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

60

(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.

61

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 (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

136

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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145 linked to upregulated CK2 and cathepsin D. European Jour­ nal of Neuroscience, 26(4), 863–874. Thiruchelvam, M. J., Powers, J. M., Cory-Slechta, D. A., & Richfield, E. K. (2004). Risk factors for dopaminergic neu­ ron loss in human alpha-synuclein transgenic mice. European Journal of Neuroscience, 19(4), 845–854. Tofaris, G. K., Garcia Reitbock, P., Humby, T., Lambourne, S. L., O’Connell, M., Ghetti, B., et al. (2006). Pathological changes in dopaminergic nerve cells of the substantia nigra and olfactory bulb in mice transgenic for truncated human alpha-synuclein(1-120): Implications for lewy body disorders. Journal of Neuroscience, 26(15), 3942–3950. Tofaris, G. K., Layfield, R., & Spillantini, M. G. (2001). Alpha­ synuclein metabolism and aggregation is linked to ubiquitin­ independent degradation by the proteasome. FEBS Letters, 509(1), 22–26. Tofaris, G. K., Razzaq, A., Ghetti, B., Lilley, K. S., & Spillan­ tini, M. G. (2003). Ubiquitination of alpha-synuclein in lewy bodies is a pathological event not associated with impairment of proteasome function. The Journal of Biological Chemistry, 278(45), 44405–44411. Ulusoy, A., Sahin, G., Bjorklund, T., Aebischer, P., & Kirik, D. (2009). Dose optimization for long-term rAAV-mediated RNA interference in the nigrostriatal projection neurons. Molecular Therapy, 17(9), 1574–1584. Uversky, V. N., Li, J., & Fink, A. L. (2001). Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular NK between Parkinson’s disease and heavy metal exposure. The Journal of Biological Chemistry, 276(47), 44284–44296. van der Putten, H., Wiederhold, K. H., Probst, A., Barbieri, S., Mistl, C., Danner, S., et al. (2000). Neuropathology in mice expressing human alpha-synuclein. Journal of Neuroscience, 20(16), 6021–6029. Vogiatzi, T., Xilouri, M., Vekrellis, K., & Stefanis, L. (2008). Wild type alpha-synuclein is degraded by chaperonemediated autophagy and macroautophagy in neuronal cells. The Journal of Biological Chemistry, 283(35), 23542–23556. von Coelln, R., Thomas, B., Andrabi, S. A., Lim, K. L., Savitt, J. M., Saffary, R., et al. (2006). Inclusion body formation and

neurodegeneration are parkin independent in a mouse model of alpha-synucleinopathy. Journal of Neuroscience, 26(14), 3685–3696. Wakamatsu, M., Ishii, A., Iwata, S., Sakagami, J., Ukai, Y., Ono, M., et al. (2008). Selective loss of nigral dopamine neurons induced by overexpression of truncated human alpha-synu­ clein in mice. Neurobiology of Aging, 29(4), 574– 585. Waxman, E. A., & Giasson, B. I. (2008). Specificity and reg­ ulation of casein kinase-mediated phosphorylation of alpha­ synuclein. Journal of Neuropathology and Experimental Neu­ rology, 67(5), 402–416. Webb, J. L., Ravikumar, B., Atkins, J., Skepper, J. N., & Rubinsztein, D. C. (2003). {Alpha}-synuclein is degraded by both autophagy and the proteasome. The Journal of Biolo­ gical Chemistry, 278(27), 25009–25013. Weinreb, P. H., Zhen, W., Poon, A. W., Conway, K. A., & Lansbury, P. T., Jr. (1996). NACP, a protein implicated in alzheimer’s disease and learning, is natively unfolded. Bio­ chemistry, 35(43), 13709–13715. Wu, K. P., Kim, S., Fela, D. A., & Baum, J. (2008). Character­ ization of conformational and dynamic properties of natively unfolded human and mouse alpha-synuclein ensembles by NMR: Implication for aggregation. Journal of Molecular Biology, 378(5), 1104–1115. Xilouri, M., Vogiatzi, T., Vekrellis, K., & Stefanis, L. (2008). Alpha-synuclein degradation by autophagic pathways: A potential key to Parkinson’s disease pathogenesis. Autop­ hagy, 4(7), 917–919. Yamada, M., Iwatsubo, T., Mizuno, Y., & Mochizuki, H. (2004). Overexpression of alpha-synuclein in rat substantia nigra results in loss of dopaminergic neurons, phosphoryla­ tion of alpha-synuclein and activation of caspase-9: Resem­ blance to pathogenetic changes in Parkinson’s disease. Journal of Neurochemistry, 91(2), 451–461. Yamin, G., Uversky, V. N., & Fink, A. L. (2003). Nitration inhibits fibrillation of human alpha-synuclein in vitro by for­ mation of soluble oligomers. FEBS Letters, 542(1-3), 147–152. Yang, Y., Nishimura, I., Imai, Y., Takahashi, R., & Lu, B. (2003). Parkin suppresses dopaminergic neuron-selective neurotoxi­ city induced by pael-R in drosophila. Neuron, 37(6), 911–924.

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

153

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

154 (a)

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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

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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).

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+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

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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

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