein complexes consisting of D1-NMDA receptor subunits exhibited a similar re-distribution in chronically L-DOPA-treated dyskinetic rats (Fiorentini et al., 2006). The phenomenon was attributed to an altered interaction of NR2B with post-synaptic scaffolding proteins in striatal neu­ rons [see Gardoni et al. (2010), this volume]. Fol­ lowing DA denervation, the synapse-associated scaffolding proteins, PSD-95, SAP-97 and SAP102, appear to be shifted from the post-synap­ tic membrane into other subcellular compartments (Gardoni et al., 2006; Nash et al., 2005). Although chronic L-DOPA treatment tends to restore the synaptic levels of these proteins, the restoration is more pronounced in non-dyskinetic animals com­ pared to dyskinetic ones (Gardoni et al., 2006). Recently, an altered trafficking of AMPA receptor subunits also has been associated with LID. In a study performed on MPTP-lesioned and L-DOPA-treated macaques, dyskinetic ani­ mals were found to exhibit a re-distribution of AMPA receptors, and particularly of the GluR2/ 3 subunit, from the vesicular fraction into the post­ synaptic membrane (Silverdale et al., 2010). The increased relative abundance of AMPA receptor subunits in the post-synaptic membrane was sug­ gested to render striatal neurons more sensitive to glutamate (Silverdale et al., 2010), providing a rationale to the use of AMPA receptor antago­ nists in the treatment of LID. This suggestion is indeed supported by independent behavioural pharmacological studies in both rat and non­ human primate models of PD (Kobylecki et al., 2010; Konitsiotis et al., 2000). Further studies are, however, required to clarify how an altered sub­ cellular distribution of ionotropic glutamate receptor subunits affects the intrinsic excitability and synaptic responses of striatal neurons in LID. Moreover, it will be important to identify key common upstream mechanisms and downstream

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effectors. These studies will guide the choice of the most appropriate target in the design of future anti-dyskinetic therapies.

Altered intracellular signaling Stimulation of supersensitive DA receptors by L-DOPA would be expected to result in a large activation and inhibition, respectively, of cAMP­ dependent signaling pathways in D1- and D2-rich striatal neurons. While the contribution of D2-rich, ‘indirect pathway’ MSN to LID has not yet been elucidated, the overactivity of D1­ mediated signaling in ‘direct pathway’ MSN has indeed been shown to play a major role in LID across all animal models so far examined (mouse, Darmopil et al., 2009; Santini et al., 2007; rat, Lindgren et al., 2009; Westin et al., 2007; and macaque, Aubert et al., 2005). Chronically L­ DOPA-treated, dyskinetic rats and mice show increased striatal phosphorylation of DA- and cAMP-regulated phosphoprotein of 32 KDa (DARPP-32) at the threonine-34 residue (Picconi et al., 2003; Santini et al., 2007). Phosphorylation of DARPP-32 at threonine 34 is induced by L­ DOPA through the stimulation of D1 receptors and PKA (Lebel et al., 2010). Because phospho­ Thr34-DARPP32 is a potent inhibitor of protein phosphatase-1 (PP-1) (Svenningsson et al., 2004), it is not surprising that the striatal levels of sev­ eral phosphorylated substrates are elevated in the ‘dyskinetic’ striatum. Particularly important sub­ strates include subunits of NMDA (Chase and Oh, 2000; Dunah et al., 2000) and AMPA recep­ tors (Santini et al., 2007), ERK1/2 (Pavon et al., 2006; Santini et al., 2007; Westin et al., 2007) and the downstream targets of ERK1/2, including mole­ cules involved in the regulation of protein transla­ tion (Santini et al., 2009) and gene transcription (Santini et al., 2009; Westin et al., 2007). As exten­ sively discussed below (see Section ‘Studies of sig­ naling pathway activation in dyskinetic rodents reveal a common pattern of alterations’), the phos­ phorylated forms of all these molecules are

significantly increased in abundance in L-DOPA­ treated dyskinetic animals compared to non-dyski­ netic cases (see also Fig. 1). A large body of recent studies has addressed the role of Ras-ERK1/2 signaling in LID, pointing to this pathway as an important target for future anti-dyskinetic treatments. Extracellular signalregulated kinases 1 and 2 (ERK1/2) belong to the MAPK family of signaling cascades, which share the motif of three serially linked kinases regulating each other by sequential phosphorylation (Seger and Krebs, 1995). The MAPK signaling system was originally discovered as a critical regulator of cell division and differentiation and was later found to be recruited in mature neurons by different types of extracellular stimuli inducing long-term synaptic and behavioural adaptations (Sweatt, 2001). In both rat (Westin et al., 2007) and mouse models of PD (Santini et al., 2007), phosphorylated ERK1/2 is detected in striatal MSN following the administra­ tion of L-DOPA, and the levels of this phosphopro­ tein correlate positively with the L-DOPA-induced AIMs scores. The time course of ERK1/2 phos­ phorylation in dyskinetic animals indicate that the kinase is activated in striatal MSN by each dose of L-DOPA and remains active for at least 120 min post-dosing (Westin et al., 2007), which is an unu­ sually long interval (cf. Valjent et al., 2000). An involvement of ERK1/2 in LID is demonstrated by the anti-dyskinetic effects of treatments that inhibit upstream components of the Ras-ERK sig­ naling cascade (Lindgren et al., 2009; Santini et al., 2007; Schuster et al., 2008). Moreover, in a recent comparison of compounds targeting different types of glutamate receptors, we found that the only pharmacological agents exerting significant antidyskinetic effects were those capable of reducing L-DOPA-induced phospho-ERK1/2 levels in the striatum (namely, selective antagonists of group I metabotropic glutamate receptors, Rylander et al., 2009). The most potent pharmacological means to suppress L-DOPA-induced phospho-ERK1/2 is represented by D1-like receptor antagonists (Westin et al., 2007), indicating that the activation of ERK1/ 2 by L-DOPA is mediated by D1 receptors.

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Unfortunately, D1 antagonists do not have any clin­ ical utility in PD, because they would interfere with the therapeutic action of L-DOPA. It is therefore important to identify non-dopaminergic treatments that can normalize ERK1/2-mediated signaling with­ out compromising the beneficial effects of L-DOPA. How does Ras-ERK signaling contribute to LID? As a mediator of synaptic and behavioural plasticity, this pathway is assumed to contribute to persistent neural adaptations that maintain the brain in a dyskinesia-prone state (Cenci and Lindg­ ren, 2007). Indeed, Ras-ERK1/2 signaling has been shown to mediate the following, long-lasting cellu­ lar adaptations in animal models of LID: (1) nuclear signaling responses in striatal neurons, such as activation of histone kinases, histone mod­ ifications and induction of nuclear transcription fac­ tors (Santini et al., 2007, 2009; Westin et al., 2007); (2) striatal regulation of the mammalian target of rapamycin complex 1 (mTORC1), which is in turn involved in the control of protein translation and synaptic plasticity (Santini et al., 2009) and (3) endothelial proliferation and angiogenic activities in the basal ganglia (Lindgren et al., 2009). In addition to mediating long-lasting effects of L-DOPA treatment, the activation of Ras-ERK1/2 signaling seems to contribute to the acute expres­ sion of dyskinesia. Indeed, using a short drug treat­ ment regimen (3 days) in 6-OHDA-lesioned rats, we found that an inhibitor of the ERK1/2 upstream kinase (mitogen-activated protein kinase kinase, MEK) acutely reduced the severity of L-DOPA­ induced AIMs (Lindgren et al., 2009). The acute consequences of ERK1/2 activation may rely on the phosphorylation of membrane-bound recep­ tors, ion channels and synaptic proteins (Sweatt, 2001), but the targets involved are as yet unknown. This gap of knowledge is not surprising. While long-term adaptations contributing to the develop­ ment of LID have been intensely investigated, we know virtually nothing about the molecular and electrophysiological activities mediating the acute expression of dyskinetic movements after each dose of L-DOPA. Further investigations are thus needed to unravel the ‘electrophysiological

signature’ of LID in the striatum, ranging from changes in conductances and synaptic responses in D1- versus D2-positive MSN, to activity patterns at the single-unit and neuronal ensemble level.

Altered expression and regulation of transcription factors As explained above (see Section ‘DA receptor sig­ naling in the neurons of the intact striatum: modula­ tion of cyclic AMP/PKA pathways and noncanonical signaling’), phosphorylation of the nuclear protein, CREB at Serine 133 mediates changes in gene expression downstream of DA receptors. Phosphorylated CREB binds to the cAMP responsive element (CRE) and to the closely related activator protein-1 (AP-1) site in the promo­ ter region of many genes, including c-fos and the opioid precursor genes, preproenkephalin and pro­ dynorphin (Cole et al., 1995; Konradi et al., 1993, 1994). Prodynorphin is of particular interest because its upregulation after chronic L-DOPA treatment is a consistent correlate of LID across species and parkinsonian conditions (Aubert et al., 2007; Cenci et al., 1998; Henry et al., 2003; Lundblad et al., 2004). The upregulation of prodynorphin mRNA by L-DOPA, which depends on the D1 receptor (St-Hilaire et al., 2005), concurs with reduced levels of opioid receptor binding in the projection targets of ‘direct pathway’ MSN (Aubert et al., 2007; Johansson et al., 2001), which is indicative of a hyperactive opioid transmission along this pathway. While CREB mediates D1-dependent prodynor­ phin transcription in the intact striatum (Cole et al., 1995), the same protein is not required for the induction of prodynorphin by L-DOPA in the DA-denervated striatum (Andersson et al., 2001). In the latter situation, CRE/AP-1 elements in the prodynorphin promoter are bound and transacti­ vated by ΔFosB-related proteins and JunD (Andersson et al., 2001). This switch in transcrip­ tional control is associated with a large increase in the striatal levels of ΔFosB-like proteins, which correlates positively with dyskinesia severity

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(Andersson et al., 1999). Unlike other Fos family proteins, ΔFosB and its post-translationally modi­ fied forms are uniquely stable (Carle et al., 2007). Accordingly, the upregulation of ΔFosB-like pro­ teins and prodynorphin mRNA by chronic L-DOPA treatment shows a strikingly protracted time course. The expression of both markers rises gradually during a 2-week course of L-DOPA administration (Andersson et al., 2001; Valastro et al., 2007), maintaining high levels of expression for up to 1 year (Westin et al., 2001), which is the longest period of L-DOPA administration so far examined. Following discontinuation of L-DOPA treatment, the striatal levels of ΔFosB and prody­ norphin mRNA remain significantly elevated for weeks (Andersson et al., 2003). Intrastriatal infusion of anti-sense oligonucleotides targeting fosB/ΔfosB mRNA attenuates the gradual increase in dyskine­ sia severity induced by repeated L-DOPA adminis­ tration and the associated upregulation of prodynorphin mRNA (Andersson et al., 1999). These results indicate that ΔFosB-like transcription factors induce and maintain changes in striatal gene expression that favour the expression of dyskinesia. In keeping with this contention, molecular interven­ tions that reduce the transcriptional activity of ΔFosB have been found to attenuate the severity of already established LID in a non-human primate model of PD (Berton et al., 2009).

Altered plasticity of corticostriatal synapses The idea that LID is caused by abnormal plasticity of corticostriatal synapses was proposed 10 years ago (Calabresi et al., 2000; Calon et al., 2000) and has remained very popular ever since (Jenner, 2008). The first evidence of altered activity-dependent plasticity of corticostriatal synapses was provided by Picconi et al. (2003). This study compared the inducibility and reversal of corticostriatal long-term potentiation (LTP) in brain slices from L-DOPA-treated, dyskinetic or non-dyskinetic rats. High-frequency stimulation of cortical afferents was found to induce a nor­ mal LTP in both groups, but dyskinetic rats

showed a lack of depotentiation upon subse­ quent low-frequency stimulation of the same afferent pathway (Picconi et al., 2003). This loss of bidirectional synaptic plasticity points to aber­ rant gating of cortical inputs in the dyskinetic striatum. If corticostriatal LTP cannot be promptly reversed, striatal neurons would be unable to ‘erase’ irrelevant information when processing cortically driven motor commands. The mechanisms accounting for the loss of synaptic depotentiation in dyskinetic animals have not been completely resolved. The altera­ tion was originally attributed to an overactive signaling downstream of D1 receptors and ensu­ ing hyperphosphorylation of DARPP-32, leading to persistent inhibition of intracellular phospha­ tases (Picconi et al., 2003). Further investigations are, however, required to clarify which phos­ phorylated substrates, and upstream kinases, impede the reversal of LTP. It will also be important to verify the relative involvement of ‘direct pathway’ versus ‘indirect pathway’ MSN in this synaptic abnormality, an issue that has not been addressed thus far.

Altered gene expression patterns The pattern of striatal mRNA expression differ­ entiating L-DOPA-treated, dyskinetic animals from non-dyskinetic ones was investigated by Konradi and collaborators using Affymetrix gene chip arrays (Konradi et al., 2004). In this study, rats with 6-OHDA lesions were given a therapeutic dose of L-DOPA, or saline, for 21 days and killed 18 h after the last injection. L­ DOPA-treated rats were classified as dyskinetic or non-dyskinetic based on the AIMs scores recorded during the treatment. The most salient features of the mRNA expression profile asso­ ciated with dyskinesia indicated increased tran­ scriptional activity of GABAergic neurons, structural and synaptic plasticity, altered calcium homeostasis and calcium-dependent signaling and an imbalance between metabolic demands and capacity for energy production in the

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striatum. More specifically, genes coding for ion transporters, calcium-dependent ATPases and voltage-gated ion channels were upregulated in the dyskinetic striatum, a pattern indicative of increased neuronal activity and high ATP con­ sumption. Accordingly, dyskinetic rats showed upregulation of the mitochondrial gene, cyto­ chrome oxidase subunit I (CO-I), which is a marker of increased metabolic demands in neu­ rons. At the same time, dyskinetic animals showed significant downregulation of genes encod­ ing two key enzymes of the phosphocreatine pathway, guanidinoacetate methyltransferase and ubiquitous mitochondrial creatine kinase (Mi-CK), a finding that was later confirmed with proteomics methods (Valastro et al., 2007). Dyski­ netic rats also had reduced expression of genes coding for glyceraldehyde-3-phosphate dehydro­ genase and lactate dehydrogenase, both of which are involved in energy production through the gly­ colytic pathway. Several genes encoding ribosomal proteins were downregulated in dyskinetic rats, a pattern suggestive of cellular stress (Warner, 1999). These results were the first to indicate that dyski­ nesiogenic treatment with L-DOPA elevates the metabolic demands of striatal neurons but downregulates pathways involved in energy production in the striatum. A more recent micro-array study focused instead on a comparison between acutely and chronically L-DOPA-treated rats (El Atifi-Borel et al., 2009). Here, the L-DOPA dose was above threshold for the induction of dyskinesia in all animals. Although acute and chronic L-DOPA treatment were found to regulate a common set of genes involved in signal transduction, transcrip­ tion and synaptic transmission, a threefold larger number of genes were altered in response to repeated drug administration. The difference between acute and chronic L-DOPA treatment was particularly noticeable for genes involved in metabolism, protein biosynthesis (ribosomal genes), neurite outgrowth, synaptogenesis and cell proliferation. This study pointed to a relation­ ship between repeated L-DOPA exposure and structural cellular modifications in the striatum.

Although L-DOPA can induce dyskinesia even upon its first administration (Nadjar et al., 2009), chronic treatment with L-DOPA is required to establish a long-lasting predisposition to this movement disorder. In fact, repeated administra­ tion of L-DOPA induces a gradual increase in dyskinesia severity over time and also reduces the dyskinetic threshold dose of the drug (Cenci and Lundblad, 2006). This delayed effect, often referred to as the ‘dyskinesia-priming action of L-DOPA’, conceivably relies on structural modifi­ cations of cells and synapses in the brain.

Towards a unifying molecular interpretation of LID The literature reviewed above has revealed a large number of molecular alterations that are linked to the expression and development of LID and uncov­ ered therapeutic targets at many possible levels. Further studies are required to shed light on the mechanisms through which these molecular changes alter the function of cells and circuits within the basal ganglia. Based on the current infor­ mation, it is however possible to outline some general molecular features of LID, and these will be the object of the following paragraphs.

Studies of signaling pathway activation in dyskinetic rodents reveal a common pattern of alterations As mentioned above (cf. Sections ‘Altered intra­ cellular signaling’ to ‘Altered expression and reg­ ulation of transcription factors’), nuclear signaling responses to L-DOPA are more exuberant in dys­ kinetic animals compared to non-dyskinetic sub­ jects. How does this difference come about? Important clues have emerged from a comparison between chronically and acutely L-DOPA-treated rats [(Andersson et al., 2001; Cenci et al., 1999; Valastro et al., 2007; Westin et al., 2007), and further unpublished data by Konradi and Cenci] (Fig. 2). In these studies, rats with 6-OHDA

220 Phospho-ERK1/2

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mod. H3 total. H3 actin Fig. 2. Deficient densensitization of DA receptor-dependent signaling in the dyskinetic striatum. Acute treatment with L-DOPA results in a supersensitive activation of cAMP- and MAPK-dependent pathways signaling to the nucleus, here exemplified by the phosphorylation of ERK1/2 (on Thr202-Tyr204), MSK-1 (on Ser376) and by a modification of histone 3 that has been linked to chromatin remodelling during active gene transcription (phospho[Ser10]-Acetyl[Lys14]-H3). Chronic L-DOPA administration normalizes these supersensitive responses only in animals that remain free from dyskinesia during the treatment (non-dyskinetic group). Levels of signaling pathway activation remain significantly elevated above control values in dyskinetic animals. Thus, a shift towards normal molecular responses during chronic L-DOPA treatment is associated with a resistance to dyskinesia, while persistent supersensitivity is a correlate of LID. These data show both published and unpublished results obtained from 6-OHDA­ lesioned rats, which received chronic (10–12 days) or acute treatment with L-DOPA (L-DOPA methyl ester, 10 mg/kg/dose combined with 15 mg/kg/dose of benserazide), or physiological saline, and were killed 30 minutes post injection. Data in (a) and (b) represent counts of immunoreactive neurons in the lateral part of the DA-denervated caudate–putamen, from animals reported in Westin et al. (2007), and five chronically L-DOPA-treated non-dyskinetic rats from the same study (not reported in the paper). Data in (c) show results from Western immunoblotting analysis. The optical density on specific immunoreactive bands was normalized to the corresponding b-actin bands, and results from each group were expressed as a percentage of saline control values. A Western blot showing bands immunoreactive for phospho[Ser10]-Acetyl[Lys14]-histone 3, total histone 3 and b-actin is shown in (d). Full descriptions of the experimental procedures can be found in Westin et al. (2007) (for a, b) and Schroeder et al. (2008) (for c, d). Abbreviations in (d): s, saline control; a, acute L-DOPA; d, chronic L-DOPA/dyskinesia; nd, chronic L-DOPA/no dyskinesia). Values indicate group means + SEM from 3 to 10 animals per group, which were statistically compared using onefactor analysis of variance and post-hoc Newman–Keuls test. P < 0.05 vs.
, saline-treated 6-OHDA-lesioned controls; , acute L­ DOPA; #, chronic L-DOPA with dyskinesia.

221

lesions were given a therapeutic dose of L-DOPA either on one single occasion (acute treatment) or repeatedly for 10–14 days, during which they were monitored on the AIMs scale. As indicators of intracellular signaling activation we examined the levels of phosphorylated (Thr 34) DARPP-32 (not shown), ERK1/2 and mitogen- and stress-activated kinase-1 (MSK-1, nuclear target of ERK), and the histone modifi­ cation, phospho(Ser10)-Acetyl(Lys14)-histone 3, which has been linked to chromatin remodelling during active gene transcription (Cheung et al., 2000). A common pattern of group differences emerged from the analysis of each of the mar­ kers under investigation. Acute L-DOPA treat­ ment caused a large activation of signaling molecules in the DA-denervated striatum in all animals. Chronic administration of L-DOPA abolished this upregulation in animals that had remained free from dyskinesia during the treat­ ment. By contrast, rats that had developed dys­ kinesia showed overtly sensitized (e.g. ERK1/2) or only partially densensitized responses to the last injection of L-DOPA, with levels of phos­ phoproteins that were significantly elevated above control values in each of the comparisons. Similar results have been recently reported from a mouse model of LID (Santini et al., 2007). Overall, these data indicate that chronic treat­ ment with L-DOPA would tend to normalize supersensitive signaling responses in DA-dener­ vated striatal neurons, but fails to achieve this effect in dyskinetic subjects. Because upregula­ tion of the above phosphoproteins by L-DOPA relies on the D1 receptor (see Sections ‘Altered intracellular signaling’ and ‘Altered expression and regulation of transcription factors’), we con­ clude that the core molecular alteration asso­ ciated with LID consists in the inability of striatal neurons to desensitize adenylyl cyclasedependent and MAPK-dependent signaling cas­ cades downstream of D1 receptors. The mechan­ isms underlying such an inability have not been resolved and may be related to a deficient

internalization of D1 receptors (Berthet et al., 2009). Surface receptor availability is not, how­ ever, the only mechanism regulating the duration of downstream signaling responses, and the pos­ sibility that intracellular signaling inhibitors are less efficient in dyskinetic animals ought to be addressed in future studies. In fact, the striatal gene expression profile detected in dyskinetic rats by Konradi and collaborators (Konradi et al., 2004) pointed to an altered expression/ activity of intracellular phosphatases, while also revealing upregulation of positive upstream modulators of Ras-ERK signaling. In particular, RAS guanyl nucleotide-releasing protein 1 (RasGRP1) was strongly upregulated in dyski­ netic rats relative to non-dyskinetic rats and con­ trols (Konradi et al., 2004). RasGRP1 (also called CalDAG-GEF2) is a DAG-regulated nucleotide exchange factors that activates Ras and the Erk/ MAP kinase cascade through the exchange of gua­ nosine diphosphate (GDP) for GTP (Yang and Kazanietz, 2003). A closely related protein, RasGRP2 (CalDAG-GEFI), activates Rap1A and inhibits Ras-dependent activation of the ERK/ MAP kinase cascade (Kawasaki et al., 1998). A recent study has addressed the regulation of RasGRP1 and RasGRP2 gene expression in the rat model of LID (Crittenden et al., 2009). In the DA-denervated striatum, L-DOPA treatment pro­ duced upregulation of RasGRP1 and downregula­ tion of RasGRP2, and both of these effects correlated significantly with the severity of the AIMs. These data are consistent with an overactiv­ ity of Ras-ERK1/2 signaling in the dyskinetic stria­ tum (cf. Section ‘Altered intracellular signaling’) and point to the dysregulation of RasGRP1 (CalDAG-GEF2) and RasGRP2 (CalDAGGEFI) as being an important upstream mechanism. These data warrant a search for therapeutic strate­ gies targeting striatum-enriched Ras-ERK1/2 sig­ naling modulators. This approach may relieve LID while avoiding the many potential side effects of a complete, systemic blockade of MAPK signaling.

222

Potential consequences of dysregulated D1 receptor-dependent signaling In summary, in dyskinetic animals each dose of L-DOPA results in an abnormally large and sus­ tained activation of adenylyl cyclase-dependent and MAPK-dependent signaling cascades in striatal neurons, with downstream changes in epigenetic and transcriptional activities. Some of the latter changes are very long-lasting (See section ‘Altered expression and regulation of transcription factors’) and prone to accumulate upon repeated exposure to L-DOPA (Figs. 3 and 4). By integrating these findings with other data reported in the literature, it is possible to make some reasonable assumptions on the cellular consequences of such a response pattern. One conceivable consequence is a pronounced structural and synaptic rearrangement of striatal neurons. Beside the data provided by micro-array studies (cf. Section ‘Altered gene expression pat­ terns’), this contention is supported by novel evi­ dence linking the D1 receptor/PKA pathway to upregulation and/or increased phosphorylation of cytoskeletal proteins (Lebel et al., 2009; SgambatoFaure et al., 2005). In particular, a recent study in 6­ OHDA-lesioned rats has reported a significant increase in the striatal levels of phosphorylated tau protein following chronic pulsatile L-DOPA treat­ ment (Lebel et al., 2010). These data are interesting because abnormal tau phosphorylation can lead to cytoskeletal and synaptic alterations (Mazanetz and Fischer, 2007). Cytoskeletal modifications in LID are also suggested by the striatal upregulation of arc (activity-regulated cytoskeletal-associated gene), since arc participates in cytoskeletal rearran­ gements during synaptic plasticity (Steward and Worley, 2001). In the rat model of LID, Arc mRNA and protein levels show a sustained upregu­ lation in prodynorphin-positive MSN within the same striatal regions exhibiting high ΔFosB-like immunoreactivity (Sgambato-Faure et al., 2005). This would suggest that ‘direct pathway’ MSN undergo long-term structural and synaptic modifica­ tions in LID, a suggestion that needs to be verified by future investigations.

Another consequence of repeated and sus­ tained signaling activation is a large energy expen­ diture in striatal neurons, a hypothesis supported by the results of gene expression micro-array stu­ dies (discussed above) and biochemical investiga­ tions (Valastro et al., 2009). A large bioenergetic expenditure is also supported by the findings of endothelial proliferation in the striatum and other basal ganglia nuclei in dyskinetic rats (Westin et al., 2006), because angiogenic responses in the adult brain are usually associated with long-lasting increases in local metabolic demands. A large energy requirement, associated with impairments in certain bioenergetic pathways (Konradi et al., 2004), would be expected to make striatal MSN more vulnerable to glutamate- and DA-mediated toxicity. Indeed, MSN are very sensitive to bioenergetic deficits. Primary genetic mitochondrial defects can lead to striatal degeneration and associated dysto­ nia and dyskinesia [reviewed in Damiano et al. (2010)]. Insufficient availability of high-energy phosphates resulting in impaired activity of plasma membrane ATPases would alter the response of striatal MSN to excitatory amino acids, lowering the threshold for excitotoxicity (Calabresi et al., 1995). In addition high extracel­ lular DA levels (as induced by a L-DOPA bolus) can prime the striatum for neurodegenerative events. Indeed, DA-dependent degeneration and apoptosis of striatal neurons has been documen­ ted to occur in situations associated with high extracellular levels of DA, such as intrastriatal DA injections (Hattori et al., 1998), metampheta­ mine administration (Schmidt et al., 1985) and genetic ablation of the DA transporter (Cyr et al., 2003). In primary striatal cultures, DA induces neuronal apoptotic death via stimula­ tion of D1 receptors, leading to sustained activa­ tion and cytoplasmic retention of ERK1/2 (Chen et al., 2009). Sustained activation of ERK1/2 has been shown to render neurons more vulnerable to glutamate toxicity in some model systems (Luo and DeFranco, 2006). It is conceivable that the combination of high extracellular DA levels, sustained intracellular

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Fig. 3. Levels of FosB/FosB-like immunoreactivity and prodynorphin mRNA are persistently upregulated in the dyskinetic striatum. (a) ΔFosB-like proteins have very slow elimination kinetics and can therefore accumulate in striatal neurons during the course of chronic L-DOPA treatment. This effect is seen only in animals that develop dyskinesia. Acute L-DOPA treatment also induces the expression of FosB/ΔFos-like immunoreactivity and prodynorphin mRNA, but this upregulation is transient (Cenci et al., 1999). In the experiments shown here, acutely and chronically L-DOPA-treated animals were killed at 3 hours or 2 days post-injection to better separate the effects of the chronic treatment from those of the last drug injection. (b) Prodynorphin mRNA shows the same expression pattern as FosB/ ΔFosB like immunoreactivity (indeed, ΔFosB-like transcription factors mediate the upregulation of prodynorphin mRNA induced by L­ DOPA in the DA-denervated striatum; see Section ‘Altered expression and regulation of transcription factors’). Values in (a) represent numbers of FosB/ΔFosB-immunoreactive cells/mm2 measured in the lateral part of the DA-denervated caudate–putamen from animals in previously published studies (Cenci et al., 1999; Westin et al., 2007). Data in (b) represent the hybridization signal to prodynorphin mRNA in the DA-denervated caudate–putamen (expressed as a percentage of the values on the contralateral intact side in each group) (data from Cenci et al., 1998; Mela et al., 2007). In each data set, values indicate group means + SEM from 3 to 10 animals per group, P < 0.05 vs. saline-treated 6-OHDA-lesioned controls; # chronic L-DOPA with dyskinesia. Acutely and chronically L-DOPA-treated rats were compared with their own saline control group. (c–e) Cellular levels of FosB/ΔFosB immunostaining are larger in chronically L­ DOPA-treated dyskinetic rats compared to acutely L-DOPA-treated animals, and a similar pattern of group differences applies to prodynorphin mRNA (f–h). Photomicrographs in (f–h) show emulsion-coated autoradiographs of striatal sections visualized under dark field optics. The sections had been hybridized with a 35S-labeled oligonucleotide probe complementary to prodynorphin mRNA. Scale bar, 50 mm. Full descriptions of all the experimental procedures can be found in our original publications.

224

signaling and bioenergetic deficits may lead to a progressive deterioration of striatal neurons in LID. Although this possibility is not yet supported by experimental data, only few studies have addressed the integrity of striatal neurons in ani­ mal models of LID (Lindgren et al., 2007; Westin et al., 2006). In these studies, 6-OHDA-lesioned rats were treated with L-DOPA for a relatively short period (2–3 weeks), and the striata were examined using histological methods (total neuro­ nal cell counts and Fluorogold histochemistry) that cannot detect neuronal deterioration preced­ ing/accompanying cell death. Yet, in this animal model, dietary supplementation of creatine can reduce the severity of LID (Valastro et al., 2009), supporting a contribution of bioenergetic deficits to the pathophysiology of this movement disorder. Patients in the complicated phase of PD receive L-DOPA treatment for years despite their dyskinesias. The experimental data reviewed

above call for further investigations addressing the long-term consequences of a prolonged, dys­ kinesiogenic L-DOPA treatment on the integrity and resilience of striatal neurons.

‘Too much’ molecular plasticity leads to plasticity failure While the overactivation of D1-dependent signal­ ing pathways may cause large morphological and functional rearrangements in striatal neurons (‘too much plasticity’), it also hijacks the molecular machinery by which these neurons normally respond to incoming stimuli (Fig. 4). Striatal neu­ rons integrate convergent cortical and thalamic inputs to modify the output of the basal ganglia, playing a pivotal role in movement selection and adaptive motor control [see Wiecki and Frank (2010), this volume]. Conceivably, dyskinesia

Response intensity vector

Structural and synaptic changes, loss of cellular resilience

Long-lasting alterations

Fast signalling responses

LD LD LD LD LD LD LD LD Time vector Fig. 4. Maladaptive plasticity of striatal neurons in LID. In animal models of LID, each dose of L-DOPA (LD) causes an abnormally large and prolonged activation of cAMP- and MAPK-dependent signaling pathways in striatal neurons (fast signaling responses). Although these responses would tend to normalize during chronic drug treatment, desensitization processes are inefficient in dyskinetic subjects. Hence, in these subjects, treatment with L-DOPA disrupts the dynamics of signaling networks that are normally under tight spatiotemporal control. Signalling dysregulation has both acute electrophysiological effects and long-term consequences on the function of striatal neurons. Long-term cellular alterations associated with LID have been documented to occur in D1/prodynorphin-positive MSN, including altered regulation of CRE/AP-1-dependent transcription, increased expression of neurotransmitter-related genes and increased synthesis and phosphorylation of cytoskeletal proteins (see Sections ‘Altered expression and regulation of transcription factors’ and ‘Potential consequences of dysregulated D1 receptor-dependent signaling’). This pattern of responses would predict the occurrence of profound structural and synaptic rearrangements in these neurons, associated with a large bioenergetic expenditure. Plastic modifications affecting D2/prepronkephalin MSN in LID are far less understood and will need to be addressed in future studies.

225

represents a disorder of motor selection featuring an inability to suppress excessive, purposeless movements. Dysregulated D1-dependent signaling and sustained potentiation of corticostriatal synapses would be expected to cause abnormal gating of cortically driven motor commands, being key to this movement disorder. Moreover, if corticostriatal synapses are clogged by irrelevant information (Picconi et al., 2003), dyskinesia would be expected to go hand in hand with an impaired capacity for experience-dependent mod­ ification of action selection. This pathophysiological hypothesis is supported by a large number of clinical observations. In the non-complicated stages of PD, treatment with L-DOPA promotes ‘positive’ plasticity, result­ ing in long-lasting symptomatic benefit [reviewed in Cenci et al. (2009)], improved efficacy of non­ invasive cortical stimulation methods (Fierro et al., 2001; Rodrigues et al., 2008) and modulation of activity-dependent synaptic plasticity in basal gang­ lia output neurons (Prescott et al., 2009). By con­ trast, in the advanced, complicated stages of PD, treatment with L-DOPA loses its capacity to induce a long-lasting symptomatic improvement extending beyond the effects of single doses (Nutt and Hol­ ford, 1996). In the same disease stages, L-DOPA worsens striatum-dependent learning functions (Cools et al., 2007; Feigin et al., 2003) and nega­ tively affects cortical plasticity. Motor cortex plas­ ticity has been probed in PD patients using a paired associative stimulation protocol (Morgante et al., 2006). While L-DOPA was found to restore LTPlike cortical plasticity in non-dyskinetic subjects, it failed to achieve an effect in dyskinetic patients. The mechanisms underlying a lack of cortical plas­ ticity in LID are presently unknown, but have been suggested to depend on synchronized neuronal activities that impair information processing in basal ganglia–thalamo–cortical loops (Hammond et al., 2007; Morgante et al., 2006). Abnormal oscil­ latory patterns of neural activity have been detected in the subthalamic nucleus and substantia nigra reticulata in dyskinetic PD patients and rat models of LID, respectively [Alonso-Fench et al.,

2006; Meissner et al., 2006]. Future studies ought to address the role played by striatal neurons in the genesis of these pathological activity patterns.

Concluding remarks During the past 10 years, considerable advances have been made in unravelling the molecular mechanisms of LID. This progress has been facili­ tated by the availability of rodent models of LID, in which improved methods of behavioural analy­ sis have allowed investigators to distinguish dyski­ netic animals from those that remained free from dyskinesia despite receiving the same L-DOPA treatment [reviewed in Cenci and Ohlin (2009)]. A large body of literature using these models has shown that the molecular, neurochemical and cel­ lular effects of L-DOPA are very different depending on whether or not the treatment induces AIMs. As well as revealing novel concepts and therapeutic targets, these studies have raised awareness that plastic modifications of striatal cells and synapses are a major determinant of treatment outcome in PD. In future research, it will be important to define the interactions between DA-dependent adaptations and genetic susceptibility factors [see Gasser (2010) and Martin et al. (2010)] in determining different pro­ files of treatment response in PD. Acknowledgements The authors’ ongoing projects in this area are supported by grants from the Swedish Research Council, the Michael J. Fox Foundation for Par­ kinson’s Research, the Swedish Parkinson Foun­ dation, the EU grant contract number 222918 (REPLACES) FP7 (Thematic priority HEALTH) (MAC) and NS48235 (CK). The authors thank Elissa Strome and Joanna Kaczmarska for techni­ cal support, Elisabet Ohlin for data analysis (Fig. 2), Hanna Lindgren and Elisabet Ohlin for helpful discussion.

226

Abbreviations 6-OHDA AC5 AIMs Akt AMPA AP-1 Arc cAMP CRE CREB D1,2,3,4,5 DA DAG DARPP-32

DAT Elk-1 ERK1/2 GDP GKS3 GPCRs G-protein GRKs GTP IP3 IP3R L-DOPA LID LTP MAPK MEK

mGluR 6-hydroxydopamine adenylyl cyclase type 5 abnormal involuntary movements protein kinase B amino-3-hydroxy-5-methyl­ 4-isoxazole propionic acid activator protein-1 activity-regulated cytoskeletal­ associated protein cyclic AMP cAMP response element cAMP response elementbinding protein dopamine receptors dopamine diacylglycerol dopamine- and cAMP­ regulated phosphoprotein of 32 kDa dopamine transporter Ets like gene1, oncogene, transcription factor extracellular signal-regulated kinases 1 and 2 guanosine diphosphate glycogen synthase kinase 3 G-protein-coupled receptors guanosine triphosphatebinding protein G-protein-coupled receptor kinases guanosine triphosphate inositol trisphosphate inositol trisphosphate receptor L-3,4-dihydroxyphenylalanine L-DOPA-induced dyskinesia long-term potentiation mitogen-activated protein kinases mitogen-activated protein kinase kinase

MPTP MSK-1 MSN mTORC1 NMDA PD PET PKA PLC PP-1 PP2A Raf

Ras

RasGRP1 RasGRP2 RGS b-ARR

metabotropic glutamate receptor 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine mitogen- and stress-activated kinase 1 medium-sized spiny neurons mammalian target of rapamycin complex 1 N-methyl-D-aspartate Parkinson’s disease positron emission tomography protein kinase A phospholipase C protein phosphatase 1 protein phosphatase 2A member of the MAP kinase pathway (rapidly accelerated fibrosarcoma oncogene) oncogene of the MAP kinase pathway (RAt sarcoma oncogene) RAS guanyl nucleotidereleasing protein 1 RAS guanyl nucleotidereleasing protein 2 regulators of G-protein signaling beta arrestin

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A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 12

Effects of GPi and STN inactivation on physiological, motor, cognitive and motivational processes in animal models of Parkinson’s disease Christelle Baunez†,
and Paolo Gubellini‡ †

Laboratoire de Neurobiologie de la Cognition (LNC), UMR6155 CNRS/Aix-Marseille Université, Marseille, France Institut de Biologie du Développement de Marseille-Luminy (IBDML), UMR6216 CNRS/Aix-Marseille Université, Marseille, France

‡

Abstract: Loss of the dopaminergic input to the striatum, characterizing Parkinson’s disease, leads to the hyper-activity of two key nuclei of the basal ganglia (BG): the subthalamic nucleus (STN) and the internal segment of the globus pallidus (GPi). The anatomo-physiological organization of the BG and their output suggested that interfering with such hyper-activity could restore motor function and improve parkinsonism. Several animal models in rodents and primates, as well as clinical studies and neurosurgical treatments, have confirmed such hypothesis. This chapter will review the physiological and behavioural data obtained by inactivating the GPi or the STN by means of lesions, pharmacological approaches and deep brain stimulation. The consequences of these treatments will be examined at levels ranging from cellular to complex behavioural changes. Some of this experimental evidence suggested new and effective clinical treatments for PD, which are now routinely used worldwide. However, further studies are necessary to better understand the consequences of GPi and STN manipulation especially at the cognitive level in order to improve functional neurosurgical treatments for Parkinson’s disease by minimizing risks of side-effects. Keywords: Basal ganglia; deep brain stimulation; dopamine; globus pallidus; lesion; substantia nigra; electrophysiology; behaviour glutamatergic inputs from the cortex and the tha­ lamus, mainly via the striatum (caudate/putamen nuclei) and in a lesser extent via the subthalamic nucleus (STN). BG are mainly implicated in motor behaviour and learning, as well as in cog­ nitive and motivational processes. In 1989, Albin et al. synthesized the data available regarding the anatomo-physiological organization of the BG

Introduction The basal ganglia (BG) are a group of interconnected deep brain structures receiving massive
Corresponding author. Tel.: 33 4 88 57 68 76; Fax: 33 4 88 57 68 72 E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83012-2

235

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and proposed a model functioning via two segre­ gated pathways going from the striatum to the output BG nuclei, that is, the direct and indirect pathways. The output BG nuclei include the internal segment of the globus pallidus (GPi), or entopeduncular nucleus (EP) in rodents, and the substantia nigra pars reticulata (SNr). GPi/EP and SNr are GABAergic structures innervating mainly the motor thalamic nuclei and receiving inputs from the striatum via two major pathways, one directly from the striatum (the direct path­ way) and the other (the indirect pathway) via the external globus pallidus (GPe, or GP in rodents) and the STN. This organization has been described for five parallel loops originating from various cortical areas and innervating different sectors of each structure, defining functional seg­ regated loops: the motor, oculomotor, dorsolat­ eral prefrontal, lateral orbitofrontal and limbic loops (Alexander et al., 1986). DeLong (1990) further improved this model of the motor loop by introducing the dysfunctions associated with the loss of substantia nigra pars compacta (SNc) neurons producing dopamine (DA), and the ensuing striatal DA depletion characterizing Parkinson’s disease (PD). This model, illustrated in Fig. 1 suggested that both the STN and the GPi are hyper-active in PD, leading to akinetic-like symptoms (DeLong, 1990). It became then obvious that an interesting alterna­ tive strategy to DArgic treatments for PD could be to reduce this hyper-activity at the level of either the STN or the GPi. This chapter will thus review the physiological and behavioural data obtained using this strategy, using various means of inactivation, that is lesions, pharmaco­ logical inactivation or deep brain stimulation (DBS) at high-frequency stimulation (HFS). This latter technique, first applied in the STN of PD patients by the group of Benabid in Grenoble, France (Limousin et al., 1995), is currently used worldwide with great success. However, there are still remaining questions regarding its mechanism of action (Gubellini et al., 2009).

Cortex +

GLU +

– GABA Enk Indirect pathway –

GLU

Striatum

+

+

+ GABA SP Direct pathway DA

Thalamus

+ GPe Brain Stem Spinal Cord GABA

-

SNc

STN

GABA –

– GLU +

EP/SNr Fig. 1. Schematic diagram of the basal ganglia organization after a DA depletion as proposed by DeLong (1990). This diagram was clearly indicating a hyper-activity of the STN and the GPi, suggesting therefore that normalization of STN or GPi activity could be a beneficial treatment for parkinsonism. STR, striatum; STN, subthalamic nucleus; GPe, external segment of the globus pallidus; EP, entopeduncular nucleus (=GPi: internal segment of the globus pallidus); Pf, parafascicular nucleus of the thalamus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; GLU, glutamate; Enk, enkephalin; SP, substance P.

During the last 50 years, several different ani­ mal models of PD have been developed to better understand the pathophysiological mechanisms of this neurodegenerative disorder. Acute models were the first to be introduced by using monoa­ mine depleting agents, such as reserpine (that blocks the vesicular monoamine transporter), and later by using DA receptor antagonists, such as haloperidol. Nowadays, the two most common

237

and relevant PD models are based on toxins that impair oxidative phosphorylation by inhibiting the complex I of the mitochondria, leading to DAer­ gic neuron loss: 6-hydroxydopamine (6-OHDA), which is injected into the SNc or the striatum of rodents and selectively kills DAergic neurons (after blocking the noradrenaline transporter), 1-methyl-4-phenyl-1,2,3,6-tetrahy­ dropyridine (MPTP), which is injected systemi­ cally in non-human primates and certain mice strains and is transformed into the toxic product 1-methyl-4-phenylpyridinium that is introduced into DAergic neurons by the DA transporter (Gubellini et al., 2010).

GPi manipulation in PD Neurons of the EP recorded in vitro show a spon­ taneous action potential discharge activity at fre­ quencies of 4–10 Hz at membrane potentials around –50 mV (Nakanishi et al., 1990; Shin et al., 2007). In primate PD models (MPTP lesion), the discharge activity of GPi neurons changes towards a more irregular pattern characterized by bursts of action potentials, which is consistent with findings in PD patients (Hutchison et al., 1994). There is no consensus about the change in their mean firing rate, which is described as increased (Boraud et al., 1996; Filion and Tremblay, 1991; Wichmann and DeLong, 2003), as well as decreased (Raz et al., 2000), while there is agreement on the appa­ rition of a synchronized low-frequency oscillatory activity (Bergman et al., 1994; Eusebio and Brown, 2007; Filion and Tremblay, 1991; Leblois et al., 2006; McCairn and Turner, 2009; Raz et al., 2000).

Neurophysiological effects The first experimental report regarding the neu­ rophysiological effects of GPi inactivation was obtained in MPTP-treated macaques, in which GPi neurons became hyper-active. GPi HFS could significantly reduce such hyper-activity,

restoring a frequency of action potential discharge similar to that observed in normal animals, and this change was correlated with an improvement of motor symptoms (Boraud et al., 1996). More precisely, the firing of the majority of GPi neurons become time-locked with GPi HFS, showing a first excitatory phase with ~3 ms latency, followed by inhibition (~4.5 ms) and a second excitation (~6.5 ms) (Bar-Gad et al., 2004). Such temporal locking has been also found during GPi recordings in PD patients (Dostrovsky et al., 2000) and sup­ ported by computational models (Johnson and McIntyre, 2008). On the other hand, no clear time-lock has been observed in another study on MPTP-treated monkeys (McCairn and Turner, 2009), where the majority of GPi and GPe neu­ rons responded to repeated periods of 30 s GPi HFS with a phasic peristimulus modulation in fir­ ing, towards both increases and decreases. A min­ ority of pallidal neurons responded with sustained responses (more common in the GPi) that could last up to the next stimulation period, and nearly all these sustained responses were significant decreases in firing rate. Such differences between findings on the effects of GPi HFS on spike fre­ quency rate could be attributed to the experimen­ tal set-up, especially the duration of HFS application. However, the interesting contribution of McCairn and Turner paper is about the role of GPi HFS in suppressing the oscillatory low-fre­ quency activity of pallidal neurons due to DA depletion that characterizes parkinsonian state (Utter and Basso, 2008). Regarding the effects of GPi HFS in other BG structures, Anderson and colleagues (2003) showed a reduction of discharge frequency in tha­ lamic neurons responding to stimulation applied in intact monkeys. These findings seem in contrast to the schematic functioning of BG, since this treatment should inactivate the GPi and thus disinhibit thalamic activity, but they are supported by evidences from patients receiving GPi HFS for dystonia (Montgomery, 2006). A recent study has also shown that GPi HFS applied in MPTP monkeys time-locks the firing rate of neurons in

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the primary motor cortex to the stimulus, increas­ ing the response specificity to passive limb movement (Johnson et al., 2009). This latter observation is in line with the findings in PD patients showing that GPi HFS increased regional cerebral blood flow in the premotor cortex detected by positron emission tomography (PET) (Davis et al., 1997). There is little data on the neurophysiological effects of EP inactivation in rodents. A recent electrophysiological slice study investigated the effects of EP HFS on EP neurons in the rat, show­ ing that HFS induces an elevation of extracellular Kþ, which decreases EP neuron activity by acti­ vating a depolarizing ion conductance with no synaptic involvement (Shin et al., 2007). Earlier studies focused on the effects of EP inactivation in the striatum of 6-OHDA-lesioned rats, showing that EP lesion could counteract the increase of preproenkephalin mRNA levels induced by L-3,4-dihydroxyphenylalanine (L-DOPA) treat­ ment (Perier et al., 2003) and that EP HFS had no significant effect on striatal DA transmission (Meissner et al., 2004).

Effects of manipulation of the GPi on motor behaviour Lesion and pharmacological GPi inactivation in the monkey One of the first evidences showing that the GPi could represent an interesting target for the treat­ ment of PD was provided by pharmacological experiments showing that blocking glutamatergic transmission within this structure could alleviate motor deficits in monkeys rendered parkinsonian with MPTP (Brotchie et al., 1991; Graham et al., 1990). A similar effect was observed in the unilat­ eral MPTP model of parkinsonian monkey, in which a unilateral GPi injection of MK801, an NMDA receptor antagonist, induced a contralat­ eral circling behaviour similar to that induced by DA agonists (Levy et al., 1997).

Pharmacological inactivation is most often per­ formed by means of infusions of the GABAA receptor agonist muscimol into the given cerebral structure. It was shown that a focal inactivation of the GPi with muscimol infusions impaired grasp­ ing and reaching, affecting velocity (Wenger et al., 1999). These results supported the hypothesis that GPi inhibition disrupts its ability to inhibit com­ peting motor mechanisms and to prevent them from interfering with desired voluntary move­ ment. In a later study where they tested the effects of muscimol infused into various selective areas of the GPi, Baron and colleagues showed that akinesia and bradykinesia induced by MPTP could be alleviated when muscimol was infused into the centromedial part of the sensorimotor GPi (Baron et al., 2002). The same study showed that inactivation of GPi areas outside of the motor territories did not improve parkinsonism but induced circling and behavioural abnormalities. Only a few studies reported effects of a GPi lesion on behaviour in intact monkeys. One of these studies using kainic acid lesion revealed motor deficits in arm movement performance (Horak and Anderson, 1984), while a study using kynurenic acid (a broad spectrum excitatory amino acid antagonist) showed dyskinesia (Robertson et al., 1989). In the parkinsonian monkey, recent studies using a chemical lesion of GPi confirmed the beneficial effects of GPi inactivation on motor activity and parkinsonian scores (Lieberman et al., 1999; Lonser et al., 1999). Since, according to the model of the BG (DeLong, 1990), dyskinesias were considered as the result of a decreased inhibitory influence from the GPi to the motor thalamus, it was surprising to find that GPi inactivation could have beneficial effects by reducing L-DOPA-induced dyskinesia. In the marmoset rendered parkinsonian with MPTP, it was indeed shown that a unilateral elec­ trolytic lesion of the GPi could reduce the L-DOPA­ induced dyskinesias (Iravani et al., 2005). There are numerous clinical studies dedicated to the beneficial effect of pallidotomy on L-DOPA-induced dyskine­ sia in parkinsonian patients (Alkhani and Lozano,

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2001; Dogali et al., 1994; Laitinen et al., 1992; Vitek et al., 2003). It is interesting to note that, as suggested by the pathophysiological model of the BG proposed by DeLong (DeLong, 1990), both STN and GPi were possible interesting targets for the treatment of PD. In contrast, GPe inactivation was not pre­ dicted to have any beneficial effect. Indeed, according to this model of the BG (Fig. 1), inacti­ vating the GPe would result in an enhancement of the STN hyper-activity and should thus worsen a parkinsonian state. This was indeed confirmed by a study showing that a lesion of the GPe in MPTP­ lesioned monkeys worsened their motor symp­ toms (Zhang et al., 2006).

GPi HFS in the monkey HFS has been widely applied into the GPi of PD patients but, surprisingly, there are not many ani­ mal studies supporting this therapeutical approach. One pioneering work has shown that unilateral HFS of the GPi could improve parkin­ sonian symptoms such as muscular rigidity and akinesia in unilateral MPTP-lesioned monkeys (Boraud et al., 1996). Although it has been shown that GPi DBS applied in PD patients was efficient for the treatment of L-DOPA-induced dyskinesia (Benabid, 2003; Wichmann and DeLong, 2006), there is no published study to date showing this effect in monkeys.

Lesion and pharmacological EP manipulation in the rat In the reserpinized model, injection of glutamater­ gic antagonists into the EP restores locomotor activity (Brotchie et al., 1991). In the same model, or in alpha-MPT model, NMDA antago­ nists injected into the EP also alleviate muscular rigidity (Klockgether and Turski, 1990). In unilat­ eral DA-depleted rats, lesioning the EP decreased the rotations induced by amphetamine (Olds

et al., 2001, 2003) or L-DOPA (Honey and Shen, 1998). This latter result is in contradiction with another study that has shown that EP lesion was unable to correct the circling behaviour induced by L-DOPA in unilateral DA-depleted rats (Perier et al., 2003). On the cataleptic state induced by haloperidol, it was shown that a bilateral excito­ toxic lesion of the EP had a beneficial effect (Zadow and Schmidt, 1994). In the intact rat first, we have shown that bilat­ eral infusions of the NMDA antagonist DL-2­ amino-5-phosphonopentanoic acid (AP-5) into the EP could induce an akinetic-like deficit asso­ ciated with a premature-responding deficit in a simple reaction-time (SRT) task (Baunez and Amalric, 1996). In order to measure the effects of intra-EP bilateral infusions of AP-5 in a rat model of early parkinsonism, we have used the same SRT task allowing a subtle measure of reac­ tion time (RT) (see Fig. 2). In this task, the rats are trained to press a lever down and sustain their paw on the lever until the occurrence of a light, at which they have to release the lever quickly to obtain a food pellet. The RT is the time taken to withdraw the paw from the lever after the onset of the light. Parkinsonian patients suffering from aki­ nesia are known to exhibit increased RT in these tasks. After a bilateral infusion of 6-OHDA into the dorsal striatum, the rats’ performance is impaired in terms of correct responses, mainly because of increased RT, resulting in an increased number of delayed responses (non-rewarded responses for which the RT exceeded 600 ms) (Baunez et al., 1995). In this model of rat parkin­ sonism, we have shown that the same bilateral infusion of AP-5 into the EP alleviates akineticlike behaviour in the SRT task in the rat, by reducing the number of delayed responses (Baunez and Amalric, unpublished).

EP HFS data in rodents Probably because of its small size in the rat, the EP has been rarely specifically targeted for

240 Reward

Light Correct Lever up Down

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RT limit = 600 ms Fig. 2. The simple reaction-time task used in the rat. The rats are trained to press a lever down and sustain their paw on it until the occurrence of a visual stimulus (a light) that may happen at either 500, 750, 1000 or 1250 ms. At the presentation of the light, the rat have to withdraw their paw from the lever as quickly as possible (i.e. reaction time, which has to be below 600 ms) to get a food pellet as a reward. Three types of responses are possible: (1) correct, (2) premature responses when the rat withdraws its paw from the lever before the presentation of the light and (3) delayed responses when the rat withdraw its paw from the lever after the presentation of the light, but with a reaction time exceeding 600 ms.

behavioural studies on EP HFS effects. Only one micro-dialysis study has reported that EP HFS increases DA levels in the striatum concomitantly with DAergic drugs administration (Meissner et al., 2004). It was also shown that EP HFS reduces the number of dystonic attacks in the dystonic dtsz hamster (Harnack et al., 2004).

Effects of manipulation of the GPi on cognition Unfortunately, no study testing the effects of GPi/ EP manipulation on cognitive functions has been published to date, either in monkeys or in rats. Given the clinical reports after pallidotomy or GPi DBS, it would be very interesting to investigate further attentional and executive functions as well as motivation.

This first part dedicated to the GPi revealed that a large body of evidence supported the ben­ eficial effect of pallidotomy or GPi HFS for the treatment of motor deficits in parkinsonism. How­ ever, our review of the literature revealed as well a serious lack in investigations of the non-motor functions. Although clinical application of palli­ dotomy or GPi DBS in the treatment of PD seems to induce modest cognitive side-effects (Rettig et al., 2000; Scott et al., 2002; Trepanier et al., 1998), there are reports of mood changes and weight gain that might be related to the direct consequence of GPi manipulation (Dalvi et al., 1999; Fukuda et al., 2000; Okun et al., 2003, 2009; Ondo et al., 2000). It would therefore be useful to investigate further these observations in animal models.

STN manipulation in PD The STN belongs to the indirect pathway of the BG, as well as to the so-called hyper-direct path­ way from the cortex to the BG output structure through the STN itself. STN is a glutamatergic structure innervating mainly the GPi/EP and the SNr, but also the GPe, the ventral pallidum, the pedunculopontine nucleus, and to a lesser extent the striatum and nucleus accumbens, and also the DAergic nuclei (ventral tegmental area and SNc). The major inputs to the STN arise from various cortical areas (i.e. the hyper-direct pathway), the ventral pallidum and the GPe (the indirect path­ way), the parafascicular nucleus of the thalamus, the pedunculopontine nucleus, the dorsal raphe, the ventral tegmental area and the SNc (Parent and Hazrati, 1995a, 1995b). Recent evidence for a direct STN-cortex loop circuit has also been pro­ vided (Degos et al., 2009). STN neurons are spon­ taneously active both in vitro and in vivo, and fire action potentials at a frequency ranging from <10 Hz up to 20–25 Hz at membrane potentials around –50 to –60 mV, reaching 300–500 Hz at more depolarized potentials. Approximately half of STN neurons have a tonic firing activity, also

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called single-spike mode. The other half switch from tonic to burst-like firing pattern, or ‘burst’ mode, when hyper-polarized. To note that at hyper­ polarized membrane potentials (–60 to –70 mV) most STN neurons become silent (Beurrier et al., 1999, 2000; Bevan and Wilson, 1999; Nakanishi et al., 1987; Overton and Greenfield, 1995). These spontaneous firing activities result from phases of cyclic and alternate activation/inactivation of depo­ larizing and hyper-polarizing currents, with a con­ tribution of pallidal GABAergic inputs (Beurrier et al., 1999; Bevan et al., 2002). In animal models of PD, a general increase in spike frequency and a shift to a more bursting pattern have been observed in vivo in STN neu­ rons, both in 6-OHDA-lesioned rats (Hassani et al., 1996; Hollerman and Grace, 1992; Kreiss et al., 1997; Ni et al., 2001; Tai et al., 2003; Vila et al., 2000) and in MPTP-treated monkeys; in the latter, a low-frequency oscillatory activity in the b band – that in humans is highly correlated with tremor – has also been detected (Bergman et al., 1994, 1998; Bezard et al., 1999; Meissner et al., 2005). Interestingly, the suppression of such oscil­ latory b activity by STN HFS in parkinsonian patients correlates with the improvement of motor performance (Kuhn et al., 2008).

Neurophysiological effects Electrophysiology Electrophysiological studies performed in vitro in brain slices of naïve rats have shown that STN HFS decreases and even blocks firing activity of STN neurons (Beurrier et al., 2001) or induces an initial increase in action potential discharge fol­ lowed by a longer-lasting inhibition (Lee et al., 2003; Magarinos-Ascone et al., 2002). Successive work in slices from reserpine-treated mice showed that spontaneous STN neuron discharge was com­ pletely replaced by a stimulation-driven one (mediated by Naþ and L-type Ca2þ channels) at the same frequency of stimulation up to 130 Hz

(Garcia et al., 2003). However, it should be con­ sidered that the stimulation parameters (pulse duration and intensity) used in these slice studies were adjusted to obtain an electrophysiological response, rather than to be relevant to those used in clinical treatment. Overall, these works support the concept that STN HFS can disrupt the abnormal low-frequency oscillations of STN neurons triggered by DA depletion by imposing a stimulation-driven pattern of spike activity. We have shown that, in brain slices of 6-OHDA-treated rats, spontaneous glutamate activity recorded from striatal medium spiny neu­ rons was significantly increased (Gubellini et al., 2002), and that 5 days of STN HFS (applied using clinically relevant parameters) could completely reverse these changes and even reduce such activ­ ity below control levels (Gubellini et al., 2006). Interestingly, striatal glutamatergic hyper-activity induced by 6-OHDA lesion is also reversed by STN lesions (Centonze et al., 2005), suggesting that similar mechanisms might underlie the synap­ tic effects of STN lesion and STN HFS. In MPTP-treated monkeys, STN HFS has been shown to inhibit the mean firing rate of STN neu­ rons and, in parallel, to reduce their low-fre­ quency oscillatory activity (Meissner et al., 2005). STN HFS also evoked spikes in these cells, which were not time-locked to the electrical stimulus, as observed in vitro. Concerning the pallidal com­ plex, STN HFS in MPTP-treated monkeys chan­ ged the spontaneous irregular firing pattern of both GPe and GPi into a high-frequency and reg­ ular pattern (Hahn et al., 2008; Hashimoto et al., 2003). In opposition to these findings, it has been shown that STN HFS regularized and reduced neuronal firing activity in the motor thalamus (Dorval et al., 2008; Xu et al., 2008), suggesting that STN HFS could increase STN output and thus produce inhibitory changes in the thalamus. Electrophysiological studies performed in vivo in 6-OHDA-treated rats have shown that STN DBS, in general, dramatically reduced the firing activity of the majority of neurons of the STN (Shi et al., 2006; Tai et al., 2003), the SNr (Benazzouz

242

et al., 2000; Tai et al., 2003) and the pedunculo­ pontine nucleus (Florio et al., 2007) and had little effect in the GP (Shi et al., 2006). Concerning the SNr, another study in rats treated with antagonists of DA receptors showed that STN HFS regular­ ized the firing pattern of SNr neurons and normal­ ized their response to cortical stimulation, suggesting that the stimulation restored the bal­ ance between inhibitory and excitatory influences on this structure (Degos et al., 2005). Another brain target examined for STN HFS effects in 6-OHDA-treated rats is the dorsal raphe nucleus (DRN), a midbrain structure providing extensive 5-hydroxytryptamine (5-HT) innervation to the limbic forebrain. In parkinsonian rats, the basal firing of 5-HT neurons was increased, and STN HFS reduced it by more than 50%, providing support for a functional link between STN and DRN neurons (Temel et al., 2007). Regarding the cerebral cortex, STN HFS has been shown to activate antidromically the neurons of layer V/VI and dampen the cortical slow-wave oscillations (recorded by EEG and local field potentials from rats under deep anaesthesia), possibly by activat­ ing local excitatory and inhibitory cortical net­ works. Intracellular recordings showed that a small group (~16%) of layer V/VI neurons responded to STN HFS with an antidromic spike, whose frequency reflected that of DBS and with a latency of ~2 ms, while the remaining neurons responded with a reduction of membrane poten­ tial fluctuations (Li et al., 2007). These findings support the idea that cerebral cortex is involved in the mechanisms of action of STN HFS, as pro­ posed by several studies in patients showing that this treatment produces evoked potentials in the frontal cortex (Ashby et al., 2001; Baker et al., 2002) and that direct stimulation of the motor cortex alleviates parkinsonian symptoms in both primates and humans (Drouot et al., 2004; Lefau­ cheur et al., 2004). Antidromic activation of the cortex has also been reported in awake cataleptic rats during STN HFS (Dejean et al., 2009). Besides antidromic mechanism, however, STN HFS could act at cortical level also by the recently

described direct subthalamocortical projection (Degos et al., 2009).

Molecular biology and metabolism Molecular studies in 6-OHDA-lesioned rats have shown that STN HFS (2–4 h) induced the expres­ sion of the transcription factors c-fos, c-jun and Krox-24 (Schulte et al., 2006) in STN neurons and, at the same time, reduced the expression of cytochrome oxidase subunit I (COI) mRNA that normally is increased by DA depletion (Salin et al., 2002). Decrease of COI mRNA expression was also observed in the SNr after its increase triggered by DA lesion. Such reduction of COI mRNA in the STN and SNr after STN HFS is consistent with a reduction or normalization of neuron firing rate. Interestingly, COI mRNA levels in the cortex (layer V neurons), which were reduced by 6-OHDA lesion, could be nor­ malized by STN HFS (Oueslati et al., 2007), further supporting an effect of STN manipulation at cortical level. Another marker of neuronal activity, glutamic acid decarboxylase (GAD) mRNA, was decreased in the EP, GP and SNr by prolonged (4 days) STN HFS, suggesting a reduced glutamatergic input from the STN to these GABAergic structures (Bacci et al., 2004; Benazzouz et al., 2004; Salin et al., 2002; Tai et al., 2003). Conversely, 10 days STN HFS in MPTP-treated monkeys resulted in an increased COI expression in the GPi, suggesting that longer period of STN stimulation would, on the contrary, increase GPi activity (Meissner et al., 2007). On the other hand, micro-dialysis experiments showed that STN HFS increased extracellular GABA in the SNr, which could arise from the concomitant stimulation of pallido-nigral fibres (Windels et al., 2005), suggesting a potential role of GABA originating from the GP in the inhibi­ tion of BG output structures during STN stimulation. The metabolic effects of STN HFS have been studied by measuring 2-deoxyglucose (2-DG)

243

uptake in MPTP monkeys (Meissner et al., 2007). Such DA lesion induced a decrease of 2-DG accu­ mulation in the STN that was reversed by 10 days STN HFS. Despite the significance of 2-DG uptake is still not clear in terms of excitatory or inhibitory influence and cellular elements involved, this study concluded that STN HFS could normalize the abnormal responses of BG structures to DA lesion resulting in STN hyper­ activity.

Neuroprotective effects STN HFS, applied 1 h per day, starting a week after 6-OHDA injection and during a period of 3 months, has been shown to enhance the survival of midbrain DAergic neurons in 6-OHDA-treated rats (Temel et al., 2006), and a similar work showed that continuous STN HFS (for 2 weeks and initiated 5 days after 6-OHDA lesion) pre­ served 30% of nigral neurons expressing tyrosine hydroxylase (Harnack et al., 2008). Another study also indicated that STN HFS in MPTP-treated monkeys provided about 20% neuroprotection to DAergic cells (Wallace et al., 2007). Thus, although clinical findings reported that STN HFS failed to improve DA outflow in PD patients or increase the survival of DAergic cells (Hilker et al., 2003; Thobois et al., 2003), most of the studies in animal models with partial DA lesion are in agreement with an activation/preservation of the DAergic system by STN HFS. However, this effect is unlikely to participate to the thera­ peutic action in late stages of PD, when patients usually undergo HFS, due to the already extensive loss of DAergic neurons. HFS of the STN is nowadays the main surgical treatment for PD, and thus it has received high attention by researchers. Overall, experimental data in PD models indicate that, while the activity of STN itself seems to be reduced by HFS, still the consequences of this treatment are much more complex than inhibition and, most importantly, they are widespread – directly or indirectly – to

the other BG structures, to the thalamus and to the cerebral cortex (Gubellini et al., 2009). In vitro electrophysiological studies show that STN HFS interferes with the pacemaker-like activity of STN neurons resulting in short-term inhibition of firing discharge and, at long term, in the replacement of spontaneous firing activity by a stimulus-driven one. These evidences suggest that STN HFS can disrupt the abnormal synchronized oscillatory activity of the subcortical–cortical loops in parkin­ sonian state, thus restoring a more physiological functioning of these structures and improving motor symptoms.

Effects of manipulation of the STN on motor behaviour Lesion, pharmacological and molecular STN inactivation in monkeys STN lesions in intact monkeys were first reported to induce a characteristic transient hyper-kinetic syndrome called ‘ballism’ or ‘hemiballism’ (Whit­ tier and Mettler, 1949). The first paper showing anti-parkinsonian effects of STN lesions in MPTP monkeys was published by Bergman and colla­ borators (Bergman et al., 1990), who showed that serious motor impairments induced by MPTP could be alleviated by STN lesions. The study was performed by means of general obser­ vation of gross motor behaviour, with no measure of controlled operant responses. This pioneer study was confirmed later (Aziz et al., 1991). In line with these reports, it was also shown that subthalamotomy performed in MPTP monkeys had a beneficial effect on certain motor deficits, but could also be detrimental by inducing hyper­ kinetic movements and hemiballism (Guridi et al., 1994, 1996; Wichmann et al., 1994). In the hemiparkinsonian marmoset, it was also shown that unilateral STN lesion reversed the bias in head position and decreased latencies to initiate reaching on the contralateral side in the staircase grasping task. However, slight deficits in skilled

244

movements persisted (Henderson et al., 1998). Akinesia and bradykinesia were strongly amelio­ rated by discrete inactivation of the lateral part of the sensorimotor territory of STN performed with muscimol infusions (Baron et al., 2002). More recently, another way of reducing STN activity in hemiparkinsonian monkeys has been developed using transfection with an adeno-asso­ ciated virus containing the gene for GAD. Chan­ ging the glutamatergic phenotype into GABA of STN neurons allowed motor recovery into a cer­ tain extent and was thus considered as beneficial for the treatment of PD (Emborg et al., 2007). All these beneficial effects of STN inactivation in parkinsonian monkeys are in line with the report showing that pharmacological blockade of STN by lidocaine or muscimol improves bradyki­ nesia, limb tremor and rigidity in parkinsonian patients (Levy et al., 2001).

STN HFS in monkeys Benazzouz and colleagues were the first to show that unilateral STN HFS applied in monkeys ren­ dered hemiparkinsonian with MPTP alleviated the muscular rigidity observed in the contralateral forelimb (Benazzouz et al., 1993). This pioneer work was actually at the origin of the idea to apply STN HFS in PD patients. In the intact mon­ key, it was also shown that STN HFS could induce hyper-kinetic movements similar to the hemibal­ lism observed after STN lesions (Beurrier et al., 1997). In contrast to what was described after STN lesions, STN HFS does not seem to induce hyper­ kinetic movements when applied to MPTP mon­ keys and when compared to L-DOPA effects (Benazzouz et al., 1996).

Lesion, pharmacological and molecular STN inactivation in rats In intact rats, unilateral lesion of the STN only produces transient hyper-kinetic movements of

the contralateral paw. This behaviour has been quantified by measuring spontaneous circling behaviour (Kafetzopoulos and Papadopoulos, 1983). When the lesion is bilateral, this beha­ vioural effect was rarely described. Only a trend to hyper-locomotion has been reported, as well as premature responses in the RT procedure illu­ strated in Fig. 2 (Baunez et al., 1995). In rat models of PD, it was first shown that STN lesion alleviated the cataleptic state induced by a high dose of haloperidol (Zadow and Schmidt, 1994). When performed unilaterally, STN lesion can reduce circling behaviour induced by either a DA D2 receptor agonist or apomorphine in hemi­ parkinsonian rats (Anderson et al., 1992; Blandini et al., 1997; Burbaud et al., 1995). These were the first studies showing that STN lesion had a bene­ ficial effect in alleviating gross motor deficits induced by DArgic depletion. In line with these beneficial effects of STN lesion on these types of motor behaviour, it was also shown that unilateral STN lesions could alleviate postural asymmetry induced by unilateral DA depletion (Phillips et al., 1998). In order to measure the effects of bilateral STN lesions in a rat model of early PD, we have tested their effects in parkinsonian rats performing the SRT task described above. As shown in Fig. 3, bilateral lesions of the DA terminals in the dorsal striatum increased the number of delayed responses, as well as the mean RT for correct responses, characterizing an akinetic-like pattern of performance. Consecutive bilateral lesions of the STN alleviated this akinetic-like deficit, but the rats maintained a poor level of performance in the SRT task due to the appearance of a pre­ mature-responding deficit (Baunez et al., 1995). Although this study confirmed the beneficial effect of STN inactivation on motor disabilities in PD, it also revealed for the first time possible sideeffects that might be related to the involvement of STN in non-motor behaviour. These results were confirmed by a similar study carried out with uni­ lateral STN lesion (Phillips and Brown, 1999). In another study, it was also confirmed that STN

245 100

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Mean number of responses/session

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10 0 Mean RT (ms)

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300

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Pre Post Post + STN

Fig. 3. Effects of STN lesions in a rat model of parkinsonism on the performance in the SRT task (Baunez et al., 1995). The performance is illustrated in terms of number of correct responses/100 trial session before surgery (Pre), after 6-OHDA lesion (Post) and after STN lesion consecutive to 6-OHDA lesion (postþSTN). The dopaminergic depletion of the dorsal striatum induced an akinetic-like deficit characterized by an increased number of delayed responses (responses with a RT above 600 ms) and an overall increased RT for correct responses. Performing a bilateral lesion of the STN in these animals alleviated these two major deficits, but affected further the performance in terms of correct responses because of a dramatic premature-responding deficit.
,

, significantly different from pre-operative performance; ¥,¥¥: significantly different from post-operative performance (6-OHDA lesion effect), p < 0.05 and 0.0,1 respectively.

lesion alleviates some of the deficits induced by DA depletion, but induces side-effects and is unable to correct some deficits such as a paw reaching deficit assessed with a stair case (Hen­ derson et al., 1999). Other means of STN inactivation have been investigated for anti-parkinsonian therapy, nota­ bly addressing GABAergic transmission. The clas­ sic GABA agonist muscimol was shown to reduce circling behaviour induced by apomorphine and limb-use asymmetry in hemiparkinsonian rats (Mehta and Chesselet, 2005). The therapy by GAD gene transfection in the STN led to motor improvement in parkinsonian rats (Luo et al., 2002), so did GABAergic cell grafts into the STN (Mukhida et al., 2008). Some of the beneficial effects observed after inactivation of the STN might be mediated via a specific system such as the 5-HT system. Indeed, the STN receives an important 5-HT innervation from the dorsal raphe (Parent and Hazrati, 1995b) and therefore affecting this transmission may result in behavioural changes, as those described after STN inactivation. It has been recently shown that targeting specifically 5-HT1A receptors into the STN could alleviate L-DOPA-induced dyski­ nesia (Marin et al., 2009), confirming the possible critical influence of the 5-HT innervation to the STN in the functioning of the BG.

STN HFS in rats The first study published on STN HFS in freely moving rats performing behavioural tasks used unilateral stimulation as well as unilateral SNc lesion. In this work we assessed both basic motor tasks such as haloperidol-induced catalepsy, apomorphine-induced circling behaviour, as well as a choice RT task (Darbaky et al., 2003). The parameters were set at 130 Hz, 60–70 ms pulse width and intensity set just below the threshold of hyper­ kinetic movements of the contralateral paw. We

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showed that both the cataleptic state induced by haloperidol and the circling behaviour induced by apomorphine in unilateral DA-depleted rats could be alleviated by unilateral STN HFS. However, in a choice RT task, only a few animals remained able to perform the task after the DA depletion and the STN HFS did not help the severely impaired animals. Thus, in contrast to the specta­ cular effect of STN HFS in PD patients, the sti­ mulation applied in the rat could not overcome the profound deficit preventing the animals to perform the task. Interestingly, however, for those able to perform the task, STN HFS alle­ viated the deficit expressed as a decreased ability to initiate a response towards the side contralat­ eral to the DA lesion (Darbaky et al., 2003). Our conclusion was that STN HFS could be beneficial for the treatment of motor deficit, but non-effi­ cient when the cognitive load was higher, leading to further cognitive studies that will be developed in the next paragraph. Later the same year, another group showed that STN HFS had a ben­ eficial effect on treadmill walking in parkinsonian rats (Chang et al., 2003) and reduced asymmetry when STN HFS was applied in hemiparkinsonian rats (Shi et al., 2004). We also showed that STN HFS could restore the use of the contralateral paw that was impaired after unilateral 6-OHDA lesion, but was not efficient to alleviate L-DOPA-induced dyskinesia (Gubellini et al., 2006), in line with a bilateral STN lesion study (Marin et al., 2004) and, possibly, because of the well-known effect of STN HFS itself in inducing dyskinesia (Boulet et al., 2006). When applied to intact rats, unilateral STN HFS induces contralateral circling behaviour that can be reduced by DA receptor antagonists (Bergmann et al., 2004). The first study testing the effects of bilateral STN HFS was carried out in intact rats performing a RT task. STN HFS in that study decreased the premature responses depending on the stimula­ tion parameters applied (Desbonnet et al., 2004). The same group confirmed such effect on prema­ ture responses at different parameters than those alleviating RT deficits in parkinsonian rats (Temel

et al., 2005) and also showed improvement on locomotion (Vlamings et al., 2007). On many aspects of motor behaviour, there is consensus around a beneficial effect of STN HFS on parkinsonian motor deficits, although this treat­ ment is not applied always in the same manner (unilateral vs. bilateral, monopolar vs. bipolar elec­ trodes, individual adjusted parameters or not). How­ ever, the question of a possible detrimental effect, or at least a lack of effect on cognitive processes, has been raised by several studies and needs to be further investigated. The evidences gained from ani­ mal models (Darbaky et al., 2003; Temel et al., 2005) seem thus to confirm that STN HFS at parameters inducing beneficial effects on motor functions does not always correlate with beneficial cognitive effects, as reported in human patients (Perriol et al., 2006). Effects of manipulation of the STN on cognition and motivation When considering cortico-BG-thalamocortical connectivity as comprising five parallel loops (Alexander et al., 1986) (reviewed above), it becomes apparent that both GPi and STN are involved in each loop, including the associative and the limbic ones. These structures should not therefore be considered as contributing to motor behaviour only. Indeed, as illustrated in Fig. 4, the STN receives direct connections from the prefron­ tal cortex. Therefore, manipulation of the STN should have consequences on frontal functions, as much as it has on motor processes. The STN is also connected more or less directly with struc­ tures such as the nucleus accumbens and the ven­ tral pallidum, well-known for their involvement in motivational processes. These anatomical consid­ erations lead us to investigate the involvement of the STN in non-motor behaviour. STN lesion or STN HFS in monkeys Only a limited number of groups study the effects of STN HFS in animals and none have published

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Prefrontal Cx (cingulate,orbitofrontal)

n. accumbens GLU

DA

VP GABA STN

VTA

Basal ganglia outputs

Fig. 4. The STN in the limbic loop. The STN receives direct inputs from the prefrontal cortex and indirectly connected with the nucleus accumbens via the ventral pallidum (VP). It receives inputs from the DA nuclei: ventral tegmental area (VTA) and substantia nigra pars compacta.

yet any study investigating its possible effects on cognitive processes in monkeys. The number of investigations focusing on cognitive processes in patients has increased in the recent years and might explain why there is little interest for these studies applied to non-human primates. However, it has been shown that STN neurons respond to reward (Darbaky et al., 2005), suggesting that STN manipulations may affect motivation.

STN lesion in rats There are only a few studies dedicated to the involvement of STN in learning and memory pro­ cesses. It has been shown that STN lesion does not seriously affect learning processes, but can affect working memory (El Massioui et al., 2007), in line with a former study showing working memory deficits in a choice RT task (Baunez et al., 2001). In our study using a SRT task in 1995, we had suggested that premature responses could reflect an attentional impairment (Baunez et al., 1995). We have used the ‘5-choice serial RT task’ in which the animals are trained to wait and detect a brief visual stimulus that can be presented in five

possible various locations. The animals have to divide their attention between these five possible choices and then go and respond by a nose poke in the appropriate location to obtain a food reward in a food magazine and then initiate the next trial (see Fig. 5). Using this specific visual attentional task, we have studied the effects of STN lesions first, and then of STN lesions combined with a bilateral DA depletion in the dorsal striatum. We first showed that bilateral excitotoxic lesions of the STN-induced multiple independent deficits in the task, such as impaired accuracy suggestive of an attentional deficit; an increased level of prema­ ture responses suggestive of increased impulsivity; an increased level of perseverative responses towards the response locations and the magazine where the animals collect the food reward, sugges­ tive of deficit in response control and an increased level of motivation for the reward (Baunez and Robbins, 1997). These results were the first to highlight the involvement of STN in cognitive functions. These results were replicated after blockade of the GABA receptors into the STN with muscimol (Baunez and Robbins, 1999b). When lesioning the DA inputs to the dorsal stria­ tum, we did not affect dramatically the level of performance in the attentional task: although there was a slight impairment in visual attention, most of the deficits were more motor related (omis­ sions, increased latencies). Interestingly, when com­ bining this lesion with STN lesions, the performance was further impaired. One of the most striking effects was observed on perseverative responses towards the food magazine, suggesting an increased level of motivation for the reward (Baunez and Robbins, 1999a). In a study using a disconnection between the medial prefrontal cortex and the STN, by lesioning the prefrontal cortex on one side and the STN on the other side, we have given the first evidence of a functional role for the hyper-direct pathway in the attentional and perseverative defi­ cits observed in this attentional task (Chudasama et al., 2003). Further studies have confirmed the role of STN in impulse control. It was indeed shown that STN lesions prevent the animals to be

248 5-choice task

Hole

Food magazine Start of a trial Stimulus (rat pushes the (in one of the 5 holes) panel of the magazine)

5 sec.

Correct response

Reward

Incorrect response Omission

Timeout

Premature

response

Fig. 5. The 5-choice serial reaction-time task (5-CSRTT): The rats initiate a trial by a nose poke in the food magazine. After a 5 s delay, a brief light (500 ms) is presented in one of the five holes. The rats have to detect and respond by a nose poke in the illuminated hole within 5 s to obtain a reward, collect it in the magazine and then start the next trial. In case of an early response in a hole before the presentation of the light, the response is recorded as a premature response and punished by a time-out (extinction of the houselight). The same punishment occurs if the rats respond in the wrong hole (incorrect response) or do not respond within 5 s (omission). After the first response has been given, additional nose pokes in the various holes are recorded as ‘perseverative responses’. Detection of the rats’ nose in the food magazine other than the first one after reward delivery are recorded as ‘perseverative panel pushes’ and characterize inappropriate visits to the magazine.

able to stop an ongoing action in a stop-signal RT task (Eagle et al., 2008). However, when tested in a behavioural task where the animals are given the choice between a small but immediate reward and a large but delayed reward, the STN-lesioned animals were able to overcome their impulsivity and wait for a bigger reward (Winstanley et al., 2005). This latter result was confirmed by another group (Uslaner and Robinson, 2006). These results suggest a specific role of STN in the control of inhibition that can be under the influence of the outcome (Eagle and Baunez, 2010).

STN HFS data in rats We have previously developed the idea that a premature response in a RT task may reflect

some cognitive deficit that relates to either an attentional deficit or a deficit in inhibition control. DAergic depletion of the dorsal striatum can sometime induce an increased number of prema­ ture responses (Turle-Lorenzo et al., 2006). Temel and colleagues also reported this type of deficit in parkinsonian rats performing a choice RT task, together with increased RT and movement time (MT) (Temel et al., 2005). Interestingly, they have shown that bilateral STN HFS could alleviate the premature-responding deficit at lower current intensity (3 mA) than that reducing RT and MT (30 mA). As mentioned above, this study provides the evidence that cognitive and motor deficits may require a different threshold of HFS to be treated. In intact and parkinsonian rats, we have tested the effects of bilateral STN HFS and could therefore compare them to those induced by bilateral

249

6-OHDA 6-OHDA-HFS Premature responses 20

(a)

Mean number of responses/session

15 10

#

##

##

##

Post

stim1

5 0 Pre

stim2 stim OFF

Perseverative panel pushes 120 (b) 100

80

60 40 20 0 Pre

££ ## ** $$

££ ## * $$ # #

#

Post

stim1

stim2

stim OFF

Fig. 6. Effects of bilateral high-frequency stimulation (HFS) of the STN in the 5-CSRTT (see Fig. 5) applied in 6-OHDA-lesioned rats (taken from Baunez et al., 2007). The performance in the 5-CSRTT is illustrated here for premature responses and perseverative responses into the food magazine (panel pushes) in the 6-OHDA-lesioned animals remaining OFF STN HFS (grey) and 6-OHDA­ lesioned animals subjected to STN HFS (black) at the different stages of the experiment: during a block of 6 sessions before surgery (Pre), during a block of 6 sessions after surgery without stimulation (Post), during the first block of 6 sessions under STN HFS (stim 1), during the second block of 6 sessions under STN HFS (stim 2) and during a block of 6 sessions during which the stimulation was turned OFF (stim OFF). Vertical bar: SEM.
,

: p < 0.05 and p < 0.01, respectively, compared with sham group. #, ##: p < 0.05 and p < 0.01, respectively, compared with pre-operative performance. $,$$: p < 0.05 and p < 0.01, respectively, compared with 6-OHDA group. : p < 0.01 compared with post-operative performance.

excitotoxic STN lesions in the visual attentional task described above. For both intact and parkin­ sonian animals, the effects of STN HFS were slightly different to those induced by STN lesions (Baunez et al., 2007). Accuracy of performance as well as latency to make a correct response was only transiently affected, while no effect on pre­ mature responses could be seen. Interestingly, the perseverative responses on both response location and reward magazine were found, in line with the lesion study. In parkinsonian rats, the subtle defi­ cits recorded in the 5-choices RT task were neither further deteriorated by bilateral STN HFS nor alleviated. The most striking effect was observed

on the perseverative responses recorded in the food magazine, suggesting that STN HFS increases motivation for the food reward (Fig. 6) (Baunez et al., 2007). These results are in line with recent studies focusing on the role of STN in motivational pro­ cesses and suggest that inactivating the STN in parkinsonian animals should affect their motiva­ tional state. We have first shown that bilateral STN lesion does not increase hunger or affect primary pro­ cesses of motivation whatever the internal state of the animals (deprived or sated) or the reward (standard animal food, palatable food, alcohol or

250

i.v. injection of cocaine). STN lesion does not affect these consummatory processes (Baunez et al., 2002, 2005; Lardeux and Baunez, 2008). When assessing motivation by measures of reac­ tivity to stimuli predicting food, we found that STN lesions increase responses to these stimuli (Baunez et al., 2002). This result was further con­ firmed by another group (Uslaner et al., 2008). We also showed that STN lesion increases will­ ingness to work on a lever to obtain food pellets and increases the score of preference for an envir­ onment previously associated with food. In con­ trast to these results, we found the opposite effects when the reward was cocaine, highlighting a pos­ sible role for STN to modulate the reactivity of the reward system with regard to the nature of the reward involved (Baunez et al., 2005). When test­ ing the effects of bilateral STN lesion on motiva­ tion for alcohol, we have further shown that it could also affect motivation in an opposite manner depending on the initial preference of the animals for the reward (Lardeux and Baunez, 2008). Very recently, we have shown that bilateral STN HFS reduces motivation for cocaine, while increasing that for food (Rouaud et al., 2010), in line with the results described after bilateral STN lesion (Bau­ nez et al., 2005). Furthermore, electrophysiologi­ cal recording of STN neurons in rats revealed that they can encode the value of the reward (Lardeux et al., 2009). It was shown that STN neurons can be categorized into sub-populations responding differently to reward. One sub-population responded exclusively to a cue predicting a 4% sucrose solution, but did not respond to the cue predicting the other reward (32% sucrose solu­ tion). The other sub-population responded to the cue predicting 32% sucrose, but not to the cue predicting 4%. In another study, we further showed that this dissociation also is observed when sucrose and cocaine are the two different rewards (Lardeux et al., 2008). Whether or not this encoding of the value of reward is dependent on the integrity of the DA system and could there­ fore be different in a rat model of PD remains to be elucidated.

Although there are no data available about the effects of STN manipulation on motivation in ani­ mal models of PD, these results that we have obtained in intact rats are in line with some clinical observations in PD patients after STN DBS, reporting craving for sweet food in some cases, or decreased addictive behaviour towards DAer­ gic treatment (Knobel et al., 2008; Lim et al., 2009; Witjas et al., 2005). In conclusion for this section on the STN, it has been shown that most of the effects observed were in line with a beneficial effect of STN inactivation for the treatment of motor symptoms in PD. The studies in rats have raised the issue of non-motor involvement of STN and lead to a better consid­ eration of these aspects in clinical studies and patients’ management: the current interest for motivational and emotional effects of STN DBS in PD patients reflects also the recent interest for these processes in animal models.

General conclusion In conclusion, this review of the literature leads to the following comments: At the cellular level, electrical stimulation of the GPi and the STN has a profound effect on the firing activity of their neurons. Rather than a mere inhibition of action potential discharge, HFS time-locks the activity of STN neurons at frequencies correlated to those of HFS. On the other hand, GPi stimulation seems also to exert an overall inhibitory effect. At neurophysiological level, it is now clear that the action of STN HFS spreads to surrounding brain structures that are directly or indirectly connected to this nucleus: the cortex, the striatum and other BG nuclei. Simi­ larly, GPi HFS affects the activity of the striatum and the motor cortex activity. Regarding the GPi or STN inactivation by lesion procedures, too little experimental data are available to draw any con­ sistent conclusion. When investigating the motor behaviour, numerous studies carried out in animal models

251

have provided data supporting the role of GPi or STN as suitable targets for the treatment of par­ kinsonism. Almost all of these studies confirmed the beneficial effects of surgical interventions tar­ geting GPi or STN on motor behaviour. However, it is important to note that there are much more studies focusing on STN than on GPi or EP, possibly in line with the predomi­ nance of STN surgery in PD over pallidotomies or GPi DBS.. However, the possible cognitive and motivational side-effects observed after STN inactivation could lead to a revival of GPi as the target of choice. Although the clinical reports indicate only mild cognitive impairment after GPi manipulation, studies on cognitive and motivational processes in animals are needed. They could lead to a better profile of what should be investigated in these behavioural pro­ cesses in patients. In general, there is a poor investigation of beha­ vioural consequences of HFS in either GPi or STN carried out in monkeys, possibly due to the fact that numerous clinical reports are published every year and might thus reduce the interest in proving behavioural efficacy of this surgical strategy in non-human primates. Most of the available studies using HFS in monkeys aimed at understanding the mechanisms of DBS. It would, however, be of great interest to also study behavioural effects in order to better understand the functional role of GPi and STN in the non-human primate, espe­ cially regarding non-motor behaviour. When it comes to cognitive and motivational processes, mainly rat data are available. These studies high­ lighted the integrative function of the STN, pla­ cing it at the interface between motivation and action. There was often a parallel to these findings in clinical observations of PD patients with STN DBS, but further studies in monkeys would be important to perform, especially because they could allow specific investigation of the sub-terri­ tories within the STN (limbic, associative and motor areas) that are impossible to perform in the rat given the small size of the STN in this species.

A better knowledge of the possible conse­ quences of GPi or STN inactivation in animals on various types of behaviour involving motor, cognitive and motivational processes was impor­ tant for the treatment of PD patients and has lead to a more cautious attitude towards the criteria of selection for surgery. Indeed, with the increasing interest in cognitive and psychiatric consequences of STN DBS, the psychiatric examination of the patients has been taken more seriously in order to anticipate and avoid possible untoward effects of this treatment.

Acknowledgements This work has been supported by grants from the Centre National de la Recherche Scientifique (CNRS) to CB and PG, the Université de la Méd­ iterranée to PG, the Université de Provence to CB, the Agence Nationale pour la Recherche (ANR-05-JC05_48262 and ANR-09-MNPS-028­ 01 to CB and the ANR-05-NEUR-021 to PG), the Fondation de France to PG, the MILDT­ InCa-INSERM grant to CB and the Fondation pour la Recherche sur le Cerveau to CB.

Abbreviations 5-CSRTT 5-HT 6-OHDA BG DBS GPe/i HFS L-DOPA

MPTP PD RT

5-choice serial reaction-time task 5-hydroxytriptamine or serotonin 6-hydroxydopamine basal ganglia deep brain stimulation external/internal segment of the globus pallidus high-frequency stimulation L-3,4-dihydroxyphenylalanine 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine Parkinson’s disease reaction time

252

SNc/r SRT STN

substantia nigra pars compacta/ reticulata simple reaction time subthalamic nucleus

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258 Temel, Y., Boothman, L. J., Blokland, A., Magill, P. J., Stein­ busch, H. W., Visser-Vandewalle, V., et al. (2007). Inhibition of 5-HT neuron activity and induction of depressive-like behavior by high-frequency stimulation of the subthalamic nucleus. Proceedings of the National Academy of Sciences of the United States of America, 104(43), 17087–17092. Temel, Y., Visser-Vandewalle, V., Aendekerk, B., Rutten, B., Tan, S., Scholtissen, B., et al. (2005). Acute and separate modulation of motor and cognitive performance in parkinso­ nian rats by bilateral stimulation of the subthalamic nucleus. Experimental Neurology, 193(1), 43–52. Temel, Y., Visser-Vandewalle, V., Kaplan, S., Kozan, R., Dae­ men, M. A., Blokland, A., et al. (2006). Protection of nigral cell death by bilateral subthalamic nucleus stimulation. Brain Research, 1120(1), 100–105. Thobois, S., Fraix, V., Savasta, M., Costes, N., Pollak, P., Mertens, P., et al. (2003). Chronic subthalamic nucleus stimulation and striatal D2 dopamine receptors in Parkin­ son’s disease- -A [(11)C]-raclopride PET study. Journal of Neurology, 250(10), 1219–1223. Trepanier, L. L., Saint-Cyr, J. A., Lozano, A. M., & Lang, A. E. (1998). Neuropsychological consequences of posteroventral pallidotomy for the treatment of Parkinson’s disease. Neu­ rology, 51(1), 207–215. Turle-Lorenzo, N., Maurin, B., Puma, C., Chezaubernard, C., Morain, P., Baunez, C., et al. (2006). The dopamine agonist piribedil with L-DOPA improves attentional dysfunction: Relevance for Parkinson’s disease. The Journal of Pharma­ cology and Experimental Therapeutics, 319(2), 914–923. Uslaner, J. M., Dell’Orco, J. M., Pevzner, A., & Robinson, T. E. (2008). The influence of subthalamic nucleus lesions on sign-tracking to stimuli paired with food and drug rewards: Facilitation of incentive salience attribution? Neu­ ropsychopharmacology, 33, 2352–2361. Uslaner, J. M., & Robinson, T. E. (2006). Subthalamic nucleus lesions increase impulsive action and decrease impulsive choice – mediation by enhanced incentive motivation? Eur­ opean Journal of Neuroscience, 24(8), 2345–2354. Utter, A. A., & Basso, M. A. (2008). The basal ganglia: An overview of circuits and function. Neuroscience and Biobe­ havioral Reviews, 32(3), 333–342. Vila, M., Perier, C., Feger, J., Yelnik, J., Faucheux, B., Ruberg, M., et al. (2000). Evolution of changes in neuronal activity in the subthalamic nucleus of rats with unilateral lesion of the substan­ tia nigra assessed by metabolic and electrophysiological mea­ surements. European Journal of Neuroscience, 12(1), 337–344. Vitek, J. L., Bakay, R. A., Freeman, A., Evatt, M., Green, J., McDonald, W., et al. (2003). Randomized trial of pallidot­ omy versus medical therapy for Parkinson’s disease. Annals of Neurology, 53(5), 558–569. Vlamings, R., Visser-Vandewalle, V., Koopmans, G., Joosten, E. A., Kozan, R., Kaplan, S., et al. (2007). High frequency stimulation of the subthalamic nucleus improves speed of

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A. Bjorklund and M. A. Cenci (Eds.)

Progress in Brain Research, Vol. 183

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 13

Computational physiology of the basal ganglia in

Parkinson’s disease

Michal Rivlin-Etzion†,‡, Shlomo Elias‡, Gali Heimer‡ and Hagai Bergman†,‡,
† The Interdisciplinary Center for Neural Computation, The Hebrew University, Jerusalem, Israel

Institute for Medical Research Israel-Canada (IMRIC), Department of Medical Neurobiology (Physiology), The

Hebrew University, Jerusalem, Israel

‡

Abstract: The normal activity of basal ganglia neurons is characterized by Poisson-like (random) firing patterns. Correlations between neurons of the same structure are weak or non-existent. By contrast, synchronous oscillations are commonly found in the basal ganglia of human patients and animal models of Parkinson’s disease. The frequency of these oscillations is often similar to that of the parkinsonian tremor, but their role in generating the tremor or other parkinsonian symptoms is still under debate. The tremor is intermittent and does not appear in all human patients. Similarly, primate models tend to develop tremor as a function of species of monkey. African green (vervet) monkeys usually demonstrate a high-amplitude, lowfrequency (4–7 Hz) tremor beyond their akinesia and bradykinesia, whereas macaques tend to be akinetic rigid and rarely demonstrate a low-amplitude high-frequency (10–12 Hz) action–postural tremor. We took advantage of this fact and studied the appearance of the synchronicity and oscillations in six monkeys, three vervets and three macaques, before and after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) systemic treatment and induction of parkinsonism. Multiple extracellular recordings were conducted in the primary motor cortex of two monkeys and in the globus pallidus (GP) of all six monkeys. All the monkeys became akinetic and bradykinetic as a result of the MPTP treatment, but only vervets demonstrated prolonged episodes of low-frequency (4–6 Hz) tremor, whereas macaques were non-tremulous. The GP population exhibited ~5 Hz oscillatory activity in all six monkeys, whereas ~10 Hz neural oscillations were only detected in the tremulous monkeys. The activity of the cortical neurons became strongly oscillatory at ~10 Hz in one of these monkeys, but not the other, although both were tremulous and exhibited comparable pallidal oscillatory activity. Finally, synchronous oscillations, when present, were centred around the higher frequencies of oscillations. These findings suggest that there is a correlation between high-frequency GP neural oscillations and tremor. Furthermore, these pallidal 10 Hz oscillations are probably transferred to the periphery through cortical and brainstem pathways.




Corresponding author.

Tel.: (þ) 972-2-6757388; Fax: (þ) 972-2-6439736;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83013-4

259

260

Keywords: Basal ganglia; Parkinson’s disease; MPTP; oscillations; synchronization

Introduction The motor symptoms of Parkinson’s disease (PD) are characterized by two opposing hypo- and hyper-kinetic features. The hypo-kinetic features of PD include a poverty of movement, for exam­ ple a reduction in spontaneous movements and difficulty in initiating a voluntary movement (aki­ nesia) and slowness of movement (bradykinesia). The hyper-kinetic features of PD are character­ ized by involuntary movement which emerges as a low-frequency (4–7 Hz) tremor. In addition, PD patients suffer from muscle rigidity and postural deficits, but the classification of these motor symp­ toms as hyper- or hypo- kinetic has yet to be firmly established. The major cellular event leading to the motor symptoms of PD is the death of midbrain dopami­ nergic neurons, resulting in dopamine (DA) depletion in the input nuclei of the basal ganglia, the striatum (Jellinger, 1987). It is still not known how DA depletion leads to the conflicting hypoand hyper-kinetic symptoms of the disease. Recordings from different nuclei of the basal ganglia in primate as well as rodent models of parkinsonism reveal that the symptoms of the dis­ ease are accompanied by several changes in neural activity, including the emergence of an oscillatory and bursty neuronal pattern (Bergman et al., 1994; Dejean et al., 2008; Filion and Trem­ blay, 1991; Heimer et al., 2006; Mallet et al., 2008; Miller and DeLong, 1987; Nini et al., 1995; Raz et al., 2000). These periodic burst oscillations can generate some or all of the motor symptoms of the disease either via the cortico-basal ganglia-tha­ lamo-cortex loop (Bergman et al., 1998; Gerfen and Wilson, 1996; Mink, 1996; Parent and Hazrati, 1995) or via the direct projections of basal ganglia output nuclei to brainstem motor centres (Parent et al., 1999). Despite the fact that the majority of GPi axons (estimated at 70%) branch to both the

thalamus and the brainstem (Parent et al., 1999), most information processing models of the basal ganglia ignore the latter projections and focus on the cortex-basal ganglia-cortex loop (Albin et al., 1989; Bar-Gad et al., 2000; DeLong, 1990; Mink, 1996). Recently, it was hypothesized that basal ganglia output projections to the brainstem may be involved in generating the motor deficits of the disease (Stein, 2009). Due to their common periodic nature, tremor is the primary symptom traditionally thought to originate from basal ganglia oscillatory activity. Yet, despite the existence of neurons with tre­ mor-frequency activity (‘tremor cells’) in the glo­ bus pallidus (GP) of PD patients (Hutchison et al., 1997), significant correlations between pal­ lidal activity and tremor are rare and intermittent (Hurtado et al., 2005; Lemstra et al., 1999). Moreover, low-frequency oscillations (1–7 Hz) are present in the local field potential of the subthalamic nucleus (STN) of predominantly aki­ netic-rigid patients. There are reports that the power of the STN low-frequency oscillations even increases during ongoing STN-deep brain stimulation (DBS) and following dopaminergic treatment (Priori et al., 2004; Rossi et al., 2008). These findings are in line with 1-methyl-4-phenyl­ 1,2,3,6-tetrahydropyridine (MPTP) primate stu­ dies: in addition to akinesia and rigidity, African green monkeys treated with the MPTP neuro­ toxin usually develop a low-frequency (~4–7 Hz) tremor resembling the classic resting tremor of PD. Similar to human patients presenting tremor, consistent relations between tremor and oscilla­ tions have not been found in these tremulous monkeys (Raz et al., 2000). Furthermore, lowfrequency oscillations have been shown to persist in the basal ganglia of MPTP-treated monkeys after amelioration of tremor (Wichmann et al., 1994). Finally, MPTP-treated macaques, which tend to be akinetic rigid and develop only rare

261

short episodes of high-frequency (~10–12 Hz) action–postural tremor, also exhibit basal ganglia neuronal oscillations (Bergman et al., 1998; Miller and DeLong, 1987). Even though PD is usually thought to originate in the basal ganglia due to its dopaminergic loss, tremor may not be generated by oscillations in the basal ganglia, but rather be cortical or thalamic in origin. In fact, motor and somatosensory cortices of PD patients also tend to oscillate in correlation with the tremor (Alberts et al., 1969; Timmer­ mann et al., 2003; Volkmann et al., 1996), and ‘tremor cells’ that oscillate in coherence with the tremor have often been found in the thalamus of PD patients (Hayase et al., 1998; Lenz et al., 1988; Magnin et al., 2000). Nevertheless, oscillatory activity in any neuronal structure may merely reflect sensory ascending pathways from the periphery, or an oscillatory activity originating in another neuronal structure, and does not necessa­ rily indicate the specific origin of the tremor (Rivlin-Etzion et al., 2006a). Moreover, the par­ kinsonian tremor may not be generated by a cen­ tral oscillator, but rather have a spinal origin, as was suggested in early studies (Teravainen, 1980; Teravainen et al., 1979). This chapter re-examines periodic oscillatory activity (or simply oscillatory activity) in the GP of six MPTP-treated tremulous as well as non-tremulous monkeys. Analysis of these pre­ viously published studies (Arkadir et al., 2004; Elias et al., 2007; Heimer et al., 2006; Morris et al., 2004; Rivlin-Etzion et al., 2008) demon­ strates that periodic oscillatory activity appears in all of them. However, the distribution of oscil­ lation frequencies differs across the monkeys depending on the existence of tremor. In addi­ tion, we explore the neuronal pattern of the motor cortex of two of the tremulous monkeys and show that oscillatory activity of the motor cortex is not a pre-condition for tremor to emerge. Thus, tremor may not be mediated by cortical periodic oscillations, but rather by basal ganglia oscillations transformed through the brainstem–spinal pathways.

Methods Animals This study is based on data collected from six monkeys: three vervets (African green, Cerco­ pithecus aethiops aethiops, T, W and C; females; weighing 3, 3.5 and 3.8 kg, respectively) and three macaques (one Macaca mulatta, R; female; weigh­ ing 5.7 kg and two Macaca fascicularis, H and P; females; weighing 3.2 and 3 kg, respectively). Before any procedures were carried out, the mon­ keys were trained to sit in a primate chair, to permit handling by the experimenter and became familiarized with the laboratory setting. Monkeys C and R were trained to perform a simple visuo­ motor task (Heimer et al., 2006). Monkey H was trained on a self-initiated probabilistic delayed visual-motor task (Arkadir et al., 2004; Morris et al., 2004). However, the control (before MPTP) recordings in these monkeys (C, R and H) were conducted during a ‘quiet-wakeful’ state. None of the other monkeys (T, W, P) were engaged in any behavioural task (Elias et al., 2007; Rivlin-Etzion et al., 2008), and recordings (before and after the MPTP treatment) were car­ ried out in the ‘quiet-wakeful’ state. The monkeys’ health was monitored by a veterinarian, and their weights and clinical status were checked daily. All experimental protocols were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Hebrew University guidelines for the use and care of laboratory animals in research and were approved and supervised by the Institutional Animal Care and Use Committee.

Surgery and recording procedures After training, a recording chamber was attached to the monkeys’ skulls. The recording chamber was tilted 40–50 laterally in the coronal plane in all monkeys except monkey H where it had a 0 tilt (and therefore permitted recordings from both

262

hemispheres). All chambers were positioned by stereotaxic procedures to cover most of the GP territory. In monkeys T and W the position of the chambers allowed access to the arm-related area of the primary motor cortex (MI) as well. The final position of the chamber was verified by a magnetic resonance imaging (MRI) scan and elec­ trophysiological mapping. Surgical and MRI pro­ cedures were carried out under full (isoflurane, N2O) anaesthesia and sedation (IM Dormitor, Ketamine), respectively. Extracellular simultaneous recordings were per­ formed using four to eight electrodes in the GP and four electrodes in MI (monkeys T and W). Details of the surgery, identification of neurons and data-recording methods were described pre­ viously (Elias et al., 2007; Heimer et al., 2002; Rivlin-Etzion et al., 2008). Unlike the situation in the normal monkey, the electrophysiological dif­ ferences (mainly discharge pattern) between the internal and the external segments are less clear after MPTP and the induction of the PD symp­ toms (Schiff et al., 2002). An initial study con­ ducted by our group (Raz et al., 2000) failed to find differences between pallidal segments, although a second study (Heimer et al., 2002, 2006) did find variations between GPe and GPi. In this study we did not differentiate between neurons located in the two segments of the GP. However, we attempted to cover most of the GP area (both internal and external segments) in the recordings. In monkey H we started recordings from the right hemisphere. After a month of recordings in the normal state the monkey developed a poverty of movement in its contralateral limbs. We stopped the recordings and started treatment with steroids for several days following which the monkey regained most of its motor abilities on the left side, though not fully. Recordings restarted from the left hemisphere from which we recorded throughout the remainder of the experiment. Histology of monkey H revealed that it had devel­ oped a medium-sized haematoma in its right hemi­ sphere. We did not observe significant anatomical

changes (beyond the MPTP-induced dopaminer­ gic degeneration and the electrode tracks) in the histological examination of any of the other monkeys.

MPTP treatment and perfusion After a period of recording in the normal state, Parkinsonism was induced by five intramuscular injections of 0.4 mg/kg of MPTP-HCl (Sigma, Rehovot, Israel). The MPTP injections were given under light intramuscular ketamine hydro­ chloride (10 mg/kg) anaesthesia and over a period of 4 days (three injections in the first 24 h). The clinical state of the monkeys was assessed daily according to a primate scale of Parkinsonism (Benazzouz et al., 1995). In addition, each monkey was given a total subjective score between 0 and 4 (4 being the most severe) on four cardinal motor features: akinesia and bradykinesia, flexed pos­ ture, rigidity and tremor. Upon termination of the recording days in the MPTP state the monkeys were treated with DA replacement therapy to verify the diagnosis of Parkinsonism by clinical improvement resulting from DA replacement treatment (L-Dopa and direct DA receptor ago­ nists). Doses for monkeys T, H and P were 0.5 × 25/250 mg of Dopicar (L-3,4-dihydroxyphe­ nylalanine and carbidopa; Teva Pharmaceutical Industries Ltd.) twice a day. The starting doses for monkeys C and R were 0.5 × 25/250 mg of Dopicar (Merck Sharp and Dohme, Haarlem, The Netherlands) in the morning and 5 mg of Parlodel (Bromocriptine; Sandoz, Basel, Switzer­ land) divided equally between morning and evening. The drugs were administered orally as crushed powder dissolved in liquid. In monkeys T, H, C and R recordings were also conducted after treatment with DA replacement therapy (data not reported here). Monkey W died before it was given any dopaminergic treatment. At the end of the experiment, the monkeys were deeply anaesthetized with a lethal dose of pentobarbital and perfused through the heart with

263

saline followed by a 4% paraformaldehyde solu­ tion. Monkey W was perfused in a similar way within 30 min of her death. The brains of the monkeys were removed, serial sections of 50 mm were cut on a freezing microtome and every 12 section was processed for Nissl or tyrosine hydro­ xylase (TH) immunohistochemistry [for details, see Heimer et al. (2006); Rivlin-Etzion et al. (2008)].

Accelerometers In monkeys C, R, T and W we used uniaxial accelerometers (8630C5; Kistler, Amherst, NY) to assess limb tremor. The monkeys had the accel­ erometer fastened to the back of their nonrestrained wrist (contralateral to the recording hemisphere), whereas in monkeys T and C accel­ erometers were fastened to all four limbs. For details regarding accelerometer recordings, see Heimer et al. (2006) and Rivlin-Etzion et al. (2008).

Data analysis Cells were selected for recording as a function of their signal-to-noise ratio and real-time assess­ ment of their isolation quality. Only isolated units (as judged by the experimenters in real

time), which are stable (minimum 5 min, off-line verification of the stability of the neurons’ firing rates throughout the recording session; we dis­ carded any neuron that demonstrated a trend of decaying or increasing firing rate since this is indi­ cative of possible neuronal injuries or unstable electrode position) were included in the analysis database of this study. Details regarding recording durations and the total number of units in each monkey that met the above criteria and were included in this study are shown in Tables 1 and 2. We re-ran quantitative analyses of oscillatory firing patterns of all cells included in the study. Periodic neuronal oscillations are neural activity (spikes or burst of spikes) that repeat in a periodic manner (e.g. every 200 ms). Periodic activity of single neurons can be studied by auto-correlation and auto-spectrum analysis, whereas pair-wise periodic activity is studied by cross-correlation function and coherence analysis. Oscillatory activ­ ity of the cells was estimated using the power spectrum density (PSD) of the spike trains (fre­ quency resolution, 0.25 Hz). The oscillatory activ­ ity of pallidal neurons was assessed using the shuffling method (Rivlin-Etzion et al., 2006b) in order to compensate for the spectral distortion that arises due to the refractory period of neurons with a high discharge rate. Briefly, the spectrum of the original spike train is divided by the mean spectrum of the locally (T=~175 ms) shuffled spike trains (n = 20 shuffled trains), resulting in a

Table 1. Pallidal recording (the neuronal data base)

GP single

Normal MPTP

GP pairs

Normal MPTP

T

W

C

R

H

P

12.60 + 4.60 (n = 114) 12.45 + 3.72 (n = 138) 11.44 + 4.56 (n = 125) 11.69 + 3.93 (n = 254)

9.63 + 2.45 (n = 163) 10.34 + 3.01 (n = 75) 8.75 + 2.32 (n = 202) 9.63 + 2.96 (n = 131)

29.61 + 12.56 (n = 244) 28.73 + 7.72 (n = 268) 25.11 + 11.04 (n = 786) 24.89 + 8.85 (n = 1165)

23.71 + 11.31 (n = 48) 19.82 + 9.65 (n = 97) 18.98 + 9.22 (n = 111) 15.07 + 7.77 (n = 195)

19.4+ 14.43 (n = 115) 15.28 + 6.76 (n = 157) 16.7 + 13.04 (n = 105) 14.15 + 6.15 (n = 168)

11.04 + 4.69 (n = 79) 10.12 + 4.21 (n = 232) 9.23 + 3.20 (n = 51) 9.68 + 4.07 (n = 312)

The table depicts the analysis duration (in minutes) of neurons/structure/state/monkey. Times are given in minutes as mean+SD of the analysis duration of neurons/structure/state/monkey. n stands for the number of neurons on which the values are based.

264 Table 2. M1 and M1–GP recordings (the neuronal data base)

MI single

Normal MPTP

MI pairs

Normal MPTP

MI-GP pairs

Normal MPTP

T

W

14.95 + 5.03 (n = 230) 11.84 + 3.71 (n = 176) 14.54+4.75 (n = 568) 11.04 + 3.69 (n = 434) 12.71 + 4.35 (n = 695) 10.75 + 3.56 (n = 846)

10.5 + 2.35 (n = 358) 11.45 +2.52 (n = 122) 10.04+2.41 (n = 947) 10.58 + 2.65 (n = 363) 9.03 + 2.38 (n = 1073) 9.80 + 2.79 (n = 573)

The table depicts the analysis duration (in minutes) of neurons/struc­ ture/state/monkey. Times are given in minutes as mean+SD of the analysis duration of neurons/structure/state/monkey. n stands for the number of neurons on which the values are based. GP, globus pallidus; MI, primary motor cortex; MPTP, 1-methyl-4­ phenyl-1,2,3,6-tetrahydropyridine.

ratio termed ‘compensated PSD’. Due to the low discharge rate of cortical neurons (Goldberg et al., 2002; Rivlin-Etzion et al., 2008), the oscillatory activity in the cortex could be evaluated based on the original PSD. A confidence level (p < 0.001, normalized to the total number of bins) was constructed based on the high-frequency range of 240–300 Hz, at which the PSD was flat. A cell was considered oscillatory if its compensated (GP) or original (cortex) PSD contained at least two consecutive bins within the range of 4–15 Hz that crossed the p = 0.001 confidence level. For the analysis of synchronous oscillations we used pairs of neurons that were simultaneously recorded and showed stable and isolated overlap­ ping activity for at least 5 min (Tables 1 and 2). Only neuronal pairs that were recorded by differ­ ent electrodes were included in this study to avoid possible artefacts attributable to a shadowing effect of high discharge rates in cells recorded from the same electrode (Bar-Gad et al., 2001). Synchronous oscillations within GP pairs as well as between MI and GP neurons were assessed using the shuffling method (Rivlin-Etzion et al., 2006b) due to the high GP discharge rate. The

cross-spectrum (representing the common oscilla­ tions frequencies of the two neurons) of the origi­ nal spike trains was divided by the mean crossspectrum of the globally shuffled (n = 20) spike trains. A confidence level (p < 0.001, normalized to the total number of bins) for the compensated spectrum was constructed based on the high-fre­ quency range of 240–300 Hz, at which the spec­ trum was flat. A correlogram was considered to have significant periodic oscillations if its compen­ sated spectrum contained at least two consecutive bins within the range of 4–15 Hz that crossed the p < 0.001 confidence level. For cortical pairs, we used the conventional significance criterion for the coherence function as a confidence level (Bloomfield, 1976; Brillinger, 1981):

1  ð1  �Þ

1 L1

ð1Þ

where a is the level of confidence (here a = 0.999), and L is the number of windows used in the cal­ culation (length of the data divided by the window size, which in our case was 4096). All analyses were carried out on custom devel­ oped software using Matlab 7.1 tools (The MathWorks, Natick, MA). The same procedures and thresholds were applied to all monkeys and all clinical states.

Results Animals’ clinical states Table 3 summarizes the PD scores of the six MPTP-treated monkeys. Three of the monkeys were macaques and three were African green monkeys. All monkeys developed the first signs of Parkinsonism by the first day after the last MPTP injection. parkinsonian symptoms contin­ ued to evolve over the following days and stabi­ lized within 6 days or less. They remained stable until the last recording day included in this study (before any DA treatment was given, see Table 3).

265 Table 3. Clinical scores and recording history of the MPTP-treated monkeys

Monkey

Species

Akinesia/ bradykinesia

R P H C T W

Macaque Macaque Macaque AGM AGM AGM

4 4 4 4 4 3

Flexed posture

Rigidity

4 4 4 4 4 3

4 2–3 2–3 1–2 3.5 1

Tremor

First recording day

Last recording day

Response to DA treatment

0 0 0 2.5–3 2.5 3

4 5 4 4 7 3

18 23 21 18 21 7

Full Partial Partial Full Full NA

For all scores, 0 – normal, 4 – most severe. First recording day – after last MPTP injection, Last recording day – after last MPTP injection (before any DA treatment was administered). AGM, African green monkey. Full, Full response to the DA treatment including regaining of ability to self-feed, an increase in amount and velocity of movements and straightening of posture; Partial, Partial response to the DA treatment including regaining of ability to self-feed, a moderate increase in amount and velocity of movements and a partial straightening of posture; NA, Not available (DA treatment not given); DA, dopamine; MPTP, 1­ methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

All monkeys developed severe akinesia and bra­ dykinesia, as well as flexed posture. The monkeys differed in their rigidity scores. Four monkeys were severely rigid, and monkey W presented the lowest rigidity score. This monkey died 7 days after last MPTP injection and exhibited stable akinesia, bradykinesia and tremor by the fourth day after the MPTP injections. As was previously reported (Heimer et al., 2006), the tremor score differed between the two monkey species and was high in all African green monkeys, but not in the maca­ ques [though monkey R developed high-frequency (~9–10 Hz) tremor after dopaminergic treatment, see Heimer et al. (2006) for details]. Responsiveness to DA treatment was tested to verify the diagnosis of Parkinsonism (see Section ‘Methods’). All monkeys responded to the treat­ ment by regaining the ability to self-feed, an increase in amount and velocity of spontaneous movements and straightening of posture. Although monkeys T, C and R demonstrated a full response to the DA treatment, monkeys H and P responded to a lesser extent, with fewer voluntary movements and only partial stable posture (Table 3). Histology All the MPTP-treated monkeys had an almost complete loss of TH staining in the striatum

except the shell area of the ventral striatum. Cell loss in the midbrain was almost complete in the ventral tier and the lateral portions of the substan­ tia nigra pars compacta in all animals. Monkey W showed a less severe loss of TH midbrain staining, probably due to its early death. As expected (Song and Haber, 2000), the cells in the ventral tegmental area of the midbrain DA system remained relatively spared. Spontaneous neuronal activity in MI and GP: example An example of the spontaneous neuronal activity recorded by extracellular electrodes located in the MI and the GP of monkey T before and after MPTP injection is illustrated in Fig. 1. PSDs of the spike trains revealed that cortical activity became periodic at 10 Hz in the parkin­ sonian state, and coherences between cortical neurons indicated that these oscillations were synchronized (Fig. 2). There was 5 Hz activity in the pallidum in the parkinsonian state, but the coherence analysis showed that these oscilla­ tions were synchronized to a lesser extent (note the different Y scales in the figure). Finally, the cortical and the pallidal oscillations were not coherent.

266 (a)

Normal

2 2 3

GP

1

4

3

CTX

1

c60710_3_1s_stabil; time 6714.5

0.5 s (b)

MPTP

2 4

3

GP

2

1

3

CTX

1

c51019_2_1s_stabil; time 11773.96

0.5 s Fig. 1. Example of motor cortex and pallidal neuronal activity before and after MPTP treatment. Extracellular simultaneous recordings (bandpassed filtered at 300–6000 Hz) from electrodes located in the motor cortex (CTX, upper traces) and globus pallidus (GP, lower traces) in the normal (a) and parkinsonian (b) states. Normal recordings are taken from monkey W, and MPTP recordings are of monkey T. Trace duration = 3 s, time scale of 0.5 s is indicated by the horizontal bottom line.

Emergence of neuronal oscillations in the parkinsonian state: population analysis Figure 3 depicts the fraction of neuronal oscillations of single neurons (upper row) and synchronous

oscillations (pair-wise correlations, lower row) before and after MPTP treatment. In the MPTP state, significant (p < 0.01, �2 test) oscillatory activity of single neurons emerged in the motor cortex of two monkeys (2/2) and in the GP of four

267

pairs comparable to reports in earlier studies of vervet monkeys (Raz et al., 2000). The fraction of synchronized MI–GP pairs increased significantly in the MPTP state in both monkeys T and W (p < 0.01), but did not reach the 2.5% cutoff.

monkeys (4/6). Pallidal oscillatory activity also emerged in the parkinsonian state in the two other monkeys (H and P), but as they demon­ strated some oscillatory activity in the normal state, the differences between the two states were not significant. Significant synchronized neuronal oscillations (p < 0.01, �2 test) of the motor cortex of monkeys T and W emerged in the parkinsonian state, although synchronous oscillations were more robust in monkey T: almost 40% of the recorded pairs. All monkeys exhibited synchronous oscilla­ tions in the parkinsonian GP, but only monkey C demonstrated high percentages of synchronized

Frequency distribution of MPTP neuronal

oscillatory activity

The distributions of the frequencies of neuronal oscillations in the parkinsonian state are depicted in Fig. 4. Interestingly, despite the fact that animals Normal

CTX1

(a)

–45

–55 0.2

CTX2

–40

–50

0.2

–40

0 0.2

0 0.2

–50

0.2

–50

0 10

0 10

0 10

–60

10

10

0 10

0 10

0 10

0 10

0 10

2

0 10

0 10

0 10

0 10

0 10

0 10

GP2

GP1

CTX4

CTX3

0 0.2

GP3

4

0

510

CTX1

0

510

CTX2

0

510

CTX3

0

510

CTX4

0

510

GP1

0

510

GP2

0

510

GP3

Fig. 2. Example of power spectral densities and cross-spectrums of neural activity before and after MPTP treatment. Auto-spectral histograms (bold framed, right diagonal) and cross-spectral histograms of the spike trains shown in Figure 1, (a) in the normal state and (b) in the MPTP state. The spectra were calculated for the entire recording periods of the units included in the analysis database. Identification of the trigger electrode appears at the bottom and identification of the reference electrode on the left. Significance (p = 0.001) levels are depicted by the dashed lines. In cases where more than one single unit was detected by the electrode, a single spike train was chosen for the illustration. YScales are the normalized logarithmic power spectral density of the activity. Y-scale is chosen to maximize the visibility of the oscillation frequencies (peaks of spectrograms) in each plot. CTX, arm-related primary motor cortex; GP, globus pallidus.

268

MPTP

CTX1

(b) –40

–50 1

0 1

–45

1

–40

0 10

0 10

–50

10

2

0 10

0 10

0 10

0

10

4

0 10

0 10

0 10

0 10

0

10

2

0 10

0 10

0 10

0 10

0 10

0

10

GP3

GP2

GP1

CTX3

CTX2

–40

GP4

2

0

5 10

CTX1

0

510

CTX2

0

0

5 10

CTX3

510

0

GP1 Frequency (Hz)

510

GP1

0

510

GP2

0

510

GP3

Fig. 2. (Continued)

T and W were tremulous monkeys, only the MI of monkey T exhibited oscillatory activity that was centred around a single frequency – 10 Hz, which was twice the tremor frequency (5 Hz, as indicated by the accelerometers, data not shown). Moreover, although all monkeys, regardless of their species or tremulous activity, had dominant ~5 Hz oscillatory activity in the GP, only tremulous vervet monkeys displayed oscillatory activity confined to ~10 Hz as well. GP synchronous oscillations, if present (mainly in monkey C), were centred around the 10 Hz – double-tremor frequency.

Discussion It is very important to address abnormal patterns of oscillatory activities in the basal ganglia. This

approach has overcome the limitations of ‘box and arrow’ models of the basal ganglia which are lim­ ited to static changes of discharge rate and there­ fore cannot explain the dynamic nature of tremor and other disorders. Oscillatory activities can be detected with LFP recordings and/or at the level of single neurons and groups of neurons within the same structure or between different structures and therefore can provide a framework for better understanding of the changes in the computational processes in the basal ganglia networks. The computational goals and algorithms of the basal ganglia networks are still unknown. The motor deficits of PD, and especially PD akinesia, suggest that the basal ganglia are involved in action initiation or selection. Nevertheless, studies of neuronal activity in the basal ganglia of behav­ ing animals have failed to reach firm conclusions

269 GP

MI

Oscillatory cells (%)

40

**

30

**

**

20

** **

**

T

R

10 0

T W

W C

H

P MI-GP

Oscillatory pairs (%)

30

20

**

10

** *

** 0

Normal MPTP

**

T

W Monkey

*

T W C R Monkey

** ** H

P

T

W Monkey

Fig. 3. Fraction of neuronal oscillations in the motor cortex and GP. Fraction of single (upper row) and coupled (lower row) neuronal oscillations in the normal (white) and parkinsonian (black) states. Left column depicts fractions in the motor cortex of two monkeys, middle column depicts the fraction in the GP of six monkeys, and the right column presents the fraction of coupled MI–GP oscillations.

about the computational physiology of the basal ganglia networks. This is partially because of the limits of primate studies, which are commonly carried out on small samples (two to three primates), and tend to employ highly diverse behavioural paradigms. This review is based on data collected during several studies by our research group. In these studies we recorded the basal ganglia and cortical activity of six MPTP-treated monkeys: three tremulous African green monkeys and three non-tremulous macaques. Our main aim was to use the same analysis tools to verify the changes in the neuronal firing pattern and synchronization following MPTP treatment and striatal DA depletion. The manifestation of 5 Hz neuronal oscillations in the GP of all monkeys suggests that these

pallidal low-frequency oscillations, although shar­ ing a similar frequency with the parkinsonian tre­ mor, do not generate the tremor and are not driven by it. By contrast, high-frequency pallidal oscillations were only found in tremulous mon­ keys, which might indicate a relationship between these 10 Hz oscillations and the tremor. Interestingly, these oscillations may be filtered out at the thalamus and/or cortex since the cortical activity in only one but not the other African green mon­ key became strongly oscillatory at 10 Hz. The fre­ quencies of oscillations in the cortex of the other monkey were more uniformly distributed, despite the fact that both monkeys demonstrated a similar fraction of oscillations in the GP, which were comparably distributed bi-modally around 5 and 10 Hz. These filtration properties were also

MI

Oscillatory cells (%)

MI

Oscillatory pairs (%)

GP

25 20 15 10 5 0

Oscillatory cells (%)

GP

25 20 15 10 5 0

10 8 6 4 2 0

Oscillatory pairs (%)

270

20 15 10 5 0

T (n = 176)

W (n = 122)

5 10 T (n = 434)

5 10 W (n = 363)

10 5 T (n = 138)

5 10 W (n = 75)

C (n = 268)

R (n = 97)

H (n = 157)

P (n = 232)

5 10 T (n = 254)

5 10 W (n = 131)

5 10 C (n = 1165)

5 10 R (n = 195)

5 10 H (n = 168)

5 10 P (n = 312)

10 5 10 5 10 5 5 10 5 10 5 10 Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz) Frequency (Hz)

Fig. 4. Distribution of single and coupled neuronal oscillations in the motor cortex and GP in the parkinsonian state. Fraction of single (oscillatory cells) and coupled (oscillatory pairs) neuronal oscillations/frequency in the parkinsonian state. Two upper rows depict fractions in the motor cortex of two monkeys and lower rows depict the fraction in the GP of six monkeys.

revealed using electrical stimulations delivered to the parkinsonian GP: only bursts delivered at 1 and 2 Hz frequencies, but not those delivered at 5 Hz or at higher frequencies were reflected in the motor cortex (Rivlin-Etzion et al., 2008). Thus, GP high-frequency oscillations may be trans­ formed via the brainstem pathways and generate the parkinsonian tremor in a 2:1 or other complex relationship. We do not know why the 5 Hz pallidal oscilla­ tions do not result in a 2.5 Hz tremor as well, but a reasonable explanation is that most of the syn­ chronous oscillatory activity in the GP, if present, is located at the 10 Hz higher frequencies and not

at the lower ones [Fig. 4, as well as Heimer et al. (2006); Raz et al. (2000)]. In fact, magnetoence­ phalography (MEG) recordings of PD patients reveal that although there is significant coherence activity between different cortical areas at tremor frequency it is even stronger at double-tremor frequency (Timmermann et al., 2003). The percentages of pallidal oscillations did not exceed 20% in most of the monkeys (Fig. 3). Moreover, the 10 Hz oscillations in the GP in two out of the three tremulous monkeys were found only in 5–6% of the recorded neurons. This may be due to the fact that we attempted to cover most of the pallidal area in the experiments. Had we

271

restricted our recordings to the motor part of the GP (Francois et al., 2004), the percentages of oscillations might have significantly increased. Associating pathological neural activities and motor symptoms is one step in the search for PD treatment and for understanding different disease subtypes. We believe that our finding of 10 Hz pallidal activity which discriminates tremulous and non-tremulous parkinsonian monkeys contri­ butes to finding such an association. Future stu­ dies will address the origins and/or the neurochemical and neurophysiological mediators of this activity. Acknowledgements This research was supported in part by the ‘Fight­ ing against Parkinson’ and the Max Vorst Family Foundations of the Hebrew University Nether­ lands Association (HUNA).

Abbreviations DA DBS GP MI MPTP PD STN

dopamine deep brain stimulation globus pallidus primary motor cortex 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine Parkinson’s disease subthalamic nucleus

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A. Bjorklund and M. A. Cenci (Eds.) Progress in Brain Research, Vol. 183 ISSN: 0079-6123 Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 14

Neurocomputational models of motor and cognitive deficits in Parkinson’s disease Thomas V. Wiecki and Michael J. Frank
Department of Cognitive, Linguistic, and Psychological Sciences, Department of Psychiatry and Human Behavior, and Brown Institute for Brain Science, Brown University, Providence, RI, USA

Abstract: We review the contributions of biologically constrained computational models to our understanding of motor and cognitive deficits in Parkinson’s disease (PD). The loss of dopaminergic neurons innervating the striatum in PD, and the well-established role of dopamine (DA) in reinforcement learning (RL), enable neural network models of the basal ganglia (BG) to derive concrete and testable predictions. We focus in this review on one simple underlying principle – the notion that reduced DA increases activity and causes long-term potentiation in the indirect pathway of the BG. We show how this theory can provide a unified account of diverse and seemingly unrelated phenomena in PD including progressive motor degeneration as well as cognitive deficits in RL, decision making and working memory. DA replacement therapy and deep brain stimulation can alleviate some aspects of these impairments, but can actually introduce negative effects such as motor dyskinesias and cognitive impulsivity. We discuss these treatment effects in terms of modulation of specific mechanisms within the computational framework. In addition, we review neurocomputational interpretations of increased impulsivity in the face of response conflict in patients with deep-brain-stimulation. Keywords: Parkinson’s Disease; Dopamine; Basal Ganglia; Computational Model; Cognition

prominent in the motor domain and progressively manifests itself as bradykinesia, akinesia and tremor. More recently, however, cognitive and learning deficits have received increased recognition and interest (e.g. Cools, 2005; Cunha et al., 2009; Frank, 2005; Grahn et al., 2009; Moustafa et al., 2008b). Although traditionally cognitive deficits are often interpreted as resulting from decline in prefrontal cortical function, these reviews have highlighted a more central role for the BG in cognitive function.

Introduction Early onset of Parkinson’s disease (PD) is characterized by loss of dopaminergic neurons innervating the striatum in the basal ganglia (BG) (Kish et al., 1988). The symptomatology is most


Corresponding author. Tel.: (401) 863-6872; Fax: (401) 863-2255; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)83014-6

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From a computational and cognitive neuroscience point of view, PD is a highly intriguing disorder. Because PD results in depleted striatal dopamine (DA) levels, but increased striatal DA levels following DA medication (Pavese et al., 2006; Tedroff et al., 1996), researchers can directly test the influence of different BG DA configurations in human subjects. Further, in early disease stages, cognitive deficits in PD are linked to depleted striatal DA levels, with frontal DA levels spared (Nobukatsu et al., 2008). Similarly, cognitive deficits in healthy ageing are correlated with striatal DA depletion rather than frontal DA (Bckman et al., 2000, 2006; Kaasinen and Rinne, 2002). Better understanding of this system will ultimately lead to better treatment options for PD, but also to other diseases involving DA in the BG such as addiction, schizophrenia and Tourette’s syndrome (TS). The BG consists of multiple interconnected nuclei (Mink, 1996) that are part of several complex anatomical/functional loops (Gerfen and Wilson, 1996; Graybiel et al., 1994; Haber, 2004; Haber et al., 2000; Nakano et al., 2000). The inherent complexity of this dynamic system, the role of learning and the existence of feedback loops often let classic box-and-arrow diagrams fall short in their predictive capabilities. Moreover, data about the BG (and PD) are contributed from across different domains reaching from psychology to cellular neurobiology. Although not without caveats, biologically constrained computational models offer a disciplined approach to (1) integrate data from different domains and (2) derive novel and unintuitive predictions which can then be tested experimentally to possibly refine the model. These models are inherently dynamic and are governed by concrete activation and learning rules. One example of where these models furthered our understanding was to reject the notion that under chronic DA depletion most synaptic plasticity in the striatum would be lost (Calabresi et al., 2007b; Kreitzer and Malenka, 2007). The computational model by Frank (2005) challenges this assumption by hypothesizing that only one class of striatal cells – those that are activated in

response to positive reinforcement – would lose synaptic plasticity; another class of cells activated in response to negative outcomes would actually show increased synaptic plasticity. This computational prediction has subsequently been confirmed behaviourally (Frank et al., 2004) and neurobiologically (Shen et al., 2008). This review is structured as follows. First, we introduce basics of neural network models of the BG, focussing on an intuitive understanding of principles rather than mathematical formulations (which can be found elsewhere). We then establish the simple notion of an activation and learning imbalance of the facilitatory and suppressive pathway in the BG and their implication in PD. By this account, the diverse symptomatology of unmedicated and medicated PD (caused by a lack and excess of DA in the striatum, respectively) represent two sides of the same coin. Increased activation and learning in the suppressive pathway (i.e. unmedicated PD) accounts for progressive decline of motor functions, increased avoidance learning and reduced updating of working memory (WM). Conversely, increased activation and learning in the facilitatory pathway (i.e. medicated PD) accounts for excess of motor functions (i.e. dyskinesias), increased anticipatory learning and excessive updating of WM. Thus, PD is not only a motor disorder, but rather a more general disorder of action selection, exacerbated by a learning process that induces a bias in the system to avoid selecting actions. This process can lead to a poverty of movement, but also of more cognitive actions. Note that we focus this review mainly on the predictive power of this dopaminergic account. While this is sufficient for the data we describe, the neurotransmitters noradrenalin, serotonin and acetylcholine have also been implicated with cognitive deficits of PD (Calabresi et al., 2006, 2007a).

Neural network models of basal ganglia Computational models in systems neuroscience (sometimes also called mechanistic or neural

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Fig. 1. (a) Box-and-arrow diagram of the basic anatomy of the BG. Frontal cortex projects to striatonigral neurons (Go) of the direct pathway and to striatopallidal neurons (NoGo) in the indirect pathway. Dopaminergic projections from the SNc innervate the striatum and excite and inhibit Go and NoGo neurons, respectively via simulated D1/D2 receptors. Fast-spiking GABAergic interneurons (g-IN) regulate striatal activity via inhibitory projections. Activation of striatonigral neurons disinhibits the thalamus by inhibiting tonically active GABAergic neurons in the GPi. Activation of striatopallidal neurons removes inhibition of the GPi by inhibiting the GPe – thus ultimately inhibiting the thalamus. The STN is part of the hyperdirect pathway which dynamically activates BG output, and thereby suppresses behaviour, as a function of cortical response conflict. (b) Implementation of the box-and-arrow diagram in form of a neural network model by Frank (2006). Cylinders represent individual simulated neurons, their height and colour encodes their activity level. The computational model complies with current anatomical and physiological BG data.

network models) consist of layers of simulated neurons (i.e. units) that are interconnected according to the anatomy of the brain. The units used in different models – though varying in their degree of biological plausibility – generally try to focus on the computational properties of real neurons and not on all aspects of their anatomy (like biophysical models do). As such, they are often implemented as point-neurons with the dendritic tree and the soma shrunken to a tiny point. The influence of presynaptic inputs is controlled via weights which model synaptic efficacy (receptor affinities, densities, number of presynaptic vesicles released, etc). The Leabra framework, for example, computes the units’ voltage according to excitatory, inhibitory and leak conductance channels (O’Reilly and Munakata, 2000). Individual excitatory and inhibitory channel conductances are

computed by multiplying the presynaptic input activity with the respective synaptic weight. Once the unit exceeds a certain voltage threshold, it communicates output to other downstream units, in the form of either a rate-coded variable (normalized firing rate), or discrete spiking.

Architecture of basal ganglia models The BG is generally conceptualized as an adaptive action selection device gating information flow from and to cortex via the thalamus (Graybiel, 1996). Its basic anatomy can be appreciated in Fig. 1. Two opposing pathways – the direct and indirect pathway – dynamically and selectively facilitate and suppress action representations in the frontal cortex, respectively (Alexander and

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Crutcher, 1990; Brown et al., 2004; Frank, 2005; Frank et al., 2001; Mink, 1996). In the context of motor control, the BG were suggested to selectively facilitate a single motor command via the direct pathway while suppressing all others via the indirect pathway (Chevalier and Deniau, 1990; Mink, 1996). The computational models described below retain the basic functionality of the direct and indirect pathway proposed in the classic model, while also extending the static model to incorporate dynamics, plasticity and updated aspects of anatomical and physiological data (Cohen and Frank, 2009). As one example, while the original model suggested that the subthalamic nucleus (STN) was a key part of the indirect pathway, the updated model places the STN as another input nucleus from cortex, forming a third ‘hyper-direct’ pathway (Miller, 2008; Nambu et al., 2000) that is functionally distinct. Below we discuss the relevance of this distinction for PD. At the heart of BG models is the striatum, a large structure that consists collectively of the caudate, putamen and nucleus accumbens. Almost all mechanistic BG models include at least the direct pathway originating in the striatum, projecting through BG output nuclei to the thalamus. The main effect of striatal activity in these models is to facilitate excitatory thalamic responses, which in turn amplifies cortical activity associated with the corresponding action plan. The striatum receives input from multiple cortical areas and consists mainly of medium spiny neurons (MSNs) (Gerfen and Wilson, 1996). Direct pathway MSNs (i.e. striatonigral neurons) express excitatory dopaminergic D1 receptors and send inhibitory projections to the substantia nigra pars reticulata (SNr) and to the internal segment of the globus pallidus (GPi). In the absence of striatal firing, neurons in SNr and GPi are tonically active and inhibit the thalamus, preventing a frontal action plan from being executed. Activation of the direct pathway leads to disinhibition of the thalamus. Disinhibition implies that thalamic units are not directly excited by direct pathway activity, but are

instead enabled to get excited if they also receive excitatory glutamatergic input (i.e. from descending cortical signals) (Chevalier and Deniau, 1990; Frank et al., 2001). Striatal MSNs of the direct pathway are sometimes labelled as ‘Go’-neurons (e.g. Frank, 2005; O’Reilly and Frank, 2006), because they act to gate or facilitate frontal action plans, the details of which are specified by cortical representations. The role of the indirect pathway is more contentious, and is sometimes omitted altogether in computational models (e.g. Arthur et al., 2006; Bogacz and Gurney, 2007). Although debated for several years, methodological advances have now confirmed the original suggestion that D1 and D2 receptors are largely segregated in MSNs, with D1 receptors predominating in the direct pathway and D2 receptors in the indirect pathway (Gerfen et al., 1990; Gong et al., 2003; Matamales et al., 2009; Surmeier et al., 2007; Valjent et al., 2009). Striatopallidal neurons expressing D2 receptors send inhibitory projections to the external segment of the globus pallidus (GPe). The GPe sends focussed inhibitory projections to GPi/SNr (Bolam et al., 2000; Kincaid et al., 1991; Smith and Bolam, 1989, 1990). Due to this additional inhibitory projection, activity in the indirect pathway ultimately results in inhibition of the thalamus and thus suppression of frontal action plans. Because of this motor suppression property (Albin et al., 1989), striatal MSNs of the indirect pathway are sometimes labelled as ’NoGo’-neurons (Frank, 2005). Electrophysiological studies from different domains support the existence of both, facilitatory and suppressive pathways (Apicella et al., 1992; Jiang et al., 2003; Kimchi and Laubach, 2009a, 2009b; Samejima et al., 2005; Watanabe and Munoz, 2009). Further, selective ablation of striatopallidal (indirect pathway) cells leads to increased locomotion (Pierre et al., 2009). Moreover, actions coded specifically in the striatal region in which the striatopallidal ablation was administered are selectively increased (Sano et al., 2003). These results support the notion that the indirect pathway acts to suppress

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behaviours, such that when ablated, these behaviours are expressed more readily (Miller, 2008). How are only certain actions facilitated or suppressed depending on the context? First, neurons in these pathways are highly structured according to the actions they encode (Deniau et al., 1996; Fger and Crossman, 1984; Mink, 1996). Striatal neurons that receive from a particular cortical region (e.g. encoding hand movements) reciprocally, via the loop through BG output and thalamus, project back to influence activity in that same cortical region (Kelly and Strick, 2004; Middleton and Strick, 2000). Evidence for this ‘closed-loop’ has also been reported in humans (Draganski et al., 2008). Second, striatal neurons receive diffuse projections from posterior cortical areas (Frank, 2005). These corticostriatal projections represent the input to most models and are implemented in the form of units which code for abstract properties of the environment (e.g. stimulus colour or context) (Frank, 2005; Guthrie et al., 2009; Wiecki et al., 2009). This many-to-many connection pattern enables the model to represent all possible stimulus–response pairs and to learn facilitation or suppression for each action in response to stimulus properties. In addition, action selection may be further contextualized by the cognitive state encoded in prefrontal cortex (PFC). Indeed, there appears to be some hierarchical structure to BG– PFC circuits: in addition to closed loops among BG and particular frontal regions, it is also the case that PFC areas in a particular loop can innervate striatal areas in more posterior loops (Haber, 2004; Haber and Calzavara, 2009). In this way, cognitive action plans in PFC can provide additional contextual input to lower level actions, for example, to influence motor control. As mentioned above, multiple cortico-striatal loops innervate the striatum. The ventral pathway, innervating the ventral striatum (nucleus accumbens), represents the motivational loop. It plays a major part in the development of addiction (Dagher and Robbins, 2009). The dorsal pathway, innervating the dorsal striatum (i.e. caudate and putamen), represents the motor loop. It plays a

major role in habit formation (Everitt and Robbins, 2005; Henry et al., 2004; Tricomi et al., 2009). In PD, nigrostriatal dopaminergic projections innervating the dorsal striatum are strongly affected, while mesolimbic dopaminergic projections innervating the ventral striatum are relatively spared (Kish et al., 1988).

Dopamine as a reinforcement learning signal Recordings of midbrain dopaminergic neurons in awake behaving monkeys reveal phasic firing patterns in response to unexpected rewards and punishments (Bayer et al., 2007; Ljungberg et al., 1992; Montague et al., 1997; Pan et al., 2005; Roesch et al., 2007; Schultz, 1998; Waelti et al., 2001). Specifically, a DA burst is observed whenever an outcome of an action is better than expected, and, conversely, a drop below tonic DA firing (i.e. DA dip) whenever the outcome is worse than expected. Importantly, the same patterns were observed in human PD patients who receive abstract (financial) rewards and punishments (Zaghloul et al., 2009). Computational models show that this DAmediated reward prediction error signal can be used to efficiently learn reward contingencies and to maximize reward intake in simple reinforcement learning (RL) environments (Barto, 1995; Friston et al., 1994; Montague et al., 1997; Schultz et al., 1997; Sutton and Barto, 1990). Based on this insight, mechanistic models explore how such action selection and contingency learning to maximize rewards is implemented in the anatomy of the BG. As mentioned above, synaptic strengths are implemented as weights that can change dynamically over time. Specifically, co-activation of two connected units results in an increase of their connection’s weight [corresponding to long-term potentiation (LTP)], otherwise the weight remains stable or is decreased [corresponding to long-term depression (LTD)]. In the corticostriatal pathway, this plasticity is strongly modulated by DA, leading to a ‘3-factor’ Hebbian learning rule (Berke and Hyman, 2000;

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Calabresi et al., 1997, 2000; Kerr and Wickens, 2001; Reynolds and Wickens, 2002; Reynolds et al., 2001; Shen et al., 2008). Importantly, DA effects on postsynaptic activity and plasticity depend on the receptor class. Active Go neurons expressing D1 receptors are depolarized by DA (Hernandez-Lopez et al., 1997), whereas NoGo neurons expressing D2 receptors are inhibited by DA (Hernandez-Lopez et al., 2000). Thus a DA burst in response to reinforcement further activates Go neurons (particularly those that are concurrently excited by corticostriatal glutamatergic input), but inhibits NoGo neurons. Conversely, a DA dip in response to punishment or lack of reward activates NoGo neurons by removing tonic inhibition of DA onto postsynaptic D2 receptors (Frank, 2005). [See Cohen and Frank (2009) for a detailed discussion of the plausibility of this mechanism.] In the model, the above plasticity dynamics are adaptive. Simulated DA activity depends on whether the network selected the correct response according to the task at hand. If the network chose correctly, a DA burst will further activate those Go neurons encoding that action in the current environmental state (stimulus). This increased activity is associated with synaptic potentiation, such that the corticostriatal weights from active inputs are increased. The next time the same stimulus is presented, and the corresponding motor action represented in cortex, striatal Go activity encoding that action will be stronger, increasing the probability that the rewarded action will be gated. Conversely, if the network is chosen incorrectly, a DA dip will increase weights between active cortical units and corresponding NoGo units, ultimately decreasing the probability that the punished action will be repeated. Across multiple trials of experience, this system is able to learn to gate actions that are most likely to produce positive outcomes and to suppress those that are most likely to yield negative outcomes – a corner stone of adaptive behaviour. A recent study provides direct support for this model by showing that direct and indirect pathway cells are required for reward/approach and punishment/

avoidance learning, respectively (Hikida et al., 2010).

Cognitive learning deficits Parkinson’s disease: too much ‘NoGo’ learning? PD patients are impaired in cognitive tasks that require learning from trial-and-error feedback (i.e. RL) (Ashby et al., 1998; Knowlton et al., 1996; Shohamy et al., 2004). Computational explorations with the above-described mechanistic BG model (Frank, 2005) provide an explicit account for these deficits. Reduced dynamic range of DA signals in PD lead to a reduced ability to learn to distinguish between different probabilities of rewards associated with multiple actions and stimuli. Moreover, the model predicted that reduced DA levels should particularly impair learning from positive outcomes (DA bursts) but would spare learning from negative outcomes (DA dips). This prediction was supported by a subsequent experiment: unmedicated PD patients showed intact or even enhanced learning from negative outcomes, but impaired learning from positive outcomes of their decisions (Frank et al., 2004). This RL bias was established by testing models and human subjects with a novel behavioural experiment. In this task, multiple pairs of stimuli are presented to participants, who have to select one stimulus in each pair. Participants receive positive or negative feedback (i.e. winning or losing) depending on their choice, but this feedback is probabilistic. Choices of some stimuli are most often associated with positive feedback, whereas others are most often associated with negative feedback. Unmedicated patients showed better performance when avoiding choices that had been associated with a high probability of negative outcomes, but were less reliable in making choices associated with positive outcomes. Crucially, medication reversed this bias, increasing learning from positive outcomes but actually impairing learning from negative outcomes (Frank et al., 2004) (see Fig. 2). This basic pattern

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Fig. 2. (a) Probabilistic selection RL task. During training, participants select among each stimulus pair. Probabilities of receiving positive/negative feedback for each stimulus are indicated in parentheses. In the test phase, all combinations of stimuli are presented without feedback. Go learning is indexed by reliable choice of the most positive stimulus A in these novel pairs, whereas NoGo learning is indexed by reliable avoidance of the most negative stimulus B. (b) Striatal Go and NoGo activation states when presented with input stimuli A and B, respectively. Simulated Parkinsons (Sim PD) was implemented by reducing striatal DA levels, whereas medication (Sim DA Meds) was simulated by increasing DA levels and partially shunting the effects of DA dips during negative feedback. (c) Behavioural findings in PD patients on/off medication supporting model predictions (Frank et al., 2004). (d) Replication in another group of patients, where here the most prominent effects were observed in the NoGo learning condition (Frank et al., 2007b).

has since been replicated across multiple experiments, tasks and labs (Bodi et al., 2009; Cools et al., 2006; Frank et al., 2007b; Moustafa et al., 2008a; Palminteri et al., 2009; Voon et al., 2010). Here, the mechanistic model provides insight into the neurobiological underpinnings that give

rise to the pattern observed behaviourally. Depleted DA levels (both tonic and phasic) results in increased activity of NoGo units expressing inhibitory D2 receptors, while Go units expressing excitatory D1 receptors receive less excitation. As a result of Hebbian learning, fronto-striatal

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projection weights to NoGo units are increased while inactive Go units do not adapt their weights. Consequently, the system has a relative bias towards NoGo learning to avoid negative outcomes (Frank, 2005). To summarize, the model predicts that decreased DA leads to (1) over-activation of NoGo neurons and subsequent and (2) LTP in these neurons. Empirical evidence for this system level prediction comes from multiple sources. First, neurons in the indirect pathway show abnormal burst firing in parkinsonism (Albin et al., 1989; Bergman et al., 1999; Mallet et al., 2006). It is now clear that this over-excitability of striatal MSNs in the DA-depleted state is specific to the striatopallidal cells, and concomitant decreased GPe, and increased GPi, activity (Boraud et al., 2002; Day et al., 2008; Mallet et al., 2006; Miller, 2008; Miller and Delong, 1987). Moreover, as predicted by activity-dependent plasticity mechanisms in the model, DA depletion causes increased LTP in striatopallidal cells (Shen et al., 2008). The implications of this enhanced plasticity (NoGo learning) for both cognitive and motor symptoms of PD are discussed extensively below.

Levodopa and positive reinforcement learning DA replacement therapy [i.e. levodopa (L-Dopa)] is still the gold-standard treatment of PD. However, while reducing cognitive and motor deficits, L-Dopa introduces a new set of cognitive deficits that have been attributed to an ‘overdose’ of DA in regions that are relatively spared in PD (Cools et al., 2001; Gotham et al., 1988). Furthermore, chronic L-Dopa treatment has been shown to increase DA bursts (Harden and Grace, 1995; Keller et al., 1988; Wightman et al., 1988), and the expression of zif268, an immediate early gene that has been linked with synaptic plasticity (Knapska and Kaczmarek, 2004) in striatonigral (Go), but not striatopallidal (NoGo) neurons (Carta et al., 2005). As predicted by computational simulations, PD patients medicated with L-Dopa showed an increased preference to seek rewarding stimuli

and reduced preference to avoid non-rewarding or punishing stimuli (Bodi et al., 2009; Cools et al., 2006; Frank et al., 2004, 2007b; Moustafa et al., 2008a; Palminteri et al., 2009) (see Fig. 2). In the model, L-Dopa is simulated by an increase in both tonic and phasic DA levels (Frank, 2005). Consequently, active Go units receive overall more D1mediated excitation and are thus subject to more learning, while NoGo units are chronically inhibited. Thus even when a DA dip occurs, the NoGo units remain largely suppressed as exogenous medication continues to bind to D2 receptors. In other words, the system is biased to learn stronger from rewards due to over-activation of the direct pathway and less from punishments because of oversuppression of the indirect pathway. Thus this pattern is the mirror inverse of that observed in unmedicated PD patients, as described above – on a behavioural and a neuroscience level. Intriguingly, this susceptibility towards rewards and relative immunity against negative outcomes could help explain cases of pathological gambling and addiction in some PD patients medicated with L-Dopa [recently reviewed by Dagher and Robbins (2009)]. Although the probability of financial gains at a casino may be roughly 48%, the medicated PD patient’s brain may distort this learned probability to be closer to, for example, 60%, thereby reinforcing gambling behaviours. Recent data support this assumption. PD patients were tested off medication, after L-Dopa treatment, and after a D2 agonist on a gambling task (van Eimeren et al., 2009). This study found that D2 agonists and L-Dopa diminished the influence of negative reward prediction errors in the ventral striatum. Similar results were reported by Voon et al. (2010). These authors specifically showed that PD patients with compulsive disorders show distorted (abnormally increased) learning from financial gains in response to DA medications. ‘Control’ PD patients without such disorders showed reduced learning from losses, and blunted striatal responses to negative prediction errors. The authors’ conclusion from both of these studies is in agreement with the model’s prediction – D2

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agonists and L-Dopa block the effects of DA dips and thus of negative RL. Another recent fMRI study found differences in dorsal striatum activation in medicated PD patients under RL conditions (Schonberg et al., 2010).

Individual differences Why are only a minority of PD patients susceptible to pathological gambling in response to medication? It may be that this, too, is explained in accordance with the theory proposed above. PD patients with pathological gambling disorder have lower baseline striatal D2 receptor density (Steeves et al., 2009). This result might be inherently linked to RL learning differences of healthy humans carrying different polymorphisms of DA signalling genes (Frank and Hutchison, 2009; Frank et al., 2007a, 2009; Klein et al., 2007). Among the tested genes, the polymorphism of the DRD2 gene, associated with D2 receptor function, has been reliably linked to the degree of learning from negative outcomes (i.e. NoGo learning). Those genotypes associated with reduced striatal D2 receptor density (Hirvonen et al., 2005) are accordingly associated with reduced NoGo learning (Frank and Hutchison, 2009; Frank et al., 2007a, 2009; Klein et al., 2007). Thus it is possible that the PD patients who are most susceptible to pathological gambling from DA medications are those who are genetically predisposed to exhibit reduced learning from negative outcomes. This hypothesis has yet to be directly tested, but the observed reduced D2 density in pathological gambling patients is supportive, whether or not due to genetic factors. Moreover, if this predisposition is coupled with increased Go learning resulting from dopaminergic medications (Voon et al., 2010), compulsive disorders may be especially evident. This same logic may suggest that a distorted bias to learn more from positive than negative outcomes in RL may help explain other addictive personality types in otherwise healthy individuals. Polymorphism of the DARPP-32 gene relates to synaptic plasticity in response to D1 stimulation.

Carriers of the polymorphism have increased synaptic plasticity and show relatively stronger positive RL (Frank et al., 2007a, 2009). Similarly, individual differences of baseline striatal DA synthesis are predictive of the extent to which participants learn from positive versus negative reward prediction errors (Cools et al., 2009). These biological factors may predispose individuals to have a greater risk for pathological gambling and other addictions. Indeed, these same factors may also play a role in the development of addiction to L-Dopa observed in some PD patients (Borek and Friedman, 2005; Dagher and Robbins, 2009). For example, a recent review highlights similarities between methamphetamine addiction and L-Dopa sensitization (Fornai et al., 2009). Intriguingly, Palminteri et al. (2009) found similar patterns of RL deficits in patients with TS. Crucially, these patients show the opposite RL pattern – unmedicated TS patients learned better from gains than losses. While this pattern can best be explained by DA hyperactivity in TS patients, this evidence for this account of TS remains controversial (Albin and Mink, 2006; Leckman, 2002; Singer, 1995; Wong et al., 2008). Nevertheless, TS patients are treated with D2 antagonists, such that the DA system is manipulated in opposite direction to PD. Critically, TS patients treated with D2 antagonists exhibited relatively better learning from negative than positive outcomes, very similarly to unmedicated PD patients (Palminteri et al., 2009). These data are also consistent with model predictions: D2 antagonism selectively increases excitability and plasticity of striatopallidal cells (Centonze et al., 2004; Day et al., 2008; Mallet et al., 2006), thereby enhancing NoGo learning.

Motor impairments Progressive development of motor symptoms: sensitization? As described above, unmedicated PD patients exhibit increased negative RL, inducing avoidance

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behaviour (Frank et al., 2004). A stream of rodent experiments performed by Schmidt and colleagues suggest that this behaviour is not limited to environments in which behaviours are explicitly reinforced, but may underlie a fundamental aspect of the cardinal symptoms of PD – akinesia and rigidity (i.e. catalepsy). In these experiments, DA depletion was induced in rats via 6-hydroxy-dopamine lesions or administration of the D2 antagonist haloperidol. Catalepsy was assessed repeatedly on consecutive days. Initially, catalepsy expression was very low (due to partial DA depletion or subthreshold doses of the D2 antagonist). Notably, catalepsy progressively increased with each consecutive test. This progressive manifestation of symptoms is also referred to as sensitization. This sensitization is not simply due to receptor upregulation, as it did not occur in control conditions in which the drug was administered on each day only after the catalepsy test had finished. Moreover, catalepsy expression was context-dependent – it was expressed only in the environmental context in which the animal was sensitized, and not in other novel contexts (Klein and Schmidt, 2003; Wiecki et al., 2009). These data imply that in the presence of DA depletion/D2 blockade, the animal learns an avoidance response in a particular environmental state. Consistent with this depiction, after haloperidol sensitization rats continued to exhibit catalepsy in this context in a subsequent test even when the drug was replaced with a placebo, despite no residual haloperidol being present (Amtage and Schmidt, 2003). After a few days of testing with the placebo, rats’ catalepsy expression returned to baseline (i.e. it was not different from control rats that had never been administered haloperidol). Notably, however, a subsequent single administration of haloperidol yielded stronger catalepsy expression in the rats that had been sensitized than the haloperidol-naive rats. Thus, although catalepsy expression had been extinguished, these data indicate that catalepsy sensitization features a non-extinguishable component. Does the same neurobiological process underlie enhanced NoGo learning in PD patients and

catalepsy sensitization in rodents? As mentioned above, striatal DA depletion increases striatopallidal excitability and plasticity. Haloperidol also potently blocks D2 receptors, induces LTP and enhances phosphorylation of a-amino-3-hydroxy5-methyl-4-isoxazole propionic acid receptor (AMPA) receptors in striatopallidal neurons (Centonze et al., 2004; Haakansson et al., 2006). Furthermore, catalepsy induced by D2 antagonism is abolished following injection of a GABA blocker into the GPe (Ossowska et al., 1984) – suggesting that catalepsy expression results from enhanced striatopallidal inhibition of GPe (and therefore increased GPi inhibition of motor programs). Thus it is plausible that the same mechanisms underlie the two effects. To evaluate this hypothesis, we tested the Frank (2006) BG model under the influence of simulated haloperidol (Wiecki et al., 2009). To simulate the D2 antagonistic properties of haloperidol, we partially reduced the inhibitory effects of DA onto D2 receptors in striatal NoGo units. As can be seen in Fig. 3, the models produced behaviour qualitatively similar to that of rats – with each consecutive test, the time at which the network facilitated a response was progressively slowed. Again, by closely analysing the model dynamics, we can derive a prediction of the underlying neural mechanisms causing this behaviour. In this case, simulated DA depletion or D2 blockade resulted in an over-activation of NoGo units [as observed empirically (Boraud et al., 2002; Day et al., 2008; Mallet et al., 2006; Miller, 2008; Miller and Delong, 1987)]. This excitability resulted in activity-dependent plasticity such that the synaptic corticostriatal weights in the NoGo pathway increased each time the same context was presented. Thus with each consecutive test, the probability to gate a response was reduced, resulting in longer and longer response latencies (Fig. 3). Thus our results are much in line with those of Frank et al. (2004) and subsequent studies in PD, but in a completely different domain and species. Interestingly, sensitization, context dependency and resistance to extinction are all properties observed also

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Fig. 3. Striatal DA depletion or D2 blockade produces context-dependent catalepsy sensitization. (a) Repeated administration of haloperidol results in a progressive increase of catalepsy across days, the expression of which is context-dependent (see context change on day 10) (Klein and Schmidt, 2003). (b) After this sensitization, catalepsy is observed even in absence of haloperidol (extinction, days 9–14), but progressively decreases to baseline levels. However, when challenged with haloperidol on day 15, sensitized rats showed significantly elevated cataleptic response relative to haloperidol-naive rats, thereby revealing a non-extinguishable component (Amtage and Schmidt, 2003). (c) and (d) Modelling results of the basal ganglia model, adapted from Wiecki et al. (2009). Haloperidol is simulated by reducing D2 receptor inhibitory effects on the striatopallidal pathway, leading to increased excitability (see text). (c) With repeated testing, simulated haloperidol led to progressively slowed response times relative to intact networks [intact data not shown; see Wiecki et al. (2009)]. (d) This slowing is due to increases in NoGo (relative to Go) activity which is further enhanced due to corticostriatal Hebbian learning. As observed behaviourally, this learning is context-dependent, and is not seen when switched to a new context (different cortical input pattern of activation, training session 40). Networks are switched to the intact mode (normal D2 function) in training session 60 (i.e. extinction), resulting in speeded responding and declining relative NoGo activity. A nonextinguishable component is also revealed in session 100 when networks are again switched back to haloperidol mode.

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in the appetitive domain of addiction (Schmidt and Beninger, 2006). It is possible that the same principles apply in that case, but with sensitization occurring in the Go pathway. Moreover, this logic has recently been applied to a novel RL experiment in human PD patients (Moustafa et al., 2008a). In this study, rather than choosing among multiple stimuli to maximize rewards, participants had to press just a single button, but had to learn to speed or slow responses in order to maximize positive outcomes and minimize negative outcomes. As predicted by the Go/NoGo model, unmedicated patients were more adept at learning to slow down (relative to their baseline speed) to avoid negative outcomes – that is, they showed a bias towards NoGo learning from negative prediction errors leading to slowed responding. In contrast, medicated patients showed the opposite pattern, learning better to speed responses to increase positive outcomes. This same pattern naturally emerged in the computational model when PD and medications were simulated as previously (Moustafa et al., 2008a). A recent study found learning impairments in a rotarod movement task of mice with selectively denervated dorso-striatal DA (due to PITx3 genetic knockout) which could be rescued by L-Dopa administration (Beeler et al., in print). Crucially, cessation of L-Dopa treatment in trained mice did not result in an immediate performance drop, but rather a progressive decline. Relatedly, healthy mice treated with a D2 antagonist showed the same progressive decline. However, treatment with a D1 antagonist resulted in an immediate performance deficit. In light of our computational framework, this pattern can be explained in terms of learning. D1 antagonists would reduce the signal-to-noise ration in Go neurons such that those cells encoding learned motor associations in the rotarod task are relatively suppressed. This same effect would also diminish synaptic plasticity in these cells (indeed, unpublished simulations of a D1 antagonist in the same model as described above also show an immediate impairment of motor function). D2 antagonists, on the other

hand, would increase NoGo activity while still leaving Go expression of learned associations intact. Initially, learned Go activity may be sufficient to overcome the drug-induced NoGo activity. After repeated exposure, however, NoGo neurons would become progressively active due to LTP and lead to the progressive decline in performance observed experimentally. In sum, these data further support the hypothesis that synaptic plasticity in the indirect pathway is the root of sensitization under low levels of DA (Wiecki et al., 2009). The progressive worsening of symptoms in PD is generally attributed to the progressive cell death of dopaminergic neurons. However, the data reviewed above, along with modelling results, let this symptom progression appear in a different light. Even though it might sound counter intuitive, it seems that motor (and cognitive?) symptoms in PD are, at least partially, learned. To better treat PD patients, we have to explore if and how much of the motor and cognitive symptoms of PD are actually learned due to a dysfunctional learning signal. Ultimately, this could open the door to a whole new set of treatment options if we manage to find a way to unlearn these symptoms.

Dyskinesia and Go learning While L-Dopa is quite effective in the beginning of treatment, following progressive treatment, L-Dopa-induced dyskinesia (LID) begin to appear in certain patients. LID are characterized by excessive and uncontrollable movements. Chronic L-Dopa treatment has been shown to increase DA bursts (Harden and Grace, 1995; Keller et al., 1988; Wightman et al., 1988), and the expression of zif-268, an immediate early gene that has been linked with synaptic plasticity (Knapska and Kaczmarek, 2004) in striatonigral (Go), but not striatopallidal (NoGo) neurons (Carta et al., 2005). In this regard, LID can be seen as the opposite of some PD symptoms (e.g. catalepsy). Like the progressive manifestation of catalepsy in parkinsonian rats, LID are also not present at first but get

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more severe with time. Is the same process underlying catalepsy sensitization at work here, just in the opposite direction (i.e. hyperactivity and LTP of the direct pathway)? Evidence supports this hypothesis. Increased DA levels promote LTP in Go neurons (Carta et al., 2005; Knapska and Kaczmarek, 2004; Shen et al., 2008), and to a higher propensity to learn from positive decision outcomes in medicated PD patients (Frank et al., 2004). Furthermore, LID development is dependent on functional D1 receptors (Lindgren et al., 2009b) and is accompanied by excessive expression and sensitization of D1 receptors in striatonigral neurons in rodent and primate models (Aubert et al., 2005; Berthet et al., 2009; Corvol et al., 2004; Gerfen, 2003). Moreover, dyskinesia development in these models is accompanied by long-term changes in intra-cellular signalling cascades involved in plasticity (Berthet et al., 2009; Crittenden et al., 2009; Gerfen, 2003; Westin et al., 2007) and, perhaps relatedly, loss of bidirectional corticostriatal synaptic plasticity (Berthet et al., 2009; Picconi et al., 2003). Crucially, these cellular changes were selectively found in those animals who had developed dyskinesias, and not others receiving chronic L-Dopa treatment. Computationally, this effect can be explained along the same lines as outlined above. Chronic high levels of DA due to L-Dopa first alleviate the symptoms by inhibiting the over-active indirect pathway and exciting (or increasing the signalto-noise ratio in) the under-active direct pathway. With time, however, LTP in now-active direct pathway neurons causes inappropriate actions to be gated seemingly at random (Bezard et al., 2001; Cenci, 2007; Cenci and Lindgren, 2007; Vitek and Giroux, 2000). This could feasibly arise via artificially elevated DA levels and fluctuations in phasic signals, leading to inappropriate reinforcement of striatonigral neurons, ultimately producing erratic behaviour. Why might there be fluctuations in phasic DA signals unrelated to environmental reinforcement? According to the false-transmitter hypothesis, in the DA denervated striatum, L-Dopa is decarboxylated

to DA and promptly released by serotonergic terminals belonging to presynaptic neurons of the dorsal raphe nucleus. Thus phasic DA signals would be released even if DA neurons themselves are not burst-firing. A similar hypothesis has been invoked to explain impairments in behavioural learning as a function of L-Dopa treatment in PD (Shohamy et al., 2006). In accordance with this theory, the serotonin system is critically involved in the development and the expression of LID (Cenci and Lindgren, 2007; Carta et al., 2007; Eskow et al., 2009). Serotonergic neurons lack DA autoreceptors and DA transporters causing unregulated DA efflux and defective DA clearance [reviewed in Cenci and Lundblad (2006)] which results in increases of extracellular DA levels following L-Dopa administration (Kannari et al., 2001; Lindgren et al., 2009a; Tanaka et al., 1999). Moreover, recent evidence shows that peak extracellular DA levels are about twice as large in dyskinetic animals compared to non-dyskinetic animals. However, high DA release alone was not sufficient to explain dyskinesias, indicating that both, high DA release in response to L-Dopa and increased responsiveness to DA must coexist for dyskinesia expression (Lindgren et al., 2009a). Future research will explore the role of these serotonin dynamics in the BG model and its role in the development of dyskinesias and other behavioural phenomena.

Response vigour Additional support for PD as a disease of action selection rather than motor function per se is provided by Niv et al. (2007). By extending an abstract model of RL to include the average expected reward rate (hypothesized to be encoded by tonic DA activity), the authors explain reduced response vigour observed in PD patients. According to their model, response latency in a free operant task is chosen according to the average reward rate: higher frequency of rewards is associated with increased vigour. Decreased tonic DA in PD lowers this effective rate and thus the

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vigour. Experiments support this hypothesis. In a grasping task, PD patients were able to achieve the same maximal movement speeds as healthy individuals, their average speed was just lower overall (Mazzoni et al., 2007). In a review highlighting the close connection between these findings, the authors conclude that ‘it is not that PD patients cannot move, it is that their DA circuitry does not “want” to’. (Niv and Rivlin-Etzion, 2007).

Working memory impairments Among movement and learning defects, PD is also characterized by WM impairments (Cools, 2005; Frank, 2005; Owen et al., 1992, 1998). Neuroimaging studies reveal that WM impairments in PD patients are associated with decreased BG activity (Lewis et al., 2003; Owen et al., 1998; Postle et al., 1997). Can the same hypothesis that explains cognitive and motor deficits (as outlined above) also explain specific WM impairments of PD? The conceptualization of the BG as a gating device of motor commands (Mink, 1996) is hypothesized to also gate information flow into WM (Frank and O’Reilly, 2006; Frank et al., 2001; Moustafa et al., 2008b; O’Reilly and Frank, 2006). The mechanism is very similar to that described above for gating action plans, only here we simulate BG circuits interacting with PFC rather than premotor cortex, and the ‘action’ is whether or not to maintain the current stimulus in PFC. In this context, Go activity indicates that a representation is task-relevant and should be stored in memory, whereas NoGo activity indicates that the stimulus should be ignored or filtered out of WM. Recent neuroimaging data provide support for this notion (Cools et al., 2007; McNab and Klingberg, 2007). According to this framework, DA depletion as in PD would lead to an increased threshold for updating WM (because of too much NoGo activity), such that most information is treated as irrelevant. In contrast, chronic DA elevations by replacement therapy would result in too much

WM updating and the gating of distracting information into WM. This specific pattern was found in a conjoint behavioural WM task (Moustafa et al., 2008b). Medicated patients were also impaired at ignoring stimulus information that had previously been relevant but is subsequently distracting, consistent with the hypothesis that Go activity for initially relevant information, combined with medication-induced suppression of NoGo activity, results in difficulty filtering out stimuli from WM. Recent evidence further supports this hypothesis. In a task where subjects had to keep certain stimuli in WM while ignoring distracting stimuli, unmedicated PD patients showed abnormally enhanced resistance to distractors (Cools et al., 2010). PD patients were impaired, however, in a task which required repeated updating of WM contents. Did DA depletion block gating of relevant and distracting information into WM? Indeed, susceptibility to distractors was reintroduced by DA replacement medication. Furthermore, a recent study showed that PD patients specifically showed reduced transient (phasic) activation of the BG during WM updating, consistent with impaired gating functionality (Petter et al., 2009).

The subthalamic nucleus, deep brain stimulation and behavioural inhibition A critical aspect of controlled cognition and behaviour is not only knowing which action to select, but also knowing when to cancel a planned response, or to slow down to take more time to make a more considered decision. The original 2005 BG model was extended to include the STN (Frank, 2006). In the model, this nucleus is conceptualized as a dynamic brake on the output structures of the BG. Rather than being part of the classical indirect pathway, the STN receives input directly from cortex and sends diffuse excitatory projections to GPi – the so-called hyperdirect pathway (Nambu et al., 2000). The computational model simulates the dynamics of

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STN activation in response to cortical activity, and how this may be adaptive. Specifically, the STN receives excitatory input from presupplementary motor area (preSMA), which in turn is most active under conditions of response conflict. In the model, preSMA represents the candidate motor actions available in a given context and conveys this information to the striatum, which then gates one of the responses and suppresses others. Response conflict occurs when multiple motor actions are represented concurrently in preSMA in response to a particular environmental stimulus. The resultant increased STN activation provides a temporary brake on action selection by exciting BG output (which then inhibits action selection in the thalamus), allowing more time to resolve conflict such that the optimal decision can be made (Frank, 2006). In PD patients, the STN is pathologically hyperactive (DeLong, 1990; Miller and Delong, 1987), leading to global inhibition of motor programmes (in addition to the NoGo pathway) deep brain stimulation (DBS) of the STN has been successfully applied in PD patients where other therapy options have failed. In this surgical procedure, a stimulating electrode is placed into the pathologically hyperactive STN, which is thought to act similarly to an STN lesion (e.g. Bergman et al., 1990). However, as predicted by the simulations and subsequently confirmed behaviourally (Frank et al., 2007b), the chronic STN stimulation comes at the cost of increased impulsivity because it prevents adaptive slowing in the face of response conflict. This was tested in a version of the probabilistic selection task as described above (Frank et al., 2004). The model and subjects were again trained to select stimuli with different probabilistic reward contingencies. In a successive test it was found that healthy individuals, PD patients on and off medication and PD patients off DBS exhibited relatively slowed responding when selecting among stimuli associated with conflict (i.e. both stimuli had been associated with similar reinforcement contingencies). In contrast, patients on DBS did not exhibit such slowing and even showed

speeded responding under conflict (Frank et al., 2007b). This same pattern was predicted when the STN was disabled in the models to simulate the DBS. Without the dynamic braking signal, models had no way to slow down in high conflict scenarios until the conflict was resolved. For the first time, a link between DBS and impulsive personality changes, so far only reported to neurologists on an individual basis, had been made. Recently, it was reported that DBS induces impulsivity to patients in their every day lives (Hlbig et al., 2009). More recent our lab has recorded EEG from both mediofrontal scalp electrodes and local field potentials in STN depth electrodes in PD patients undergoing DBS surgery. In both scalp EEG and STN local field potentials, power in the theta band (4–8 Hz) is enhanced under conditions of response conflict (Cavanagh et al., in progress). Furthermore, patients off DBS exhibit slower response times when cortical theta power is high, suggesting that cortical conflict produces controlled behaviour. When DBS stimulators were turned on, patients no longer slowed responses with increased cortical theta and the relationship between theta power and conflict was also reduced. These data support the hypothesis that mediofrontal cortical signals recruit the STN to slow behavioural responding under conditions of conflict, and that DBS disrupts this mechanism by preventing the STN from responding naturally to its cortical inputs. In the future, computational models might aid the development of a new generation of DBS systems, which, instead of disabling the STN, will stimulate the STN dynamically, depending on the task at hand. One could imagine, for example, a closed-loop system in which STN stimulation is set according to recorded electrophysiological activity correlating with response conflict. The STN does not solely respond to response conflict. A recent rat study identified neural subpopulations in the STN that respond to different reward types available to the animal (Lardeux et al., 2009). Theoretically, if multiple rewards would be made available, multiple subpopulations

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should get activated simultaneously and thus result in higher overall STN activation. In light of the putative response inhibition role of STN, this would mean that the STN not only halts action selection in the BG during motor response conflict, but also when multiple rewards are present. This conceptualization is still hypothetical and needs further exploration, but may also be adaptive to enable controlled selection of action plans that would produce the most desirable reward.

Conclusion and outlook Neural network models allow us to bridge the gap between the behavioural and neuronal level. By integrating data from different domains into one conglomerate model, we might start to see the ‘bigger picture’. For this approach to be successful, it must stay close to empirical data and provide concrete predictions which have to be tested experimentally to possibly refine the model. These models pose an advantage to the classic box-andarrow diagrams: neural network models provide a more disciplined approach that is grounded by mathematics and allows exploration of more complex dynamics than are considered by static anatomical diagrams. As the research described above has hopefully shown, this approach has already proven to be very valuable in understanding the BG and associated disorders. Nevertheless, we look forward to revising the models to incorporate other existing and future biological data. Acknowledgements This work was funded by the Michael J Fox Foundation for Parkinson’s Research.

Abbreviations BG DA

basal ganglia dopamine

DBS GPe GPi L-Dopa

LID LTD LTP MSN PD PFC preSMA RL SNr STN TS WM

deep brain stimulation external segment of the globus pallidus internal segment of the globus pallidus levodopa L-Dopa-induced dyskinesia long-term depression long-term potentiation medium spiny neuron Parkinson’s disease prefrontal cortex presupplementary motor area reinforcement learning substantia nigra pars reticulata subthalamic nucleus Tourette’s syndrome working memory

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Subject Index

Note: The letters ‘f ’ and ‘t ’ following the locators refer to figures and tables respectively.

A2A antagonists for PD trials for disease modification, 197–198 therapeutic targets, purine metabolic pathway, 198f trials for symptomatic anti-parkinsonian indications, 195–197 non-selective adenosine antagonists, 195–196 selective adenosine A2A antagonists, 196–197 Abnormal burst firing, 280, 281 Abnormal involuntary movements (AIM), 174, 210, 214–219, 222 dyskinesia, 174–178, 195, 197, 209–227, 238–239, 245–246, 286–287 Accelerometers, 263, 268 Activation of diacylglycerol (DAG), 150 Activation of protein kinase A (PKA), 150, 211 Acute dopaminergic modulation of striatal MSN excitability, 150–152 D1 receptor coupling activation of protein kinase A (PKA), 150 striatal-enriched tyrosine phosphatase (STEP), 150 D2 receptor coupling, 150 activation of DAG/PKC/IP3, 150 Addiction, 170, 276, 279, 282–284 Adenosine A2A receptors and neuroprotection, 189–190 See also Neuroprotection and adenosine A2A receptors Adenosine antagonists, epidemiology and clinical trials of caffeinated beverages, 194

Health Professionals Follow-up Study (HPFS), 194 higher initial coffee intake, 194 Honolulu Heart Program, 194 Nurses Health Study (NHS), 194 rates of consuming decaffeinated coffee, 194 caffeine epidemiology, 193–194 coffee/tea, 193–194 gender differences (oestrogen interactions), 194–195 caffeine–PD link, 195 coffee exposure in women, 194 effect of post-menopausal hormone, 194–195 MPTP toxicity, 195 oestrogen replacement caffeine use, 194 PD progression, 195 CALM-PD cohort, 195 NET-PD trial cohort, 195 pramipexole vs. levodopa, 195 UPDRS, 195 Adenosine/caffeine and urate, pathophysiological roles for purines, 183–200 adenosine antagonists caffeine epidemiology, 193–194 cohort studies, 194–195 clinical trials of A2A antagonists trials for disease modification in PD, 197–198 trials for symptomatic anti-parkinsonian indications, 195–197 functional interactions with dopamine receptors, 183–184 localization of adenosine receptors, 183–184 299

300

Adenosine/caffeine and urate, pathophysiological roles for purines (Continued) neuroprotection and adenosine A2A receptors, 189–190 glial A2A receptors and neuroprotection, 192–193 neuronal A2A receptors and neuroprotection, 190–192 neuroprotection and A1 adenosine receptors, 187–189 receptor–receptor interaction, 184–187 role of adenosine receptors in neuroprotection, 187 urate, novel target for neuroprotection biology, 198–199 epidemiology, 199–200 Adenosine receptors and functional interactions with DA receptors, 183–184 A2A/CB1/mGlu5 receptors in striatopallidal neurons, 185f in neuroprotection, role of, 187 A2A receptor blockade, 188f models of ischemic and excitotoxic brain injury, 187 neuroprotective outcome of A2AR manipulation, 190t pro-neurotoxic role of adenosine, 187 Adenosine triphosphate (ATP), 32f, 49, 64–65, 67, 72, 102, 104, 219 Advances in PD, 115–140 phosphorylation, 117–126 rate of a-synuclein fibrillization, factors affecting, 117 structural and biochemical properties of a-syn, 116–117 truncations, 126–130 in vivo studies, 129–130 ubiquitination, 130–136 Aetiology of PD, 61, 64, 100–102 Affymetrix gene chip arrays, 218 Age of onset in PD, 4 Aggregated a-synuclein, 34 Aging brain disorder, 99 Akinesia, 170, 238–239, 244, 260, 262, 265t, 268, 275, 283

Akinetic-like symptoms, 236 Alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptors, 172 in experimental PD, alterations of, 172–173 activation of CRE elements, 172 GluR1 immunoreactivity, 173 levels of GluR1 subunit, 173 MPTP-lesioned monkeys, 173 6-OHDA-lesioned rats, 173 tetrameric proteins, 172 modulation in PD therapy, 175–176 ampakines, allosteric modulators, 176 BDNF in regulation of neuronal activity, 176 expression of DARPP-32, 176 high-affinity TrkB, 176 MPP(þ)-induced toxicity, 176 neurodegenerative diseases, 175 6-OHDA and MPTP rodent models of PD, 176 Alpha-synuclein (protein) (aSYN), 4–6, 16 See also a-synuclein (a-syn) Alzheimer’s disease (AD), 4, 118, 125–126, 171 Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), 150, 154f, 156, 172–173, 175–177, 211, 215–216, 284 Ampakines, 176 Amyloid precursor protein (APP), 4, 16 Amyotrophic lateral sclerosis, 4, 50 Anandamide, 152–153 “An Essay on the Shaking Palsy,” 22f Ankyrin-like repeats, 27 Anti-parkinsonian (symptomatic) indications, trials for non-selective adenosine antagonists, caffeine A1, A2A and A2B sub-types, 195 caffeine effects on parkinsonian symptoms, 196 idiopathic PD and excessive daytime somnolence, 196 parkinsonian motor symptoms, 196 non-selective adenosine antagonists, theophylline, 195–196 dykinesias, 196 non-specific adenosine antagonist, 196 selective adenosine A2A antagonists, 196–197 BIIB014, 196

301

dyskinesias, 197 istradefylline, 196–197 levodopa, 197 New Drug Application (NDA), 196 PD/monotherapy trials, 197 phase III studies, 196 phase II trial results, 196 preladenant, 196 syn-115, 196 US Food and Drug Administration (FDA), 196 xanthine and non-xanthine structure-based adenosine receptor, 196 Apoptotic pathways, 27, 87 2-arachidonylglycerol (2-AG), 152 a-synuclein (a-syn), 23–26 affects protein degradation, 26 aggregation effects of S!D and S!A substitutions on, 122 in vivo, 120–121 aggregation and toxicity, 133–136 140 amino acid protein, 24 amyloidogenic proteins, 25 tau, 25 A53T and A30P (SNCA mutations), 25 in brains MSA and synucleinopathy, 26 Ca2þ levels, 26 chaperone mediated autophagy, 26 C-terminal truncations promote fibrillization of, 126–128 duplication or triplication of SNCA, 24 enzymes in proteolysis of, 128–129 expressed in neurons, 24 fibrillization factors affecting, 117 in vitro at S129 and S87, 120 in vivo and in vitro, inclusion formation, 121 209G>A (Ala53Thr) mutation, 24 gain-of-toxic-function mechanism, 24 genetic knock-out studies, 24 Lewy bodies and Lewy neurites, 24–25 mitochondrial complex I inhibitors, 25 mutant, 26 oligomerization and fibrillogenesis, 116f oxidative and nitrative damage, 25 oxidative stress, 26

PD and AD, clinical and pathological features, 25 physiological and pathogenic activities of, 120 plasma a-synuclein levels, 24 post-translational modifications, 118f potential phosphorylation sites, 119 role in neurotransmission, 24 SNARE complex, 24 SNCA mis-sense mutations 188G>A (Glu46Lys), 24 88G>C (Ala30Pro), 24 species in brain/PD and related disorders, 126 structural and biochemical properties of, 116–117 C-terminal region, 116–117 fibril formation, 117 hexameric motif (KTKEGV), 116 non-amyloid component region (NAC), 116 N-terminal region, 116 with synaptic neurotransmission, 26 toxicity, 122–123 in Drosophila and rodent models, 123 phosphorylation-dependent, 124–125 toxicity to neurons, 25 wild-type or A30P, 26 yeast and mammalian systems, studies in, 24 a-synuclein (SNCA) protein, 44 Atypical parkinsonian syndromes, 11 Autophagic vesicles (AV), 107 Autophagolysosome, 105 Autophagy, 26, 48–49, 100, 102, 104–107 -related gene 6 (Atg6), 104 Autosomal-dominant forms of inherited PD, 4–5 erroneous classification, 5 haplotype, 5 linkage analysis, 5 lod score, 5 Autosomal-dominant parkinsonism, 5 Autosomal recessive early-onset parkinsonism, 8 Autosomal recessive juvenile-onset parkinsonism, 8, 29 Autosomal-recessive monogenic diseases, 8 Autosomal-recessive parkinsonism autosomal-recessive juvenile parkinsonism, 8 autosomal-recessive monogenic diseases, 8 PARK2 (parkin), 8

302

Axon degeneration and PD, 85–86 age-related hypokinesia, development of, 86 CEP-1347, mixed lineage kinase inhibitor, 85 loss of striatal dopaminergic markers, 85 LRRK2(G2019S) mutant, 85 MAPK signalling blocks apoptosis, 86 neurodegeneration in PD, 85 PRECEPT neuroprotection trial in PD, 85 TH immunostaining, 85 Axon degeneration, inhibition of, 86–87 C57Bl/6 mice, mutations in, 86 experiments with NMNAT3, 86 GFP in TH-GFP mice, 87 85-kb tandem triplication, 86 NAD synthesizing enzyme, 86 neurotoxin models of Parkinsonism, 86 NMNAT, 86 N-terminal 70 amino acids of Ube4B, 86 6OHDA or axotomy, 86 toxic peripheral neuropathies and genetic neuropathies, 86 WldS mutant mouse, 86 Axon initial segment (AIS), 157 Bacterial artificial chromosome (BAC), 150 Ballism/hemiballism, 243 Basal ganglia (BG), 3, 235, 260–261, 268–271, 275–276, 277–279 anatomy of, 277f architecture of, 277–279 cognitive learning deficits, 280–282 levodopa and positive reinforcement learning, 282–283 dopamine, 279–280 extracellular simultaneous recordings, 262 method, 261 accelerometers, 263 animals, 261 data analysis, 263–264 MPTP treatment and perfusion, 262–263 motor impairments dyskinesia and Go learning, 286–287 response vigour, 287 sensitization, 283–286 subthalamic nucleus, DBS and behavioural inhibition, 288–289

working memory impairments, 287–288 motor symptoms of, 260–261, 268–271 neural network models of, 276–277 neurons, 261 pallidal recording, 263t results animals clinical states, 264–265 histology, 265 MPTP neuronal oscillatory activity, 267–268 population analysis, 266–267 spontaneous neuronal activity in MI and GP, 265–266 Beclin-1, See Autophagy Behaviour, See Motor behaviour, effects of manipulation of STN; Motor behaviour, GPi on Bonferroni corrections, 13 BRET (Bioluminescence Resonance Energy Transfer) analyses, 185 Ca2þ/calmodulin-dependent protein kinase I alpha (CaMKIalpha), 101 Caffeine epidemiology coffee, 193 dietary aetiology of chronic diseases, 193 non-selective adenosine receptor antagonist, 193 reduced risk of developing PD, 193 tobacco smoking and confounding factors, 193 tea, 193–194 case–control studies, 194 consumption and PD risk, 193 CalDAG-GEF2, See RAS guanyl nucleotidereleasing protein 1 (RasGRP1) cAMP response element-binding (CREB), 90f, 184, 211, 212f, 217 cAMPresponsive element (CRE), 130, 172, 176, 190, 217, 224f Cardinal symptoms, 283 Caspases, 83, 88 Catalepsy, 245, 283–284, 285f, 286 Cdk-5 and MEF2 transcription factors, 81–82 calpains, calcium-dependent proteases, 81–82 cell death in SN, 81 chronic MPTP model, 81 MEF2, 81

303

neural differentiation, Cdk5 and p35 role in, 81 serine/threonine protein kinase, 81 Cell autonomous mediators, 80 Cell-specific risk factors in PD, 61–65 calbindin expression, 64 cortical pyramidal neurons, 62 endoplasmic reticulum (ER), 64 excitotoxicity, 64 interaction between risk factors, 66–67 aging, 66 aging-related cell death in humans, 67 aging-related decline in SNc mitochondrial function, 67 alpha-synuclein over-expression, 67 cell death, combination of cell-specific/pancellular factors, 66f inflammation, 67 selective pattern of degeneration, 67 L-DOPA administration, 62 metabotropic glutamate receptor (mGluR), 65 motor symptoms, 61 MPTP, 6-hydroxydopamine (6-OHDA), rotenone, 64 NMDA receptors, 64 regional variability, 62 for SNc DA neurons, 65 Cav1.3 calcium channels, 64 locus ceruleus (LC) neurons, 62 L-type calcium channels, 62 L-type ion channels, 62 mitochondrial density, 62 mito-roGFP, 64 physiology, 63f types of neuron, 65–66 autonomous or spontaneous activity neurons, 65 BF cholinergic neurons, 65 DA neurons, 65 DMV cholinergic neurons, 65 lateral hypothalamic neurons, 65 LC noradrenergic neurons, 65 PPN cholinergic neurons, 65 serotonergic neurons, 65 tuberomammillary neurons, 65

Cell transplantation procedures, 210 Central nervous system (CNS), 82, 107, 170–171, 187, 195, 199 Ceramide pathway, 9 Chaperone mediated autophagy, 26 Charcot–Marie–Tooth neuropathy, 16 type 2A, 101 Childhood-onset lysosomal storage disease, 9 CNS disorders (AD/PD/HD/epilepsy), 171 Cognition and motivation, effects of manipulation of STN STN HFS data in rats, 248–250 bilateral STN HFS, effects of, 250 effects of bilateral HFS of STN in 5-CSRTT, 249f perseverative responses recorded in food magazine, 249 STN in limbic loop, 247f STN lesion in rats, 247–248 5-choice serial RT task (5-CSRTT), 247–248, 248f lesioning DA inputs to dorsal striatum, 247 STN lesion or STN HFS in monkeys, 246–247 Cognition, effects of manipulation of GPi on, 240 beneficial effect of pallidotomy, 240 Cognitive learning deficits, 275–276, 280–282 levodopa and positive reinforcement learning, 282–283 COR (C-terminal of ROC), 27 Correlogram, 264 Corticobasal degeneration (CBD), 11 C-terminal of ROC (COR), 27, 44, 50 Cyclic adenosine monophosphate (cAMP) formation, 184 Cyclic AMP (cAMP), 101, 150, 184, 185f, 211, 212f, 213, 216, 221f, 224f Cyclin-dependent kinase (Cdk1/cyclin B), 81–82, 101 Cytochrome oxidase subunit I (CO-I), 219, 242 D1 and NMDA receptors, 176–178 D1 DA receptor stimulation, 177 D1/NMDA receptor complexes, 177–178 D1/NR1 interaction, 177 D1 receptor-mediated potentiation, 177

304

D1 and NMDA receptors (Continued) L-DOPA-induced dyskinesias, 177 NR2A and NR2B-containing receptors, 177 PKA- and DARPP-32-regulated phosphorylation, 177 substantia nigra pars compacta, 176 DA receptor signalling in DA-denervated striatum, 213–214 denervation-induced supersensitivity of receptors, 214 GTP-binding activity, 214 hyperactive canonical signalling, 214 ligand-binding activities, 214 non-canonical signalling pathway activation, 214 parkinsonian motor symptoms, 213 post-synaptic DAreceptor sensitivity, 213 RGS, 214 viral vector-mediated over expression of RGS9-2, 214 in neurons of intact striatum, 211–213 adenylyl cyclase enzyme(s), 211 CaM kinase II and IV, 211 canonical and non-canonical signalling, 212f cyclic AMP (cAMP), 211 DAG and IP3, 213 dephosphorylation/inactivation of Akt, 213 direct and indirect striatofugal pathways, 211 D1-like DA receptors (D1, D5), 211 D2-like receptors (D2, D3, D4), 211 formation of protein complex, 213 GSK3-mediated signalling, 213 kainate receptors and L-type Ca2þ channels, 211 methylation of CpG islands, 213 molecular cloning, 211 NMDA receptors, effects on, 211 opioid precursor genes, 211 post-translational modifications, 213 preproenkephalin (preproenkephalin-A), 211 prodynorphin (preproenkephalin-B), 211 protein kinase A (PKA), 211 DA replacement therapy, 174, 210, 262, 282 DARPP-32 gene, 283

DATATOP cohort, 199–200 Death domains (DD), 27, 88f Death effector domain (DED), 88–89 Death-inducing signalling complex (DISC), 88 Deep brain stimulation (DBS), 236, 260, 288–289 Defective genes, role of, 43–53 genetic basis of parkinsonism Mendelian genes for PD show segregation, 44–46 risk factor genes show association, 46–47 mutations in recessive genes decrease protein function, 47–49 ATP utilization by mitochondria, 49 carbonyl cyanide m-chlorophenylhydrazone, 48 direct phosphorylation of parkin by PINK1, 49 DJ-1, 49 Drosophila melanogaster homologues of PINK1 and parkin, 48 early-onset parkinsonism, 49 G411S variant, 48 kinase activity, 49 male sterility, 48 mitochondrial dysfunction/phenotypes, 48 PINK1-deficient flies, 48 recessive mutations, 48 ROS production, 49 mutations in SNCA and LRRK2 alter protein function, 49–52 amino acid sequence, 50 animal models, 51 A30P and A53T mutation, 50 A53T and E46K mutations, 50 authentic outputs of LRRK2 kinase, 51 authentic pathogenic mechanisms, 50 GTPase activity and binding, 51 kinase activity with autophosphorylation, 50–51 LRRK2, 50 mutations in ROC region, 51 ROC/GTPase domain, 51 SNCA protein chemistry in vitro, 50 soluble oligomers, 50 superoxide dismutase mutations, 50

305

risk variants found in association studies, 52 LRRK2 expression, 52 MAPT risk variants, 52 single-nucleotide polymorphism (SNP), 52 Degeneration of dopaminergic neurons (DA), 99 Dementia, 5–7, 9–11, 23t, 24–25, 31, 52, 99, 117 Dementia with Lewy bodies (DLB), 7 Dendritic excitability and synaptic plasticity, 156–158 convergent synaptic stimulation in dendritic tuft, 157 spikes in AIS, 157 Diacylglycerol (DAG), 150, 152, 212f, 213, 222 Diffuse Lewy body disease (DLBD), 8, 44 Dihydropyridines L-type channels, for pacemaking, 68–69 all-points histogram of membrane potential, 68 apparent dissociation constant (Kapp), 68 autonomous spiking, 68–69 blood–brain barrier (BBB), 68 Cav1.3 channel function, 68 Cav1.3 pore-forming subunit, 68 dissociation constant for low-affinity state, 68 dissociation constant (KD) for high-affinity state, 68 dose–response curve, 68 isradipine, 68 modulated receptor model, 68 robust network of ion channels, 69 SK and HCN channels, 69 two-photon laser scanning microscopy, 68 voltage-dependent inactivation, 68 Disease-causing mutations, 15 Disease modification in PD, trials for, 197–198 anti-parkinsonian motor effects, 197 levodopa-induced dyskinesia (LID), 197 neuroprotection trial, 197 therapeutic targets along purine metabolic pathway, 197 DJ-1, 30–31 cellular redox sensor, 31 deficiency in Drosophila, 31 evidence in mice, 31 member of ThiJ/PfpI family, 30 protective effects, 31

DNA polymorphisms, 4–5, 11, 29, 46, 52, 282–283 Dominant optic atrophy (DOA), 101 Dopamine- and cAMPregulated phosphoprotein32 (DARPP-32), 184 Dopamine (DA), 261, 279–280 depletion, 284, 285f, 288 dorso-striatal, 286 modulating structure and function of striatal circuits, 149–163 D1 and D2 MSN are differentially excitable, 151f D1 DA receptor, 150 D2 DA receptor, 150 receptors, 170 replacement medication, 288 therapy, 282 Dopaminergic neurons in PD, causes of death of, 59–73 cell-specific risk factors in PD, 61–65 interaction between risk factors, 66–67 other types of neuron, 65–66 for SNc DA neurons, 65 dihydropyridines L-type channels, for pacemaking, 68–69 epidemiological support for cell-specific risk factors, 69–71 pan-cellular risk factors, 59–61 Dopamine transporter (DAT) ligand, 134f, 200 DRD2 gene, 283 Dual leucine zipper kinases (DLK), 83 Dynamin-related protein 1 (Drp1), 101 Dyskinesia, 210 striatal DA denervation and pulsatile treatment, 210 Dyskinesia and Go learning, 286–287 Dyskinesia-priming action of L-DOPA, 219 Early-onset familial disease, 23 Early-onset parkinsonism, 8–9, 49 4E-BP phosphorylation, 27 E46K mutation, 7, 50 Electrophysiology, 241 Elusive oligomers, 6 Endocannabinoids (EC), 152

306

Endocannabinoids (EC) (Continued) anandamide and 2- arachidonylglycerol (2-AG), 152 Endoplasmic reticulum (ER) mass, 64, 72, 80, 105, 124 Epidemiological support for cell-specific risk factors, 69–71 calcium channel antagonists (CCA), 69 co-enzyme Q10, 71 creatine, 71 DHP isradipine, 69 low relative affinity of DHP, 69 nanomolar concentrations of isradipine, 70f–71f pharmacokinetic studies, 69 rasagiline or deprenyl, 71 Epilepsy, 171–172 Erroneous classification, 5 Eukaryotic initiation factor 4E (eIF4E), 27 Exogenous phosphatase and tensin homologue (PTEN), 27, 45, 60, 87, 99, 102, 104 Experimental parkinsonism, 171–173, 175, 178 Extracellular simultaneous recordings, 262, 266f Ezrin/radixin/meosin (ERM) protein family, 27–28 Familial and sporadic PD, 47 Fas-associated protein with DD (FADD), 88–89 Fission, 33, 100–103, 105–108 Fluorescence resonance energy transfer (FRET), 185–186 FosB/DFosB, 217–218, 222, 223f Frontotemporal lobar degeneration (FTLD), 11 Fusion, 33, 100–103, 105–108, 184, 186, 191, 210, 218, 238–239, 244, 262–263 Gain-of-toxic-function mechanism, 24 Gamma-aminobutyric acid (GABA) neurons, 184, 186–187, 236f, 242, 244–245, 247, 247f, 284 Gaucher’s disease, 9 Genetic basis of parkinsonism Mendelian genes for PD show segregation, 44–46 diffuse Lewy body disease (DLBD), 44 dominant mutations in SNCA and LRRK2, 45 dopaminergic neuron degeneration, 45 G2019S, mutation, 44 LRRK2 protein, 44

mutations in Htra2/omi, 46 protein aggregation, 45 recessive mutations in parkin/DJ-1 and PINK1, 45 single parkin mutations, 45 SNCA or tau proteins, 45 three point mutations, 44 risk factor genes, 46–47 ApoE4 variant and Alzheimer’s disease, 46 familial and sporadic PD, 47 GWAS, 46 MAPT/tau gene, 46 PARK16, 47 risk variants in SNCA, 46 SNCA and LRRK2, 47 variant in LRRK2 (G2385R), 47 Genetic research of PD, impact of, 21–34 dominantly inherited mutations DJ-1, 30–31 LRRK2, 26–28 mechanisms of PD pathogenesis, 31 mitochondrial dysfunction and oxidative stress, 31–33 parkin, 29–30 PINK1, 30 recessive mutations, 29 a-synuclein, 23–26 ubiquitin–proteasome system impairment, 33–34 identifying role for genetics in PD, 22–23 early-onset familial disease, 23 genes underlying familial PD, 23t narcotic use, 22 non-genetic disease, 22 parkinsonism and loss of nigral DA neurons, 23 role for genetics in PD, 23 symptoms of PD, 21 timeline of key discoveries in PD pathogenesis, 22f Genetic risk factors, 11, 16 Genetic variants causing or pre-disposing to PD, 9–10 ceramide pathway, 9 childhood-onset lysosomal storage disease, 9 Gaucher’s disease, 9 glucocerebrosidase (GBA), 9–10

307

G2385R or R1628P variants, 9 G2019S variant, 9 M mutation in GBA, 10 neuropathologic examination of 17 GBA mutation carriers, 10 N370S mutation, 10 rare variant-common disease hypothesis, 9 R1441C or Y1699C mutations, 9 ‘1000 genomes project,’ 16 Genome-wide association studies (GWAS), 4, 12–14, 46 Genome-wide transcriptome analysis, 4 Glial A2A receptors and neuroprotection, 192–193 antagonist SCH58261, 192 astroglial and microglial activation, 192 neuroinflammation, 192 persistent microglial activation, 192 striatal Olig 2 transcription factor, 192–193 Glial cell linederived neurotrophic factor (GDNF), 89–90, 90f, 188f, 191–192 Globus pallidus (GP), 150, 161, 184, 191, 236, 236f, 242, 260–265, 263f, 264f, 266f, 267–270, 267f, 269f, 270f, 278, 288 Globus pallidus (GPi) manipulation in PD, 237–240 effects of manipulation of GPi on cognition, 240 irregular pattern characterized by bursts of action potentials, 240 effects of manipulation of GPi on motor behaviour EP HFS data in rodents, 239–240 GPi HFS in monkey, improve parkinsonian symptoms, 239 lesion and pharmacological EP manipulation in rat, 239 lesion and pharmacological GPi inactivation in monkey, 238–239 neurophysiological effects, 237–238 EP inactivation in rodents, 238 temporal locking, 237 Glucocerebrosidase (GBA), 9–10, 15 Glutamate receptor subunits with post synaptic density proteins, 169–178 alterations of AMPA receptors, 172–173 alterations of glutamatergic synapse in LID, 174–175

alterations of NMDA receptor complex, 170–172 AMPA receptor modulation in PD therapy, 175–176 functional and molecular cross-talk between D1 and NMDA receptors, 176–178 NMDA receptor antagonist in experimental PD and in dyskinesia, 175 pathological synaptic plasticity in striatum, 173–174 Glutamatergic synapse in LID, 174–175 abnormal involuntary movements (AIM), 174 chronic L-DOPA therapy, 174 DA replacement therapy, 174 development of LID, 174 levels of PSD-95 and SAP97, 174 treatment of non-dyskinetic animals with synthetic peptide (TAT2B), 174 unilateral 6-OHDA model of PD in rats, 174 Glycogen synthase kinase 3 (GSK3)-mediated signalling, 213 GPi and STN inactivation, effects of basal ganglia organization after DA depletion, 236f deep brain stimulation (DBS), 236 6-hydroxydopamine (6-OHDA), 237 MPTP, 237 output BG nuclei, 236 reserpine, 236 G-protein-coupled receptor kinases (GRK), 120–122, 134f, 135t, 211, 212f, 215 G-protein-coupled receptors (GPCR), 184, 211, 214–215 Green fluorescent protein (GFP), 64, 87, 105, 122–123, 136t, 150, 162f Grif-1 and OIP106, mammals homologues, 104 Guanosine triphosphate (GTP)-binding activity, 27, 44, 50–51, 100–101, 104, 107, 212f, 214, 222 Haplotypes (H1/H2), 11–12 High-frequency stimulation (HFS) protocol, 155, 173, 218, 236–251, 249f Homeostatic plasticity in PD models, 158–161 DA depletion LTP in D2 MSN and LTD in D1, 158 reduction in spine density in D2 MSN, 159f synaptic plasticity lost, 158

308

L-type Ca2þ channels for spine and synapse elimination, 160f mechanisms, 159 MEF2 -dependent Arc expression, 162f synaptic remodelling genes Nur77 and Arc, 161 upregulation, 161 Homozygous loss-of-function mutation, 29 Huntington’s disease (HD), 4, 161, 171–172, 191 Hybridization techniques solidphase or emulsion-based short sequences, 15 6-hydroxydopamine (6-OHDA), 64, 69, 82–83, 84f, 86–87, 90–91, 171, 172f, 173–176, 184, 189, 190t, 191, 210, 215, 217–219, 221f, 222, 223f, 237–239, 241–243, 245f, 246, 249f, 283 Hyper-direct pathway, 240 Hyper-kinetic symptoms, 260 Immunoblotting, 124, 221f Inner mitochondrial membrane (IMM) fusion, 100–101, 107 Inositol trisphosphate (IP3), 72, 150, 152, 212f, 213 International Human Genome Sequencing, 15 Intracellular signalling pathways for dopaminergic axonal degeneration, 79–91 axon degeneration and PD, 85–86 inhibition of axon degeneration, 86–87 PCD in dopamine neurons, 87–91 See also PCD in dopamine neurons resistance of neuron cell bodies, 84f Kinesin isoforms kinesin-3 (KIF1B), 104 Knockdown (KO), 47, 87, 101–103, 105, 135t, 136t Kufor-Rakeb syndrome, 29 Late-onset dopa-responsive parkinsonism, 6–7 Late-onset neurodegenerative disorder, 15 LC3 in mammalian cells, See Ubiquitin-like protein Atg8 and Atg4 protease L-Dopa administration, 62, 218, 221f, 286–287 L-Dopa-induced dyskinesia (LID), 170, 174–177, 209–226, 238–239, 245–246, 286

L-DOPA (L-3,4-dihydroxyphenylalanine)

or dopaminergic agonists, 7, 22, 29, 61–62, 170, 174–177, 184, 209–226, 238–239, 244–246, 262, 282–283, 286–287 Leabra framework, 276 Lesion pharmacological and molecular STN inactivation in monkeys, 243–244 akinesia and bradykinesia, 244 ballism or hemiballism, 243 transfection with adeno-associated virus, 244 pharmacological and molecular STN inactivation in rats, 244–245, 245f anti-parkinsonian therapy, 245 DArgic depletion, 244 L-DOPA-induced dyskinesia, 245 premature- responding deficit, 244 reduce circling behaviour, 244 pharmacological EP manipulation in rat, 239 akinetic-like deficit/deficit in simple reactiontime (SRT), 239, 240f decreased rotations induced by amphetamine, 239 pharmacological GPi inactivation in monkey, 238–239 enhancement of STN hyper-activity, 239 focal inactivation of GPi with muscimol infusions, 238 kainic acid lesion, 238 STN HFS in monkeys, 246–247 STN, in rats, 247–248 5-choice serial RT task (5-CSRTT), 247–248, 248f lesioning DA inputs to dorsal striatum, 247 Leucine-rich repeat kinase 2 (LRRK2), 7, 26–28 autosomal dominantly inherited mutations, 26 cell death by apoptosis, 28 domains, 27 Drosophila (dLRRK) mutation, 27 4E-BP phosphorylation, 27 eukaryotic initiation factor 4E (eIF4E), 27 ezrin/radixin/meosin (ERM) protein family, 27 G2019S mutation, 27 G2019S or I2020T mutations, 28 280 KDa protein, 27

309

MAPK kinase (MAPKK) kinases, 27 MAPK signalling pathway, 28 moesin phosphorylation, 27 phosphorylation of JNK and c-Jun, 28 PTEN-induced putative kinase 1 (PINK1), 27 role of kinase activity, 28 Ser65/Thr70, 27 Levodopa, 282–283 Lewy body (LB) and Lewy neurites, 6, 24–25, 60 causes and evidence, 25 dementia with Lewy bodies, 25 dopaminergic and non-dopaminergic neurons, 25 parkinsonism with dementia, 25 Lewy neuritis, 6 Linkage analysis, 5 Local drug infusion experiments, 210 Lod score, 5, 12 Long-term depression (LTD) at glutamatergic synapses on MSN inhibition of DAGLa, 153 generation, endocannabinoids (EC), 152 NO signalling, 153 post-synaptic depolarization, 152 STDP in D1 and D2 MSN, 154f Long-term potentiation (LTP) at glutamatergic synapses on MSN, 155–156 activation of A2a adenosine receptors, 155 activation of STEP, 156 anti-Hebbian plasticity, 156 regulator of calmodulin signalling (RCS), 156 spike timing-dependent plasticity (STDP), 155 Loss-of-function mutations, 15, 29, 31, 32f Low-coverage genome sequencing, 16 ‘1000 genomes project,’ 16 whole genome SNP analyses, 16 Low-frequency stimulation (LFS) of corticostriatal fibers, 173–174, 218 Magnetoencephalography (MEG) recordings of PD, 270 Maladaptive striatal plasticity in LID, 209–226 DA receptor signalling in DA-denervated striatum, 213–214 in neurons of intact striatum, 211–213 molecular alterations

expression and regulation of transcription factors, 217–218 gene expression patterns, 218–219 intracellular signalling, 216–217 intracellular trafficking of DA and glutamate receptors, 215–216 plasticity of corticostriatal synapses, 218 molecular interpretation dysregulated D1 receptor-dependent signalling, 222–225 signalling pathway activation in dyskinetic rodents, 219–222 molecular plasticity leads to plasticity failure, 225 MAPK kinase (MAPKK) kinases, 27–28, 222 Mechanistic/neural network models, 276 Medium spiny neurons (MSN), 149, 211, 278 striatonigral MSN, 150 striatopallidal MSN, 150 Mendelian genes for PD show segregation, 44–46 DLBD, 44 dominant mutations in SNCA and LRRK2, 45 dopaminergic neuron degeneration, 45 G2019S, mutation, 44 LRRK2 protein, 44 mutations in Htra2/omi, 46 protein aggregation, 45 recessive mutations in parkin/DJ-1 and PINK1 deletion/truncations and point mutations, 45 gene rearrangements, 45 heterozygous mutations, 45 single parkin mutations, 45 SNCA or tau proteins, 44–45 a-synuclein (SNCA) protein, 44 three point mutations, 44 A53T/A30P and E46K, 44 triplications and duplications, 44 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 22–23, 22f, 100, 171, 210, 237, 260–269, 264f, 265f Micro-array technology, 4 Microtubule-associated protein tau (MAPTau), 10–12, 14, 46, 137 Midbrain dopaminergic neurons, 260, 279 Milton family proteins, 104

310

Mitochondrial autophagy (mitophagy), 100, 104–107 Mitochondrial complex I inhibitors rotenone and MPTP, 100 Mitochondrial dysfunction and oxidative stress, 31–33 genetic interaction between PINK1 and parkin, 33 genetic manipulations of Mfa/Opa1, 33 inhibition of mitochondrial respiratory chain complex I, 33 knock-out studies in animals, 31 loss-of-function mutations, 31 loss of PINK1 function in flies, 32 mitochondrial respiration, 33 mutations and multiplications in SNCA, 33 nigrostriatal deficits, 31 oxidative stress, 32 toxic effects of MPTP on neurons, 33 Mitochondrial fusion and fission (Mff) machinery, 100–103 autophagy, 102 autosomal DOA, 101 CaMKIalpha, 101 cAMP-dependent protein kinase, 101 carbonyl cyanide m-chlorophenylhydrazoneinduced fragmentation, 101 Cdk1/cyclin B, 101 dPINK1 KO and mutant flies, 102 Drosophila PINK1 (dPINK1), 102 dynamin-related proteins, role by, 100–101 guanosine triphosphate (GTP), 100 IMM fusion, 100 KO of Fis1 blocks mitochondrial fission, 101 Mff and Fis1, 101 mitochondrial RING finger ubiquitin ligase MARCH-5, 101 mitofusins 1 and 2 (Mfn1 and Mfn2), 100 mtDNA mutations, 101 mutations in Mfn2 and Opa1, 101 OMM fusion, 100 optic atrophy protein 1, 100 over-expression of Opa1 or Mfn, 102 over-expression of PINK1 in COS-7 cells, 103 parkin, cytosolic ubiquitin E3 ligase protein, 102 pathogenesis of PD, genes in, 102 PINK1 in HeLa cells, silencing of, 103

PINK1 (PARK6), 102 PINK1/parkin in mammals, 103 small ubiquitin-like modifier-1 (SUMO1), 101 synapse formation, 102 Mitochondrial integrity in PD, control of, 99–108 Mff machinery, 100–103 mitophagy, 104–107 motility, 103–104 See also Individual Mitochondrially targeted redox-sensitive variant of green fluorescent protein (mito-roGFP), 64 Mitochondrial motility, 103–104 abnormal mitochondrial morphology, 104 defective transport of axonal mitochondria, 103 exogenous PTEN, 104 Grif-1 and OIP106, mammals homologues, 104 KIF1B, 104 kinesin-1 (KHC, KIF5B), 104 microtubule system in neuronal process, 104 mitochondrial trafficking, 104 neurons, 103 OMM proteins (Miro1 and Miro2), 104 PI3K pathway, 104 protein adaptors, Milton family proteins, 104 translocation along actin, 104 Mitofusins 1 and 2 (Mfn1 and Mfn2), 100 Mitogen-activated protein kinase (MAPK) pathways, 27–28, 82–83, 86, 185f, 192, 212f, 214, 216, 217, 221–222, 221f, 224f Mitophagy, 100, 104–107 Atg12-Atg5-Atg16 complex, 104 autophagic vesicles (AV), 107 autophagolysosome, 105 autophagy, 104 bafilomycin A1 and in Atg5–/– MEF cells, lysosomal inhibitor, 105 co-over-expression of parkin and PINK1, 106 depolarization before autophagy, 105 endoplasmic reticulum (ER) mass, 105 formation of autophagosomes, 104 GFP-LC3-positive structures, 105 loss of Dcm, feature of mitophagy, 105 mitochondrial autophagy or mitophagy, 105 mitochondrial markers Tom20, cytochrome c and TRAP1, 105

311

multi-vesicular bodies (MVB), 107 mutations in LRRK2, 107 Nix, non-canonical BH3, 105 Omi/HtrA2, mitochondrial serine protease, 106–107 over-expression of Drp1 or Fis1, 105 PARL, mammalian homologue, 107 Rhomboid-7, inner mitochondrial membrane protease, 107 role for PINK1/parkin pathway, 106 ubiquitin-like protein Atg8 and Atg4 protease, 104 Ulk1, serine/threonine kinase, 105 VDAC1, mitochondrial target, 106 in vitro and in vivo with SUMO1, 107 Mixed lineage kinase JNK signalling cascade, 82–83 apoptotic death in 6OHDA model, 83 axotomy model, 83 calpain-Cdk5-MEF2 pathway, 83 chronic MPTP toxicity, role for JNK/c-jun signalling in, 83 c-jun protein and mRNA, 82 DLK, dominant negative forms of, 83 kainic acid-induced hippocampal neuron apoptosis, 82 MAPK cascade, 82 natural cell death and induced death in CNS, 82 neurotoxin-mediated degeneration of dopamine neurons, 83 non-phosphorylatable alanines, 82 6OHDA and by axotomy, 82 Moesin phosphorylation, 27 Molecular alterations expression and regulation of transcription factors, 217–218 AP-1 and CRE, 217 CRE/AP-1 elements, 217 D1-dependent prodynorphin transcription, 217 levels of DFosB and prodynorphin mRNA, 218 prodynorphin, 217 gene expression patterns, 218–219 acute and chronic L-DOPA treatment, 219 Affymetrix gene chip arrays, 218 cytochrome oxidase subunit I (CO-I), 219

dyskinesia-priming action of L-DOPA, 219 dyskinetic or non-dyskinetic, 218 micro-array study, 219 phosphocreatine pathway, enzymes of, 219 intracellular signalling, 216–217 anti-dyskinetic effects of treatments, 216 cellular adaptations in animal models of LID, 217 D1- and D2-rich striatal neurons, 216 direct pathway MSN, 216 dyskinetic striatum, 216 effects of L-DOPA treatment, 217 electrophysiological signature of LID, 217 ERK1/2, 216 group I metabotropic glutamate receptors, 216 MAPK signalling system, 216 phospho-Thr34-DARPP32, 216 Ras-ERK signalling, 217 intracellular trafficking of DA and glutamate receptors, 215–216 chronic L-DOPA treatment, 215 DA denervation and L-DOPA treatment, 215 distribution of ionotropic glutamate receptor subunits, 215 dyskinesiogenic L-DOPA treatment, 215 ionotropic glutamate receptors, 215 non-human primates treated with MPTP, 215 studies in 6-OHDA-lesioned rats, 215 plasticity of corticostriatal synapses, 218 direct pathway vs. indirect pathway MSN, 218 inducibility and reversal of corticostriatal LTP, 218 loss of synaptic depotentiation, 218 Molecular interpretation dysregulated D1 receptor-dependent signalling, 222–225 bioenergetic expenditure, 222 chronic pulsatile L-DOPA treatment, 222 cytoskeletal modifications in LID, 222 DA-dependent degeneration and apoptosis, 224 dietary supplementation of creatine, 225 intracellular signalling and bioenergetic deficits, 224 levels of DFosB-like proteins and prodynorphin mRNA, 223f

312

Molecular interpretation (Continued) maladaptive plasticity of striatal neurons in LID, 224f primary genetic mitochondrial defects, 224 prodynorphin-positive MSN, 222 structural and synaptic rearrangement of striatal neurons, 222 signalling pathway activation in dyskinetic rodents, 219–222 acute L-DOPA treatment, 221 chromatin remodelling during active gene transcription, 221 core molecular alteration, 221 DA receptor-dependent signalling, 220f–221f intracellular signalling inhibitors, 221 RasGRP1 (CalDAG-GEF2), 222 RasGRP2 (CalDAG-GEFI), 222 Molecular plasticity leads to plasticity failure, 225 basal ganglia–thalamo–cortical loops, 225 dyskinesia, 225 dysregulated D1-dependent signalling, 225 motor cortex plasticity, 225 non-invasive cortical stimulation methods, 225 striatal neurons, 225 Monogenic forms of PD by positional cloning strategies, identification of autosomal-dominant forms of inherited PD, 4–5 PARK1 (alpha-synuclein), 5–7 PARK8 (LRRK2), 7–8 Motor behaviour, effects of manipulation of STN lesion, pharmacological and molecular STN inactivation in monkeys, 243–244 akinesia and bradykinesia, 244 ballism or hemiballism, 243 transfection with adeno-associated virus, 244 lesion, pharmacological and molecular STN inactivation in rats, 244–245, 245f anti-parkinsonian therapy, 245 DArgic depletion, 244 L-DOPA-induced dyskinesia, 245 premature- responding deficit, 244 reduce circling behaviour, 244 STN HFS in monkeys, 244 hemiparkinsonian with MPTP, 244

STN HFS in rats, 245–246 choice RT task, 245–246 DA receptor antagonists, 246 reduced asymmetry, 246 Motor behaviour, GPi on EP HFS data in rodents, 239–240 GPi HFS in monkey, improve parkinsonian symptoms, 239–240 lesion and pharmacological EP manipulation in rat, 239 akinetic-like deficit/deficit in simple reactiontime (SRT), 239, 240f decreased rotations induced by amphetamine, 239 lesion and pharmacological GPi inactivation in monkey, 238–239 enhancement of STN hyper-activity, 239 focal inactivation of GPi with muscimol infusions, 238 kainic acid lesion, 238 Motor cortex (MI), 158, 225, 238, 242, 250, 261–262, 264, 264f, 265–268, 266f, 267f, 269f, 270, 270f Motor dysfunction of PD, 183, 196 Motor dyskinesias, 276, 287 Motor impairments dyskinesia and Go learning, 286–287 response vigour, 287 sensitization, 283–286 subthalamic nucleus, DBS and behavioural inhibition, 288–289 working memory impairments, 287–288 Motor symptoms, 237 Multifaceted cell death, 100 Multiple cortico-striatal loops, 279 Multiple sclerosis, 198 Multiple system atrophy (MSA), 7, 26, 31, 117–118, 125–126, 132 Multi-vesicular bodies (MVB), 107 Mutations in DJ-1 gene (PARK7), 9, 23t, 87 Myocyte enhancer factor 2 (MEF2), 26, 81–83, 161, 162f Myotonic dystrophy, 4 Native gel electrophoresis, 124 Neural network models of basal ganglia, 276–277

313

Neurodegenerative disorders (AD and PD), 15, 91, 158, 170–171, 173, 176, 187, 236 Neurodegenerative movement disorder, 21, 99 Neurogenetic disorders, 4 Neurological disorders (epilepsy/HD/ischemia), 103, 172 Neuronal A2A receptors and neuroprotection, 190–192 conditional knockout (Cre/loxP) system, 190 Huntington disease, 191 ischemia or excitotoxin-induced brain injury, 190 model of acute striatal neuron damage, 191 neurotoxicity induced by acute MPTP, 191 pre-synaptic level striatal A2A receptors, 191–192 sub-chronic MPTP model of PD, 190 toxin-induced striatal neuron death modelling, 191 Neuronal oscillations, 267–268 in motor cortex and GP, 269f Neuroprotection and adenosine A2A receptors A2A receptor antagonists SCH-58261 and ANR 94, 189 glial A2A receptors and neuroprotection, 192–193 MPTP administration in mice, 189 neuroinflammatory response, 190 neuronal A2A receptors and neuroprotection, 190–192 neuroprotection and A1 adenosine receptors, 187–189 8-cyclopentyl-1,3-dipropylxathine (CPX), 188 Glial A1 receptors, 189 ischemic stroke, 188 methamphetamine-induced nigrostriatal toxicity, 188 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP), 188 nerve growth factor (NGF), 189 pre-synaptic terminals and post-synaptic membranes, 187 6-OHDA-lesioned rat, 189 tyrosine hydroxylase (TH)-positive cells, 189 Neurotoxin-induced PCD, 80–81 Nicotinamide adenine dinucleotide (NAD) synthesizing enzyme, 86

Nicotinamide mononucleotide adenylyltransferase (NMNAT), 86 Nigrostriatal dopaminergic projections, 279 Nigrostriatal pathway, 33, 170 Nissl or tyrosine hydroxylase (TH) immunohistochemistry, 263 Nitric oxide (NO) signalling, 153–154, 161, 188f, 192 N-methyl-D-aspartate (NMDA) receptors, 170, 211 antagonist in experimental PD and in dyskinesia, 175 adrenergic, serotoninergic and sigma receptors, 175 anti-parkinsonian effects, 175 CP-101,606, 175 L-DOPA-induced dyskinesia, 175 NR2B-selective antagonist ifenprodil, 175 parkinsonism, 175 complex in experimental PD, alterations of, 170–172 CNS disorders (AD/PD/HD/epilepsy), 171 6-hydroxydopamine (6-OHDA), 171 long-term potentiation (LTP), 171 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 171 neurodegenerative disorders (AD and PD), 171 neurological disorders (epilepsy/HD/ ischemia), 172 post-synaptic density (PSD), 171 PSD-MAGUK, 171 structural rearrangement of glutamatergic synapse, 172f transmembrane ion flux, 171 vesicular localization of PSD-95 and SAP97, 172 NoGo neurons, 277f, 278–280, 279, 282, 286 Non-amyloid component, 116 Non-cell-autonomous mediators, 81 Non-genetic disease, 22 Oligomers, 6, 24–26, 32f, 50, 116f, 121, 123, 134f Orphan nuclear receptor Nurr1, 89 Outer mitochondrial membrane (OMM) fusion, 29, 48, 100–101, 104 OMM proteins (Miro1 and Miro2), 104 Oxidative stress, 8, 21, 26–27, 29–33, 60, 62, 71–72, 87, 100, 108, 129–130

314

Pale bodies, 132 Pallidal oscillatory activity, 259, 267 Pallidopyramidal syndrome, 29 Pan-cellular risk factors, 59–61 age, risk factor in PD, 59 best-documented pan-cellular factors, 59 Braak hypothesis, 61 declining mitochondrial function, 60 DJ-1, 60 dorsal motor nucleus of the vagus (DMV), 61 environmental toxin exposure, 60 genetic mutations, 60 grafting embryonic dopamine (DA) neurons, 61 identification of genes and disease risk, 60 inflammation and resultant oxidant stress, 60 mitochondrial dysfunction, 60 pan-cellular risk factors, 61 parkin and PINK1, 60 PINK1 deletion, 60 premature proteostatic dysfunction and LB formation, 61 proteostatic dysfunction, 60 viral or prion-like infection, 60 Paralysis agitans, 22f PARK1 (alpha-synuclein), 5–7 akinetic/rigid form of PD, 7 A30P mutation, 7 autosomal-dominant parkinsonism, 5 ‘Contursi’ kindred, 5 dementia with Lewy bodies (DLB), 7 E46K mutation, 7 elusive oligomers, 6 late-onset dopa-responsive parkinsonism, 6 Lewy body and Lewy neurites, 6 multiple system atrophy (MSA), 7 point mutations in SNCA, 5 aSYN immunopositive neuronal inclusions, 6 aSYN protein (A53T), 5 western blotting, 6 wild-type aSYN protein, 5 Parkin, 29–30 autosomal recessive juvenile-onset parkinsonism, 29 encodes protein of 465 amino acids, 29 homozygous loss-of-function mutation, 29 Lewy body pathology, 29

location, 29 mono/polyubiquitination through K48 or K63, 29 putative parkin substrates, identification of, 29 studies on Drosophila and mice, 29 ubiquitin–proteasome function (UPS), 29 Parkinsonian tremor, 259, 261, 269–270 Parkinson Study Group, 85, 199–200, 200 PARK8 (LRRK2), 7–8 cell culture studies, 8 clinical signs and symptoms, 8 diffuse Lewy body disease, 8 disease causing mutation, 7 G2385R variant, 7 G2019S mutation, 7 L-dopa-responsive parkinsonism, 7 leucine-rich repeat kinase 2 (LRRK2), 7 pathologic changes, 8 PARK2 (parkin), 8 early-onset parkinsonism, 8 recessive diseases, 8 superoxide dismutase 2 (SOD2), 8 Pathogenesis, mechanisms of PD, 32f dysfunction of UPS, 31 environmental factors, 31 neuronal a-synuclein accumulation, 31 relationships between PD and disorders, 31 PCD in dopamine neurons default activation of PCD, 89–91 classic neurotrophic theory, 89 GDNF, conditional deletion of, 89 GDNF–GFRa1 activation of Ret tyrosine kinase, 90 GDNF signalling pathways, 90f initial cellular injury, 89 orphan nuclear receptor Nurr1, 89 positive regulator of PI3K/Akt signalling, 89 Ret-dependent PI3K/Akt signalling, 90 Ret tyrosine kinase, 89 RTP801 (REDD1), gene in apoptosis, 91 SN dopamine neurons, 89 Src-family kinases, 90 survival signalling pathways in SN dopamine neurons in vivo, 90 transduction of post-natal SN dopamine neurons, 90

315

downstream vs. upstream effectors, 87–89 anti-apoptotic Akt kinase pathway, 87–88 apoptosis signalling kinase-1, activation of, 89 Bcl-2 family, Bid and tBid, 88 caspase-8 activation, 88 close, upstream relationship, example of, 88 co-expression of PI3K, 87 DD of Fas and protein Daxx, interaction between, 89 death receptors, 88 deletion mutation, 87 DISC, 88 DJ-1 knockdown in neurons, 87 Drosophila DJ-1 homologue, 87 Fas ligand (FasL), 88 intracellular DD of Fas and DD of FADD, 88 L166P mutation, 87 LRRK2-induced cell death, 89 mediation of extrinsic pathway of PCD by Fas, 88f mutations in fourth gene, LRRK2, 87 mutations in gene for DJ-1 (PARK7), 87 mutations in parkin/PINK1 and DJ-1 genes, 87 protein stabilization, 87 RNAi knockdown of DJ-1, 87 TNF and Fas, 88 major pathways of neuron destruction, 80–83 cdk-5 and MEF2 transcription factors, 81–82 mixed lineage kinase JNK signalling cascade, 82–83 PD-causing genes and genetic susceptibility factors, 3–16 autosomal-recessive parkinsonism PARK2 (parkin), 8 common risk variants for PD genome-wide association studies, 12–14 future strategies, 14–15 low-coverage genome sequencing, 16 whole genome sequencing, 15–16 genetic variants causing or pre-disposing to PD, 9–10 monogenic forms of PD by positional cloning strategies

autosomal-dominant forms of inherited PD, 4–5 PARK1 (alpha-synuclein), 5–7 PARK8 (LRRK2), 7–8 recessive forms of parkinsonism, 8–9 Peak-dose dyskinesia, 210 Phosphatidylinositol 3-kinase (PI3K) pathway, 87–90, 104 Phosphorylation -dependent a-syn toxicity, 124–125 ER–Golgi trafficking by non-phosphorylated a-syn, 124 inclusions by macroautophagy, 124 mutations at position 129 of a-syn, consequences of, 124t phosphorylated a-syn (S129-P), 125 rotenone treatment, 124 effects of S!D and S!A substitutions on a-syn aggregation, 122 AAV-mediated gene transfer in substantia nigra, 122 bi-cistronic mRNA, 122 neuronal loss caused by a-syn and two S129 mutants, 122 WT or a-syn S129A immunolabelling, 122 enhances or protects against a-syn toxicity, 122–123 Caenorhabditis elegans, 122 disease-associated a-syn mutations, 123 in Drosophila, co-expression of a-syn, 122 PLK2 over-expression, 122 in postmortem human brains and in synucleinopathy, 122 primary cultures of rat mesencephalic neurons, 122 screening in yeast, 122 toxic effect of a-syn S129-P, 122 GRK2-mediated phosphorylation at S129 or substitution of S129D, 121 inclusion formation by S129D, 121 a-syn oligomerization, 121 modulating a-syn toxicity in Drosophila and rodent models, 123 correlation between aggregation and toxicity, 123

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Phosphorylation (Continued) neuronal loss in rat model, 123 soluble oligomers, 123 toxicity of inclusions between rat and fruit flies, 123 in normal and pathological conditions, a-syn, 119f novel phosphorylation sites, 125–126 a-syn in vivo, phosphorylated at S87 and Y125, 125 a-syn (S129-P and S87-P), 125 phosphomimics S129D/E or S87E, 125f potential phosphorylation sites in a-syn, 119 protein–protein/protein–ligand and protein– metal interactions, 119 in vitro (S87/S129 and Y125/Y133/Y136), 119 in vivo (S129/S87 and Y125), 119 at S129 and S87, 120 mimic constitutive phosphorylation, 120 mutation of S!D/E, 120 phosphomimics S129D/E with S129-P, 120 serine/threonine or tyrosine residues, phosphorylation at, 120 of S129A promotes a-syn inclusion formation and fibrillization, 121 of a-syn requires identification of kinases and phosphatases in vivo, 120 casein kinase I (CKI) (S87 and S129), 120 casein kinase II (CKII) (S129), 120 G protein-coupled receptor kinases (GRK 1/2/ 5/6 and S129), 120 LRRK2 (leucine-rich repeat kinase 2) (S129), 120 PLK (polo-like kinases) (S129), 120 Syk/Lyn/c-Frg and Src tyrosine kinases, 120 Y125 by Fyn, 120 a-synuclein aggregation in vivo, 120–121 PINK1, 30 atypical clinical phenotypes, 30 kinase domain, 30 1p35–p36 chromosome, 30 protection against cell death, 30 TNF-receptor-associated protein 1(TRAP1), 30 Polymerase chain reaction, 3 Positional cloning strategies, 4–8

See also Monogenic forms of PD by positional cloning strategies, identification of Post-encephalitic parkinsonism (PEP), 22f Post-synaptic density (PSD), 169–178, 215, 263–264 Power spectrum density (PSD), 283 PRECEPT cohort, 199–200 Prefrontal cortex (PFC), 246–247, 247f, 279 Preproenkephalin (preproenkephalin-A), 211, 217, 238 Presupplementary motor area (preSMA), 288 Prodynorphin, 217 preproenkephalin-B, 211 Programmed cell death (PCD), 64, 79–83, 85–89, 91, 108 Progressive supranuclear palsy (PSP), 8, 11–12 Protein kinase A (PKA), 150–156, 162f, 177, 185f, 192, 211–213, 212f, 215–216, 222 Protein kinase C (PKC), 60, 150, 152, 177, 185, 185f Protein translation control, 27 Protofibrils, 117 PSD-MAGUK (membrane-associated guanylate kinases), 171, 178 PTEN-induced novel kinase 1 (PINK1), 8–9, 22– 23, 29–33, 45, 47–49, 60, 87, 99, 102–104, 106–108, 171 Purines roles in, adenosine/caffeine and urate, 183–200 See also Adenosine/caffeine and urate, pathophysiological roles for purines Quiet-wakeful state, 261 RasGRP2 (CalDAG-GEFI), 222 RAS guanyl nucleotide-releasing protein 1 (RasGRP1), 222 Reactive oxygen species (ROS), 25–26, 29, 31–32, 49, 52, 60, 67, 105 Receptor–receptor interaction, 184–187 A2A and mGlu5 receptors, 186 adenosine or dopamine receptors interaction with CB1 receptors, 186 A2A/D2 receptor heteromerization, 185–186 antagonistic CB1/D2 interactions, 186

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co-immunoprecipitation studies FRET and BRET, 185 D3 agonist binding sites and signalling, 186 ERK and DARPP-32 phosphorylation, 186 formation of functional receptor complexes, 184 heterodimerization, 184 molecular interactions with neurotransmitters, 184 Recessive forms of parkinsonism, 8–9 autosomal recessive early-onset parkinsonism, 8 complex phenotypes, 9 Mendelian forms of disease, 9 mutations in DJ-1 gene (PARK7), 9 mutations in PINK1 gene, 8 Recessive mutations, 29 ATP13A2 mutations, 29 loss-of-function mutations in parkin/PINK1/DJ-1, 29 role of mitochondrial dysfunction and oxidative stress, 29 Regulator of calmodulin signalling (RCS), 156, 162f Regulators of G-protein signalling (RGS), 214 Reinforcement learning (RL), 275, 279–284, 282–283, 287 Response vigour, 287 Rhot T1 and Rhot T2, See Outer mitochondrial membrane (OMM) fusion Rigidity, 21, 61, 99, 170, 239, 244, 260, 262, 265, 283 Risk factor genes, 46–47 ApoE4 variant and Alzheimer’s disease, 46 familial and sporadic PD, 47 GWAS, 46 MAPT/tau gene, 46 PARK16, 47 risk variants in SNCA, 46 SNCA and LRRK2, 47 variant in LRRK2 (G2385R), 47 Risk variants for PD atypical parkinsonian syndromes, 11 corticobasal degeneration (CBD), 11 familial and sporadic PD, 11 frontotemporal lobar degeneration (FTLD), 11 FTLD-17 or FTLD-Tau, 11

GWAS, 12–14 Affimetrix 550K mapping chips, 13 Bonferroni corrections, 13 cross-seeding of different proteins, 14 genomic and haplotype structure of SNCA locus, 14f haplotype bloc structure, 12 risk-conferring variants, identification of, 12 risk variants in PD, 13 SNCA and MAPT act, 14 two-stage GWAS, 13 30 -untranslated region (30 -UTR), 13 haplotype blocks of SNCA gene, 11 haplotypes (H1/H2), 11–12 H1 sub-haplotype (H1c), 12 microtubule-associated protein tau (MAPTau), 11 pathology of PD, 12 progressive supranuclear palsy (PSP), 11 tauopathies, 11 Schizophrenia, 170, 276 Schizophrenia and Tourette’s syndrome (TS), 276 Sensitization, 176, 215, 221, 224f, 283–286 Serine 129 with alanine (S129A), 121–125, 135, 136t Serine with aspartate (S129D), 120–125, 135, 136t Single-nucleotide polymorphisms (SNP), 4, 12, 14f, 15, 52 Single-spike mode, 241 Small ubiquitin-like modifier-1 (SUMO1), 101, 107 SNCA missense mutations 188G>A Glu46Lys), 24 88G>C (Ala30Pro), 24 Soluble NSF attachment protein receptors (SNARE) complex, 24 Spike timing-dependent plasticity (STDP), 154f, 155, 157 Spinal muscular atrophy, 4 STN-deep brain stimulation (DBS), 236, 239–242, 250–251, 260, 289 STN manipulation in PD, 240–250 direct STN-cortex loop circuit, 240 effects of manipulation of STN on cognition and motivation STN HFS data in rats, 248–250 STN lesion in rats, 247–248

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STN manipulation in PD (Continued) STN lesion or STN HFS in monkeys, 246–247 effects of manipulation of STN on motor behaviour lesion, pharmacological and molecular STN inactivation in monkeys, 243–244 lesion, pharmacological and molecular STN inactivation in rats, 244–245 STN HFS in monkeys, 244 STN HFS in rats, 245–246 neurophysiological effects antidromic activation of cortex, 242 COI mRNA expression, 242 dorsal raphe nucleus (DRN), 242 electrophysiology, 241–242 glutamic acid decarboxylase (GAD) mRNA, 242 molecular biology and metabolism, 242–243 neuroprotective effects, 243 STN HFS interferes with pacemaker-like activity of STN neurons, 243 striatal glutamatergic hyper-activity induced by 6-OHDA lesion, 241 survival of midbrain DAergic neurons in 6-OHDA-treated rats, 243 transcription factors c-fos, c-jun and Krox-24, 242 single-spike mode/burst mode, 241 suppression of oscillatory b activity by STN HFS, 241 Striatal-enriched tyrosine phosphatase (STEP), 150, 156 Striatal neurons, 150, 151f, 176–177, 186, 191, 209–210, 210, 215–219, 221–222, 223f, 224f, 225, 278 Striatopallidal GABA outflow, 184 Striatum in basal ganglia aspiny cholinergic interneurons, 170 subtypes of GABAergic neurons, 170 Substantia nigra pars compacta (SNc) DA neurons, 21, 59, 61–69, 63f, 71–73, 80, 99, 130, 154f, 170, 173, 176, 189, 190t, 236–237, 240, 245, 247, 265, 277f Substantia nigra pars compacta (SNpc), 21, 59, 61, 80, 82, 84f, 99–100, 107–108, 130, 173, 176, 189, 236, 247, 265

Substantia nigra pars reticulata (SNr), 63f, 236, 236f, 240–242, 278 Substantia nigra (SN), 7, 21, 25, 32, 43, 49, 51, 59, 61, 80–81, 81–85, 89–91, 99, 122–123, 125, 130–131, 136, 150, 173, 176, 189–192, 225, 235–236, 247, 265, 278 Subthalamic nucleus (STN), 184, 191, 225, 235–236, 235–251, 260, 277, 288–289, 289 Superoxide dismutase 2 (SOD2), 8 Symptomatology, 275–276 Synaptic plasticity in striatum, pathological, 173–174 alphaCaMKII autophosphorylation, 174 D1- and D2-like receptors, 173 glutamatergic and dopaminergic pathways, 173 high-frequency stimulation (HFS) protocol, 173 long-term depression (LTD) and long-term LTP, 173 low-frequency stimulation (LFS) of corticostriatal fibers, 173 spontaneous membrane depolarization, 173 striatal synaptic plasticity, 173 synaptic plasticity induction, 174 Synchronous oscillations, 259, 264, 266–268, 267–268 Synucleinopathies, 115–118, 125, 130, 137 Tauopathies, 11 TNF-receptor-associated protein 1(TRAP1), 30, 105 Tourette’s syndrome (TS), 276, 283 TRAK2 and TRAK1, See Grif-1 and OIP106, mammals homologues Treatment of non-dyskinetic animals with synthetic peptide (TAT2B), 174 Truncations enzymes in proteolysis of a-syn, 128–129 A53T aggregated mutations, 129 calpain-mediated cleavage, 128 caspase-like activity of 20S proteasome, 129 cathepsin D, 128 CKII expression and phosphorylation, 129 matrix metalloproteases, MMP (MMP-1/ MMP-3), 129 neurosin, trypsin-like serine protease, 128 N-terminal/C-terminal truncated fragments, 128 PD-linked mutations, 129

319

promote fibrillization in vivo, 128 proteolysis by neurosin, 128 in vitro aggregation assay, 129 in vitro cleavage, 128 promote fibrillization of a-syn, C-terminal, 126–128 of a-syn in vivo and in vitro, 127f, 128 1–87 asyn vs. 1–120 a-syn, 127 fibril formation and aggregation of monomeric a-syn, 128 initiation of asyn aggregation and fibrillogenesis in vivo, 126 initiation of a-syn fibrillogenesis in PD, 126 polyamines and C-terminal deletion, 128 variants 1–89/1–102/1–110/1–120 and 1–30, 128 in vitro fibrillization studies, 127 a-syn species in brain/PD and related disorders, 126 LB-derived and cytosolic a-syn forms, comparison between, 126 PD/DLB and MSA brain tissues, 126 in SDS- and urea-soluble fractions, 126 in vivo studies, 129–130 A53T 1–130, over-expression of, 130 Cre-dependent expression, consequences of, 130 C-terminal truncations, 130 dopaminergic neurons, 129 expression of a-syn 1–130, 130 fibril and non-fibrillar 1–120 a-syn inclusions, 130 formation of inclusions, 129 non-fibrillar and fibrillar a-syn, 129 a-syn 1–87, 129 a-syn 1–120, 129 toxicity in SH-SY5Y neuroblastoma, 130 toxicity of C-terminal truncated forms, 130 Two-photon laser scanning microscopy (2PLSM), 68, 71, 151, 157, 161 Two-photon laser uncaging (2PLU), 158, 161 Tyrosine hydroxylase (TH) immunohistochemistry, 263 immunostaining, 64, 85, 129, 136, 189, 243, 263, 265

Tyrosine kinase-linked receptor (TrkB), 176 Ubiquitination E3ubiquitin ligases of a-syn, 132 parkin, 132 seven in absentia homologue (SIAH), 132 ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), 132 -positive inclusions, 130–132 a-synuclein and ubiquitin co-staining, 132 covalently modifies a-syn, 131f double-immunostaining, 130 intra-cytoplasmic inclusions, 132 lysosomal and autophagic pathways, 132 mono- or di-ubiquitinated and tri-ubiquitinated a-syn species, 132 neuropathologic studies, 130 PD and related disorders, 130 protein clearance pathways, 132 western blotting and mass spectrometry techniques, 132 sites, 132–133 K80, K96, K97 and K103 residues, 132 15 lysine (K) residues in a-syn, 132 lysine residues, 132 major ubiquitin-conjugated sites, 132 residues to argnine (R), 132 trypsin digestion and LCMS/MS analysis, 133 of a-syn enhance or prevent aggregation and toxicity, 133–136 in animal models, 133 A30P and 453T mutations, 134 in cell culture, 134 consequences of truncation in animal models, 135t–136t dopaminergic vs. GABAergic neurons, 136 in Drosophila, 133 effects of a-syn post-translational modifications, 134f inhibition of proteasome or impairment of UCH-L1 activity, 136 lactocystin-exposed cells, 136 phosphorylation on a-syn in animal models, 135t–136t proteasome inhibitors, 136

320

in rat model, 133 studies in fly, 133 Ubiquitination (Continued) in TG mice, 133 in vitro ubiquitination of a-syn, 134 Ubiquitination factor E4B (Ube4B), 86 Ubiquitin-like protein Atg8 and Atg4 protease, 104 Ubiquitin–proteasome system (UPS) impairment, 33–34 aggregated a-synuclein, 34 molecular chaperone Hsp70 in flies, 34 mutations in parkin, 33 nigrostriatal pathway, 33 yeast chaperone Hsp104 in rats, 34 Uncoupling proteins (UCP), 71 UCP1–5, 72 Undetected population stratification, 10 30 -untranslated region (30 -UTR), 1, 13 Urate, novel target for neuroprotection biology, 198–199 evolutionary significance, 198 neuroprotective effects in cellular models of PD, 198–199 epidemiology, 199–200 clinical studies of urate and PD progression, 199–200 urate crystallization in joints and hyperuricemia, 199

urate-elevating diet, 199 Urate oxidase gene (UOx), 198 Voltagedependent anion channel 1 (VDAC1), 106 Wallerian degeneration slow (WldS) mutant mouse, 86–87 WD-40 domain, 27 Western blotting, 6, 132 Whole exome sequencing, 15 autosomal-dominant or autosomal-recessive disease genes, 15 disease-causing mutations, 15 hybridization techniques solid phase or emulsion-based short sequences, 15 late-onset neurodegenerative disorder, 15 loss-of-function mutations, 15 SNP-genotyping approaches, 15 solid phase or emulsion-based short sequences, 15 Whole genome sequencing, 15–16 Charcot–Marie–Tooth neuropathy, 16 ‘1000-dollar genome,’ 16 International Human Genome Sequencing, 15 single-nucleotide variants, 16 Wild-type aSYN protein, 5–6 Working memory (WM) impairments, 176, 247, 276, 287–288, 287–290, 288