Recent Advances in Parkinsons Disease, Volume 184: Part II: Translational and Clinical Research (Progress in Brain Research)

PROGRESS IN BRAIN RESEARCH

VOLUME 184

RECENT ADVANCES IN PARKINSON’S

DISEASE: TRANSLATIONAL AND

CLINICAL RESEARCH

EDITED BY

¨ ANDERS BJORKLUND 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
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: 978-0-444-53750-8 ISSN: 0079-6123 For information on all Elsevier publications visit our website at elsevierdirect.com

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List of Contributors

S.B. Rangasamy, Departments of Neurological Sciences and Neurosurgery, Rush University Medical Center, Chicago, IL, USA R.A.E. Bakay, Departments of Neurological Sciences and Neurosurgery, Rush University Medical Center, Chicago, IL, USA R.A. Barker, Cambridge Centre for Brain Repair, Robinson Way, Cambridge, UK C.J. Barnum, Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA A. Björklund, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, Lund, Sweden T. Björklund, Brain Repair and Imaging in Neural Systems, Department of Experimental Medical Science, Lund University, Lund, Sweden D.J. Brooks, Division of Experimental Medicine, Imperial College London, Hammersmith Hospital, London, UK J.M. Brotchie, Toronto Western Research Institute, Toronto Western Hospital, Toronto, ON, Canada P. Brundin, Neuronal Survival Unit, Wallenberg Neuroscience Center, Lund University, Lund, Sweden J.R. Cannon, Pittsburgh Institute for Neurodegenerative Diseases, Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA E.A. Cederfjäll, Brain Repair and Imaging in Neural Systems, Department of Experimental Medical Science, Lund University, Lund, Sweden K.R. Chaudhuri, National Parkinson Foundation Centre of Excellence, Kings College Hospital and University Hospital Lewisham; and Kings College and Institute of Psychiatry, London, UK M.-F. Chesselet, Department of Neurology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA M. Decressac, Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, Lund, Sweden S.B. Dunnett, Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, South Wales, UK D. Eidelberg, Center for Neurosciences, The Feinstein Institute for Medical Research; and Departments of Neurology and Medicine, North Shore University Hospital, Manhasset, NY, USA S.H. Fox, Division of Neurology, University of Toronto; and Toronto Western Research Institute, Toronto Western Hospital, Toronto, ON, Canada J.T. Greenamyre, Pittsburgh Institute for Neurodegenerative Diseases, Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA M. Guo, Department of Neurology, Department of Molecular and Medical Pharmacology, Brain Research Institute, David Geffen School of Medicine, Los Angeles, CA, USA D. Kirik, Brain Repair and Imaging in Neural Systems, Department of Experimental Medical Science, Lund University, Lund, Sweden

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J.H. Kordower, Departments of Neurological Sciences and Neurosurgery, Rush University Medical Center, Chicago, IL, USA R. Kuriakose, Pacific Parkinson’s Research Centre, University of British Columbia and Vancouver Coastal Health, Vancouver, BC, Canada E.L. Lane, Welsh School of Pharmacy, Cardiff University, Cardiff, South Wales, UK M. Lelos, School of Biosciences, Cardiff University, Cardiff, South Wales, UK A.M. Lozano, Division of Neurosurgery, University of Toronto, Toronto Western Hospital, Toronto, ON, Canada I. Magen, Department of Neurology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA P. Odin, Department of Neurology, Skane University Hospital, Lund, Sweden; and Department of Neurology, Central Hospital Bremerhaven, Bremerhaven, Germany M. Parmar, Neurobiology Unit, Wallenberg Neuroscience Center, Lund University, Lund, Sweden N. Pavese, MRC Clinical Sciences Centre and Department of Medicine, Imperial College London, London, UK P. Piccini, Centre for Neuroscience and MRC Clinical Sciences Centre, Faculty of Medicine, Hammersmith Hospital, Imperial College London, UK M. Politis, Centre for Neuroscience and MRC Clinical Sciences Centre, Faculty of Medicine, Hammersmith Hospital, Imperial College London, UK F.A. Ponce, Division of Neurosurgery, University of Toronto, Toronto Western Hospital, Toronto, ON, Canada; Division of Neurological Surgery, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix, AZ, USA K. Soderstrom, Departments of Neurological Sciences and Neurosurgery, Rush University Medical Center, Chicago, IL, USA A.J. Stoessl, Pacific Parkinson’s Research Centre, University of British Columbia and Vancouver Coastal Health, Vancouver, BC, Canada C.C. Tang, Center for Neurosciences, The Feinstein Institute for Medical Research, Manhasset, NY, USA M.G. Tansey, Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA A. Ulusoy, Brain Repair and Imaging in Neural Systems, Department of Experimental Medical Science, Lund University, Lund, Sweden C. Winkler, Department of Neurology, University Hospital Freiburg, Freiburg, Germany

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 a-synuclein 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 investi­ gations 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 modeling 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 (Volumes 183 and 184) 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 in 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 nonhuman 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 vii

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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, nonprofit 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 11, 2010 Anders Bjo¨ rklund M. Angela Cenci

SECTION I

Animal models of PD

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

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

CHAPTER 1

What have we learned from Drosophila models of Parkinson’s disease? Ming Guo
Department of Neurology, Department of Molecular and Medical Pharmacology, Brain Research Institute,

David Geffen School of Medicine, Los Angeles, CA, USA

Abstract: Parkinson’s disease (PD) is characterized clinically by motor symptoms such as resting tremor, slowness of movement, rigidity, and postural instability, and pathologically by the degeneration of multiple neuronal types, including, most notably, dopaminergic (DA) neurons in the substantia nigra. Current medical treatment for PD focuses on dopamine replacement, but dopamine replacement ultimately fails and has little effect on a variety of dopamine-independent symptoms both within and outside the nervous system. To develop new therapies, we need to aim at alleviating widespread cellular defects in addition to those focusing on DA neuronal survival. Recent observations in Drosophila have provided important insights into the cellular basis of PD pathogenesis through the demonstration that two genes associated with familial forms of PD, pink1 and parkin, function in a common pathway. In this pathway, pink1 functions upstream of parkin to regulate mitochondrial fission/fusion dynamics and normal mitochondrial function. Subsequent observations in both fly and mammalian systems show that these proteins are important for sensing mitochondrial damage and recruiting damaged mitochondria to the quality control machinery for subsequent removal. This chapter reviews these findings, as well as studies of DJ­ 1 and Omi/HtrA2, two additional genes associated with PD that have also been implicated to regulate mitochondrial function. The chapter ends by discussing how Drosophila can be used to probe further the functions of pink1 and parkin and the regulation of mitochondrial quality more generally. In addition to PD, defects in mitochondrial function are associated with normal aging and with many diseases of aging. Thus, insights gained from the studies of mitochondrial dynamics and quality control in Drosophila are likely to be of general significance. Keywords: Parkinson’s disease; Drosophila; PINK1; parkin; mitochondria; dopaminergic neurons; animal model; mitochondrial fusion and fission; mitophagy


Corresponding author. Tel.: þ1-310-2069406; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84001-4

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Parkinson’s disease is a disorder involving more than dopaminergic neuronal loss Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting 5% of people over the age of 80. N and no treatments can definitively halt the progression of the disease. Thus, understanding the disease mechanisms and identifying new therapeutic strategies are crucial for treating patients with PD. Clinical features of PD include “motor symptoms” such as resting tre­ mor, slowness of movement, rigidity, and postural instability. PD is characterized pathologically by the degeneration of multiple neuronal types including, most notably, dopaminergic (DA) neurons in the substantia nigra of the midbrain (Dauer and Przed­ borski, 2003). The mainstay of current medical treatment for PD is dopamine replacement. How­ ever, this treatment becomes less effective over time and is often associated with intolerable side effects. In addition, PD patients also present with a variety of non-motor symptoms (Simuni and Sethi, 2008). These include including dementia, which occurs in one third of patients, psychiatric symptoms, such as depression, anxiety, obsession, and sleep disruption, and symptoms outside of the nervous system, including skin lesions and musculoskeletal abnorm­ alities. Some of these non-motor symptoms may be more debilitating than the motor impairment, but they do not usually respond to dopamine replace­ ment. In addition, pathology of many non-DA neu­ rons, including olfactory and brain stem neurons, predates that of DA neurons (Braak et al., 2003). In short, PD is a multi-system disease affecting more than DA neurons. Therefore, therapies targeted to DA neurons or their targets (such as dopamine replacement, cell transplantation, and deep brain stimulation) can provide some therapeutic benefit to patients, particularly with respect to the motor symptoms. However, a true cure requires that we develop therapies that target the underlying cellular defects. A prerequisite for this work is that we understand the pathogenesis of PD at the cellular and molecular levels. As will be described below, mitochondrial dysfunction, which has

consequences in multiple tissues, is crucial for pathogenesis in at least some forms of PD.

Familial forms of PD Although once believed to be solely an environ­ mental disease, over the past decade mutations in five genes have been definitively shown to med­ iate familial forms of PD. Mutations in PARKIN (PARK2) (Kitada et al., 1998), DJ-1 (PARK7) (Bonifati et al., 2003), and PTEN-induced kinase 1 (PINK1, PARK6) (Valente et al., 2004) are asso­ ciated with autosomal recessive forms of PD, while mutations in a-Synuclein (PARK1 (Poly­ meropoulos et al., 1997) and PARK4 (Singleton et al., 2003)) and Leucine-rich repeat kinase 2 (LRRK2)/Dardarin (PARK8) (Paisan-Ruiz et al., 2004; Zimprich et al., 2004) are associated with autosomal dominant forms of the disease. Muta­ tions in ATP13A2 (PARK9), which encodes a lysosomal ATPase, have been found in an atypi­ cal, autosomal recessive parkinsonism (Ramirez et al., 2006); however, the clinical manifestations, of this disease are quite distinct from PD (Schnei­ der et al., 2010). The “PARK” here refers to genetic loci that have been identified from family linkage studies for PD (Hardy et al., 2009). Together, these single gene-mediated, Mendelian forms of the disease represent about 10–15% of all PD cases. The clinical features of at least some of these familial forms of PD bear significant similar­ ity to those of sporadic forms. Thus, the hope is that studies of familial forms of the disease will also provide insights into the more prevalent sporadic forms. Formal nomenclature has utilized the term PD for sporadic PD and parkinsonism for genetic forms of PD. This is largely based on the fact that the cause of PD was previously thought to be non-genetic and solely environmental, a notion that is clearly no longer believed to be true (Hardy et al., 2006). It is also probable that as research advances, more genes that mediate Mendelian forms of PD and multigenic forms of PD will be identified. Thus, to simplify, this

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chapter uses “PD” as an overall term for both sporadic and familial forms of PD. Drosophila as a model system to study PD The identification of genes that mediate familial PD provides an unprecedented opportunity to understand the pathogenesis of the disease. Stu­ dies of in vivo functions of these PD genes in model systems have been instrumental in under­ standing PD pathogenesis. Among the various model organisms, Drosophila melanogaster has emerged as an especially effective tool to study PD genes. Drosophila has been used extensively for investigating complex biological processes, such as cell death (Hay and Guo, 2003, 2006; Hay et al., 2004), and complex behaviors such as circadian rhythms, learning and memory, sleep, and aggression. The accumulated studies from over a century have left the modern fly field with powerful molecular genetic tools (Adams and Sekelsky, 2002; St Johnston, 2002; Venken and Bellen, 2005). Drosophila also has a compact gen­ ome size (1/30th of the human genome), limited genetic redundancy, and a short generation time (10 days). The complete sequence of the Droso­ phila genome (Adams et al., 2000) has revealed that 77% of human disease genes are conserved in the fly (Bier, 2005; Rubin et al., 2000). These features make flies an excellent model system in which to study the function of disease genes including those involved in neurodegenerative dis­ eases (Lessing and Bonini, 2009) and in which to dissect genetic pathways related to these disease genes. The adult brain of Drosophila contains clusters of DA neurons (Nassel and Elekes, 1992) and these neurons degenerate when flies are fed rote­ none (Coulom and Birman, 2004), a complex I inhibitor that also triggers DA neuronal degenera­ tion in mammals (Bove et al., 2005; Sherer et al., 2003). Among the genes that mediate familial PD, only alpha-synuclein does not have a homolog in Drosophila. Nevertheless, expression of human

wild-type and PD-causing mutant forms of alpha­ synuclein in Drosophila results in DA neuronal loss (Feany and Bender, 2000; Trinh et al., 2008). In this chapter, we will mainly focus on Droso­ phila homologs of genes associated with recessive forms of PD: PINK1, parkin, and DJ-1 as they relate to mitochondrial function. We will also emphasize the implications that these studies in Drosophila have for advancing our understanding of PD in clinical settings. It is important to note that although DA neurons do show slight degen­ eration in some Drosophila models of PD (Trinh et al., 2008; Whitworth et al., 2005), the signifi­ cance of Drosophila models of PD involves much more than DA neuron pathology. Flies show defects in multiple systems, reminiscent of the multiple system involvement in PD patients. It is the cellular basis of these defects that provides insight into the pathogenesis of PD. Pink1 and parkin function in a common pathway to regulate mitochondrial integrity The PARK2 locus encodes Parkin, which has RING-finger motifs and E3 ubiquitin ligase activity in in vitro assays. Shortly after it was cloned, a major hypothesis was that loss of parkin resulted in the aberrant accumulation of toxic proteins, perhaps as a result of a failure of the ubiquitin– proteasome system to degrade substrates of Parkin ubiquitination. Consistent with this hypothesis, Parkin catalyzes K48-mediated polyubiquitination, which targets substrates for proteasomal degrada­ tion, and multiple proteins have been shown to interact with Parkin and are ubiquitinated in a Parkin-dependent manner in vitro. In some cases, overexpression of parkin can suppress toxicity asso­ ciated with overexpression of the potential target of Parkin ubiquitination. However, few of these sub­ strates have been shown to accumulate in vivo, in PARK2 patients, or parkin knockout mice, leaving their significance unclear, though clearly worthy of further investigation (reviewed in Dawson and Dawson, 2010; West et al., 2007).

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Parkin also catalyzes monoubiquitination and K63-linked polyubiquitination (reviewed in West et al., 2007), as well as a very recently described K27-linked ubiquitination (Geisler et al., 2009). Monoubiquitination and K63-linked polyubiquiti­ nation can influence cellular processes such as signal transduction, transcriptional regulation, and protein and membrane trafficking without promoting substrate degradation (Mukhopadhyay and Riezman, 2007). Together with the fact that overexpression of parkin protects from death asso­ ciated with proteasome inhibition (Chung et al., 2004; Petrucelli et al., 2002), the above observa­ tions suggest that at least some important compo­ nents of Parkin’s neuroprotective activity when overexpressed—which may or may not be the same as those lost when parkin is absent—involve ubiquitin-dependent processes other than K48­ linked ubiquitination and proteasome-dependent protein degradation. Important clues to the endogenous functions of parkin have come from studies of Drosophila parkin mutants. Flies lacking parkin exhibit dramatic mitochondrial defects—swollen mitochondria that have severely fragmented cristae—in several energy-intensive tissues, including the male germline and adult flight muscle (Greene et al., 2003; Pesah et al., 2004). The flight muscles ultimately die and their death shows features of apoptosis (Greene et al., 2003). Flies lacking parkin also display a small but significant degeneration of a subset of DA neurons (Whitworth et al., 2005). These studies in Drosophila provided the first in vivo indication that parkin regulates mitochon­ drial integrity. Subsequently, it was reported that although severe defects in mitochondrial morphol­ ogy are not observed in parkin knockout mice, these animals do display mitochondrial functional defects including reduced mitochondrial respira­ tory activity (Palacino et al., 2004). Key studies that strengthen the idea that parkin regulates mitochondrial function have come from studies of pink1, the Drosophila homolog of PINK1, and its interaction with parkin. pink1 encodes a protein with a mitochondrial targeting

sequence and a serine–threonine kinase domain. We and others reported that a large fraction of Pink1 is localized to mitochondria (Clark et al., 2006) and Drosophila lacking pink1 show pheno­ types very similar to those of flies lacking parkin: mutants are viable but exhibit increased stress sensitivity and mitochondrial morphological defects in testes and muscle (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). pink1 mutants also show reduced ATP levels and mitochondrial DNA (mtDNA) content (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). Flies lacking endo­ genous pink1 function but expressing PD-asso­ ciated mutant forms of pink1, either by overexpression (Yang et al., 2006) or under the control of the endogenous pink1 promoter (Yun et al., 2008), show phenotypes similar to those of pink1 null mutants, consistent with PINK1-asso­ ciated disease being the result of loss of function. As in parkin mutant flies, mitochondria in pink1 mutant flight muscle are swollen with fragmented cristae and these cells ultimately undergo apopto­ tic death (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). Mitochondria within DA neurons in pink1 mutants also display aberrant morphology and there is a small but statistically significant loss of a subset of these neurons with age (Park et al., 2006; Yang et al., 2006). Mitochondrial dysfunction and oxidative stress were originally implicated in PD pathogenesis in the 1980s, based on the findings that exposure to the environmental toxin, 1-methyl-4-phenyl­ 1,2,3,6-tetrahydropyridine (MPTP), which inhibits mitochondrial respiration and promotes produc­ tion of reactive oxygen species (ROS), causes loss of DA neurons in humans and primates (Bove et al., 2005; Langston et al., 1983). It is important to note, however, that while human exposure to mitochondrial toxins kills DA neurons, resulting in a PD-like syndrome with motor symptoms, there is little effect on other tissues. In contrast, the findings that pink1 and parkin regulate mito­ chondrial integrity in multiple tissues implicate mitochondrial dysfunction as the central mechan­ ism for PD pathogenesis in both DA neurons and

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non-DA tissues. Studies of the PINK1/PARKIN pathway thus have the potential to uncover new therapies targeting the cellular defects that cause cell loss in PD, going beyond the current treat­ ment strategies of dopamine and DA neuron replacement. The similar phenotypes observed in pink1 and parkin null mutants suggested that the two genes might function in a common pathway. Indeed, parkin overexpression in flies suppresses all pink1 mutant phenotypes tested (Clark et al., 2006; Park et al., 2006; Yang et al., 2006), while pink1 overexpression does not compensate for loss of parkin function (Clark et al., 2006; Park et al., 2006). Furthermore, double mutants lacking both pink1 and parkin have phenotypes identical to, rather than stronger than, either single mutant (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). In addition, several groups have reported that Parkin and Pink1 can physically interact in at least some contexts (Kim et al., 2008; Xiong et al., 2009). Together, these observations provide com­ pelling in vivo evidence that pink1 and parkin act in a linear pathway that affects mitochondrial function, with parkin functioning downstream of pink1. Several lines of evidence suggest that these observations on pink1 and parkin function in flies are relevant to humans. First, PD patients who harbor mutations in PINK1 or PARKIN are clinically indistinguishable (Ibanez et al., 2006) and mice lacking both pink1 and parkin show phenotypes no worse than those of the single mutants (Kitada et al., 2009), consistent with the hypothesis that these genes function in a common genetic pathway. Second, expression of human PINK1 (Clark et al., 2006; Yang et al., 2006) or PARKIN in Drosophila suppresses phenotypes caused by loss of function of pink1 or parkin, respectively, suggesting that the human and fly proteins are functionally conserved. Third, recent studies of pink1 knockout mice, which do not show DA neuron loss or severe defects in mito­ chondrial morphology (though see below for other evidence of effects on mitochondrial

morphology), nonetheless show reduced respira­ tory activity (Gautier et al., 2008; Morais et al., 2009). In particular, both mouse and Drosophila pink1 mutants show defects in complex I activity (Morais et al., 2009). Finally, pathological changes and defects in mitochondrial respiration have been detected in peripheral tissues from patients with PINK1 (Hoepken et al., 2006) or PARKIN (Muftuoglu et al., 2004) mutations. The pink1/parkin pathway promotes mitochondrial fission and/or inhibits fusion How do pink1 and parkin regulate mitochondrial function? Examination of pink1 and parkin mutant phenotypes in the Drosophila male germline pro­ vided an important clue that these proteins regulate mitochondrial fission/fusion dynamics. During Dro­ sophila spermatogenesis, mitochondria undergo sig­ nificant morphological changes (Fuller, 1993). Early spermatids undergo mitochondrial aggregation and fusion, creating a spherical structure known as the nebenkern, which is composed of two intertwined mitochondria (Fuller, 1993). During subsequent spermatid elongation, the nebenkern unfurls, yielding two mitochondrial derivatives that are maintained throughout subsequent stages of sper­ matogenesis. In both pink1 and parkin mutants, however, only one mitochondrial derivative is seen, suggesting a defect in mitochondrial fission or an overabundance of fusion (Deng et al., 2008). Mitochondria are continually undergoing cycles of fission and fusion. This allows mitochondria to change shape and share components. Mitochon­ drial dynamics also plays an important role in facil­ itating recruitment of mitochondria to specific cellular compartments such as synapses where ATP or Ca2þ buffering demands are high. Mito­ chondrial fusion is promoted by mitofusin (mfn), which is required for outer membrane fusion, and Optic atrophy 1 (Opa-1), which is essential for inner membrane fusion. Mitochondrial fission is pro­ moted by dynamin-related protein 1 (Drp1), a pre­ dominantly cytoplasmic protein that is recruited to

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mitochondria during fission. The recruitment of Drp1 may involve a mitochondrial outer membrane protein Fis1 (reviewed in Chen and Chan, 2009). In addition to the spermatid morphology defects seen in pink1 or parkin mutants, several other lines of evidence support the hypothesis that the pink1/parkin pathway regulates mitochondrial dynamics. First, mitochondria in pink1 or parkin mutants are clumped in large aggregates in both DA neurons and flight muscle. They are also swollen and have disrupted cristae. Second, these cellular defects in mitochondrial morphology, as well as other defects such as the degeneration of flight muscle, cell death, locomotion defects, and a decrease in dopamine levels in fly heads, can be suppressed by increasing the expression of the pro-fission molecules drp1 or fis1 and/or decreas­ ing levels of the pro-fusion molecules mitofusin or opa1 (Deng et al., 2008; Park et al., 2009; Poole et al., 2008; Yang et al., 2008). Third, heterozyg­ osity for drp1 is lethal in a pink1 mutant back­ ground, consistent with the idea that pink1 and drp1 work in the same direction to promote fission (Deng et al., 2008; Poole et al., 2008). That said, it is important to note that the phenotypes asso­ ciated with loss of pink1 or parkin, and loss of drp1, are distinct (Deng et al., 2008), indicating that Pink1 and Parkin are not core components of the fission machinery, but instead regulators of the process. Since both cellular defects and organismal defects can be rescued by manipulating mitochon­ drial dynamics, manipulation of mitochondrial dynamics provides a novel therapeutic strategy. Observations that point to roles of pink1 and parkin in regulating mitochondrial morphology have also been obtained in mammalian systems. However, in contrast to the story in Drosophila, which is consistent across cell types and labs, in mammalian systems various effects have been observed. Enlarged mitochondria have been observed in pink1 striatal neurons (Gautier et al., 2008) and in COS7 cells in which pink1 was silenced using RNAi. In the latter system, this phenotype was suppressed by fis1 or drp1 overexpression, as in Drosophila (Yang et al., 2008).

However, others have observed that loss of pink1 results in fission, with decreased levels of drp1 resulting in suppression (Dagda et al., 2009; Exner et al., 2007; Lutz et al., 2009; Sandebring et al., 2009). The reasons for these differences are not clear but they are undoubtedly interesting and important. Screens in Caenorhabditis elegans have shown that disruption of many genes leads to changes in mitochondrial morphology, including fragmentation or elongation, indicating that mor­ phology is very sensitive to a variety of signaling pathways and physiological states (Ichishita et al., 2008). Thus, it is likely that the final mitochondrial morphology phenotype observed in any particular cell type, particularly with respect to the presence or absence of pink1/parkin, will depend on many variables. In any case, what is most important is not the specific morphology observed but the functional state of the mitochondrial population and the mechanisms by which this is influenced by pink1 and parkin. Recent observations in mam­ mals and flies detailed below have provided a number of important insights. Pink1 and parkin promotes mitophagy In a seminal work, Youle and colleagues showed that in mammalian cells Parkin is recruited to mito­ chondria whose inner membrane has been depo­ larized (an outcome common to multiple forms of mitochondrial damage) or that have been treated with the herbicide paraquat, an inducer of com­ plex-1-dependent reactive oxygen species (ROS) (Narendra et al., 2008). Recruitment of Parkin was followed by removal of these (damaged) mito­ chondria through a specialized form of autophagy known as mitophagy, in which mitochondria are specifically degraded following engulfment by autophagosomes (reviewed in Goldman et al., 2010). Recent findings suggest that mitophagy is intimately linked with changes in mitochondrial size and shape brought about through fission and fusion (reviewed in Hyde et al., 2010). Mitophagy requires both the loss of fusion and the presence of

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fission. Importantly, decreased mitophagy results in the accumulation of oxidized proteins and decreased cellular respiration, strongly suggesting that the end result of this process is the selective removal of damaged mitochondria (Twig et al., 2008). Mitophagy, but not recruitment of Parkin to depolarized mitochondria, requires Drp1, indi­ cating that Parkin-dependent mitophagy requires fission (Narendra et al., 2008). Work in flies and mammals further demon­ strates that recruitment of Parkin to mitochondria depends on Pink1 (Geisler et al., 2009; Narendra et al., 2009; Vives-Bauza et al., 2010; Ziviani et al., 2010). Parkin fails to be recruited to depolarized mitochondria in cells lacking pink1 and in some but not all pink1 disease mutant backgrounds. Pink1’s ability to recruit Parkin requires Pink1 kinase activity but how kinase activity serves to recruit Parkin is unclear. In flies, early work sug­ gested a role for Pink1-dependent phosphoryla­ tion of Parkin as important for recruitment (Kim et al., 2008). But in mammals, while Pink1 and Parkin appear to localize near each other, there is no evidence that they bind each other, that Pink1 phosphorylates Parkin, or that Parkin ubi­ quitinates Pink1 (Vives-Bauza et al., 2010). How does Pink1 promote the recruitment of Parkin specifically to damaged mitochondria? Pink1 protein levels are specifically upregulated on damaged mitochondria (Narendra et al., 2009; Vives-Bauza et al., 2010; Ziviani et al., 2010). Pink1 is a membrane protein with its C-terminus facing the cytoplasm (Zhou et al., 2008). In healthy mito­ chondria, Pink1 is constitutively cleaved, releasing its C-terminal kinase domain into the cytoplasm where it is degraded in a proteasome-dependent manner. In damaged mitochondria that have lost their membrane potential, cleavage decreases and full-length Pink1 remains anchored to the mem­ brane (Narendra et al., 2009). Mitochondrial anchorage is all that is required, because tethering of Pink1 to the mitochondrial membrane through other methods is sufficient to recruit Parkin (Narendra et al., 2009). As expected, based on these observations, overexpression of pink1 in a

wild-type background, but not a parkin mutant background, is also sufficient to promote Parkin recruitment (Vives-Bauza et al., 2010; Ziviani et al., 2010). In cultured Drosophila cells, recruit­ ment of Parkin results in the ubiquitination and removal of Mitofusin, while loss of pink1 and parkin results in accumulation of Mitofusin (Ziviani et al., 2010). Ubiquitination of Mitofusin may func­ tion to prevent outer mitochondrial membrane fusion, thus facilitating the segregation and isola­ tion of damaged mitochondria. Ubiquitinated Mitofusin may also serve as a signal for mitophagy, a role also suggested for ubiquitinated voltagedependent anion channel 1 (VDAC1), which is generated in a parkin-dependent manner in response to mitochondrial damage in mammalian cells (Geisler et al., 2009). The fate of VDAC1 has not been examined in Drosophila. Interestingly, loss of mitofusin in Drosophila, and VDAC1 in mammalian cells, results in decreased recruitment of Parkin to mitochondria, suggesting that these proteins may be involved in recruitment as well. The protease that cleaves Pink in response to stress remains to be identified. In Drosophila, the Rhom­ boid-7 protease is required for Pink1 cleavage, though it remains to be shown that this cleavage activity is regulated by mitochondrial stress (Whit­ worth et al., 2008). The mammalian ortholog, PARL, is not required for damage-dependent Pink1 cleavage (Narendra et al., 2009). While these findings are intriguing, it is important to note that the recruitment of Parkin to mitochon­ dria has been carried out in cells lines (Ziviani et al., 2010). It remains to be shown that recruit­ ment of Parkin occurs in vivo in tissues that show phenotypes when pink1/parkin are removed and that this is associated with mitophagy. The findings that the pink1/parkin pathway reg­ ulates mitochondrial dynamics and mitophagy suggest an exciting model in which a failure of mitochondrial quality control lies at the heart of PD pathogenesis. In this model when mitochon­ dria undergo damage, Pink1 senses the damage, becomes stabilized, and recruits Parkin specifi­ cally to the damaged mitochondria. Parkin then

10

mediates degradation of Mitofusin, promoting mitochondrial fission and mitophagy to remove these damaged mitochondria. In PINK1/parkin­ mediated PD, damaged mitochondria fail to be cleared, thus resulting in posing significant risk of damage to cells. Are there other components in the pink1/parkin pathway? Are there any other factors that function in the pink1/parkin pathway? Mutations in DJ-1 cause autosomal recessive forms of PD (Bonifati et al., 2003). DJ-1 has been suggested to function through various mechanisms, including as a tran­ scriptional coactivator, a protease, and a molecu­ lar chaperone (Dodson and Guo, 2007; Kahle et al., 2009), but exactly how DJ-1 exerts its pro­ tective effects remains unclear. Studies in both cell culture and animal models have demonstrated that DJ-1 deficiency increases sensitivity to cell death induced by oxidative stress, whereas overexpression is protective (Dodson and Guo, 2007; Kahle et al., 2009). These and other observations have suggested a model in which DJ-1 senses the redox state of the cell, as a result of the modifica­ tion of cysteines, and under oxidative conditions is activated to exert protective effects. Recent cellbased studies have reported physical interactions of DJ-1 with Pink1 (Tang et al., 2006) and with Parkin (Moore et al., 2005; Xiong et al., 2009), and that DJ-1 can serve as a substrate for Parkin­ dependent ubiquitination (Olzmann et al., 2007). In addition, loss of DJ-1 is associated with defects in mitochondrial integrity and respiration in mam­ malian cells (Krebiehl et al., 2010). In Drosophila, flies lacking DJ-1 (deletions) are viable, with their most prominent phenotype being increased sensitivity to oxidative stress as assayed by reduced survival upon paraquat or rotenone feeding (Menzies et al., 2005; Meulener et al., 2005, 2006). In one study, DJ-1 was proposed to promote health through regulation of phosphati­ dyl-inositol 3-kinase and AKT (Yang et al., 2005).

However, these studies utilized RNAi to silence expression of DJ-1, which also resulted in lethality (Yang et al., 2005). Since deletion of the DJ-1 coding regions or silencing of DJ-1 expression using other RNAi constructs results in viable flies (Meulener et al., 2005; M. Guo, unpublished obser­ vations), it remains possible that some of the observed interactions between DJ-1 and other genes represent off-targeting effects of DJ-1 RNAi, an issue that should be further explored. In any case, overexpression of DJ-1 fails to rescue pink1 mutant muscle phenotypes (Yang et al., 2006) or male sterility due to lack of pink1 or parkin (M. Guo, unpublished observations). Therefore, there is currently no in vivo support in Drosophila for the idea that DJ-1 acts in the same genetic pathway as pink1/parkin. Omi/HtrA2 encodes a mitochondrially localized serine protease (Vande Walle et al., 2008) and has been suggested to function downstream of PINK1 in a common pathway (Plun-Favreau et al., 2007) based on the findings that mammalian Omi/HtrA2 binds Pink1 and that the phosphorylation of Omi/ HtrA2 is dependent on Pink1 in mammalian cells (Plun-Favreau et al., 2007). One group has also reported the presence of mutations/polymorphisms in Omi/HtrA2 in sporadic PD patients (Strauss et al., 2005). Overexpression of Omi/HtrA2 pro­ motes apoptosis (Vande Walle et al., 2008) but loss of Omi/HtrA2 does not result in less apopto­ sis, though it does result in some loss of non-DA neurons in the striatum (Jones et al., 2003; Martins et al., 2004; Rathke-Hartlieb et al., 2002). Does Omi/HtrA2 function as a downstream tar­ get of pink1 in vivo? An overexpression-based genetic interaction is observed between pink1 and omi (Whitworth et al., 2008; Yun et al., 2008). How­ ever, in contrast to pink1 mutants, omi null mutants have normal mitochondrial morphology in both muscle and testes, and a normal number of DA neurons (Yun et al., 2008). In addition, extensive loss-of-function-based genetic interaction studies fail to provide any in vivo evidence supporting the hypothesis that omi functions in the same pathway as pink1, either upstream or downstream,

11

to either positively or negatively regulate pink1. They also do not provide any clear evidence that omi acts in a parallel manner to regulate mitochon­ drial morphology (Yun et al., 2008). These loss-of­ function-based analyses are more relevant to PD than are omi overexpression-based analyses because the reported Omi/HtrA2 mutations asso­ ciated with PD are proposed to represent loss-of­ function or dominant-negative mutations (Strauss et al., 2005). In addition, mutant forms of the Dro­ sophila Omi/HtrA2 analogous to the reported dis­ ease form in sporadic PD patients, as well as a phosphorylation incompetent mutation of a serine residue thought to be the direct phosphorylation target of Pink1 (Plun-Favreau et al., 2007) retain significant, if not full, Omi/HtrA2 function in vivo (Yun et al., 2008). Thus, loss-of-function studies strongly suggest that omi does not play an essential role in regulating mitochondrial integrity in the pink1/parkin pathway (Yun et al., 2008). This con­ clusion is supported by recent work that found no association between mutations in Omi/HtrA1 and PD and work showing that the PD-associated muta­ tion in Omi/HtrA2 occur with comparable fre­ quency in unaffected populations, suggesting they represent simple polymorphisms (Ross et al., 2008; Simon-Sanchez and Singleton, 2008). Finally, it has recently been reported that overexpression of 4E-BP, which suppresses capdependent translation when hypo-phosphorylated, suppresses muscle degeneration, DA neuronal degeneration, and mitochondrial integrity pheno­ types seen in pink1 or parkin mutants (Tain et al., 2009). Consistent with these findings, feeding flies rapamycin, which inhibits Target of Rapamycin (TOR) thereby activating 4E-BP, also suppresses pink1/parkin phenotypes (Tain et al., 2009). These findings implicate cap-dependent translational con­ trol of unknown targets as regulators of the pink1/ parkin pathway or pathways that can compensate for their absence. The targets of translational reg­ ulation in this context are unknown. It is worth noting that activation of 4E-BP in the context of life span extension in flies results in increased translation of a number of mitochondrial proteins,

suggesting these as obvious targets for future char­ acterization (Zid et al., 2009). Contributions of Drosophila and future directions Recent findings of the pink1/parkin pathway raise many interesting questions. What regulates the stabilization of Pink1? What is the identity of the protease that cleaves Pink1 and how is its activity regulated? How does Pink1 regulate Parkin activity and localization? How does mutation of the Pink1 kinase domain disrupt Parkin recruitment? How does Parkin recruitment serve to promote mito­ phagy? Ubiquitination-dependent recruitment of the mitophagy machinery (Geisler et al., 2010), directed movement of damaged mitochondria to the site of autophagy (Vives-Bauza et al., 2010), and disruption of mitochondrial fusion (Tain et al., 2009) have all been suggested; do other mechanisms exist as well? Work in mammals has identified independent pathways that promote mitophagy (Zhang and Ney, 2009). In cells that lack func­ tional pink1 and/or parkin, are there other ways of inducing mitophagy? Can cellular physiology be manipulated so as to decrease the consequences of losing pink1 and/or parkin signaling in order to slow down or prevent mitochondrial damage from occurring? Finally, do pink1/parkin have mito­ phagy-independent roles in regulating mitochon­ drial and/or cellular physiology? The advantages of studying the pink1/parkin pathway in Drosophila are three-fold: (1) Loss of pink1 or parkin in the fly results in robust pheno­ types at the organismal level (held-up wings, male sterility, muscle degeneration, locomotion defects) as well as the subcellular level (mitochondrial morphology) in multiple tissues. Importantly, these phenotypes are present in young adults. Thus, they can be examined (and scored for sup­ pression or enhancement) without having to carry out prolonged aging studies. In contrast, studies of knockouts of parkin and pink1 in mice have not shown DA neuronal loss and yielded subtle phe­ notypes (Fleming et al., 2005; Gautier et al., 2008;

12

Goldberg et al., 2003; Itier et al., 2003; Kitada et al., 2007; Palacino et al., 2004; Perez and Palmi­ ter, 2005; Von Coelln et al., 2004; ), which are not suitable for screens for genetic modifiers. In short, Drosophila provides robust genetic and cell biolo­ gical readouts for mitochondrial function and integ­ rity. (2) The epistatic studies of genes that mediate familial PD, which allow one to order the action of genes in a pathway, are stringent, yet rapid and straightforward to carry out in flies. (3) Unbiased forward genetic screens and compound screens for regulators of the pink1/parkin pathway, or path­ ways that can compensate for their loss, are straightforward to carry out in flies. Given that defects in mitochondria accumulate in normal aging as well as in PD, identifying multiple ways of activating mitophagy may be generally useful therapeutically in many diseases of aging. In short, Drosophila provides an indispensable and unique opportunity to contribute to the PD field. Finally, it is worth discussing the opportunities provided by the pink1 and parkin phenotypes of male sterility. These phenotypes are associated with defects in mitochondrial morphology but these phe­ notypes are only partly suppressed (the morpholo­ gical defects but not the fertility) by expression of drp1 or silencing of mitofusin. Importantly, pink1 and parkin are in this context involved in a devel­ opmental transition, not a stress response. This suggests that they may respond to different signals and could implement outcomes different from those in somatic cells. Given the evolutionary conservation of the pink1/parkin pathway, it is reasonable to propose that similar unexplored functions for these proteins may also exist in mam­ mals. These functions may also be related to those involved in protecting from PD. In summary, work in Drosophila has provided key insights in understanding PD. Flies provided the first in vivo evidence that pink1 and parkin regulate mitochondrial integrity. The stringent epistatic studies provided compelling evidence that pink1 and parkin function in a common genetic pathway, with pink1 positively regulating parkin. In contrast, loss-of-function-based studies

have not provided in vivo support for DJ-1 or Omi/HtrA2 as components of the pink1/parkin pathway. In addition, studies in Drosophila also suggest that the pink/parkin pathway promotes mitochondrial fission and/or inhibits fusion. These studies provide the first demonstration that manip­ ulation of mitochondrial dynamics can suppress the pink1/parkin phenotypes both at the cellular level (mitochondrial integrity) and at the organismal level (muscle degeneration, dopamine levels, and locomotion), thereby providing novel therapeutic targets. More recent work in cultured cells demon­ strates that pink1 and parkin regulate mitophagy in Drosophila, paralleling work in mammals, suggest­ ing the exciting possibility that failure of mitophagy, and thus mitochondrial quality control, underlies the pathogenesis of PINK1/parkin-mediated PD. The unique attributes of Drosophila (robust phe­ notypes, straightforward genetics providing oppor­ tunities for genome-wide genetic screens and drug screens) suggest that Drosophila studies will con­ tinue to move the field forward. These studies may help identify novel therapies for PD and potentially other aging-related neurodegenerative disorders.

Acknowledgments The author is grateful to Bruce A. Hay and the Guo lab members for discussions, and Bruce A. Hay and Mark Dodson for comments on the manuscript. This work is supported by grants and funds from National Institute of Health (R01, K02, P01), the Glenn Family Foundation, the Alfred P. Sloan Foundation, the Esther A. and Joseph Klingenstein Fellowship, and the McKnight Foun­ dation of Neuroscience to M.G.

Abbreviations PD pink1 Drp1 Opa-1

Parkinson’s disease PTEN-induced kinase 1 Dynamin-related protein 1 Optic atrophy 1

13

DA mtDNA ROS RNAi VDAC1

Dopaminergic Mitochondrial DNA Reactive oxygen species RNA interference Voltage-dependent anion channel 1

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16 Valente, E. M., Abou-Sleiman, P. M., Caputo, V., Muqit, M. M., Harvey, K., Gispert, S., et al. (2004). Hereditary early-onset Parkinson’s disease caused by mutations in PINK1. Science, 304(5674), 1158–1160. Vande Walle, L., Lamkanfi, M., & Vandenabeele, P. (2008). The mitochondrial serine protease HtrA2/Omi: An over­ view. Cell Death and Differentiation, 15(3), 453–460. Venken, K. J., & Bellen, H. J. (2005). Emerging technologies for gene manipulation in Drosophila melanogaster. Nature Reviews Genetics, 6(3), 167–178. Vives-Bauza, C., Zhou, C., Huang, Y., Cui, M., de Vries, R. L., Kim, J., et al. (2010). PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proceedings of the National Academy of Sciences USA, 107(1), 378–383. Von Coelln, R., Thomas, B., Savitt, J. M., Lim, K. L., Sasaki, M., Hess, E. J., et al. (2004). Loss of locus coeruleus neurons and reduced startle in parkin null mice. Proceedings of the National Academy of Sciences USA, 101(29), 10744–10749. West, A. B., Dawson, V. L., & Dawson, T. M. (2007). The role of PARKIN in Parkinson’s disease. In Parkinson’s disease: Genetics and pathogenesis. New York: Informa Healthcare USA, Inc., pp. 199–218. Whitworth, A. J., Lee, J. R., Ho, V. M., Flick, R., Chowdhury, R., & McQuibban, G. A. (2008). Rhomboid-7 and HtrA2/Omi act in a common pathway with the Parkinson’s disease factors Pink1 and Parkin. Disease Model Mechanics, 1(2–3), 168–174. 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 USA, 102(22), 8024–8029. Xiong, H., Wang, D., Chen, L., Choo, Y. S., Ma, H., Tang, C., et al. (2009). Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation. Journal of Clinical Investigation, 119(3), 650–660. Yang, Y., Gehrke, S., Haque, M. E., Imai, Y., Kosek, J., Yang, L., et al. (2005). Inactivation of Drosophila DJ-1 leads to

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 2

Neurotoxic in vivo models of Parkinson’s disease: recent advances Jason R. Cannon and J. Timothy Greenamyre
Pittsburgh Institute for Neurodegenerative Diseases, Department of Neurology, University of Pittsburgh,

Pittsburgh, PA, USA

Abstract: Animal models have been invaluable to Parkinson’s disease (PD) research. Of these, neurotoxin models have historically been the most widely utilized. The goal of this chapter is to give a brief historical description of classic PD models and then to identify the most recent important advances in modeling human PD in animals. Indeed, significant advances in modeling additional features of PD and expansion to new species have occurred in both older and newer models. The roles these new advances in modeling may have in future PD research are examined in this chapter. Keywords: Parkinson's disease; neurotoxin; animal models

in humans. Thus, the importance of animal models is immediately obvious and it is clear that the better the model, the better the understanding of the human disease will be. The ability to predict successful treatments for human disease is also dependent on the quality of the animal model. Neurodegenerative diseases are exceptionally difficult to model. While a small number of these diseases are caused by known purely genetic fac­ tors, the causes of the vast majority are unknown. Thus, most models typically focus on recapitulat­ ing the key pathological and biochemical diseases. A perfect animal model of neurodegenerative disease would recapitulate all of the pathological features observed in human patients and share the etiology of the clinical condition. Unfortunately,

Introduction Animal models of human disease are essentially utilized for two major purposes: (1) to study the pathogenic mechanisms of the human disease and (2) to test potential clinical therapeutics. Understanding pathogenic pathways provides clues to the potential etiology of a disease and may provide insights into therapeutic strategies. Drugs, gene therapy, or medical devices designed to exploit these pathways must then be tested in animal models before proceeding to clinical trials
Corresponding author. Tel.: þ1-412-6489793; Fax: þ1-412-6489766; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84002-6

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such a model does not exist. Therefore, researchers continually strive to improve the quality of models. Animal models will continue to be a major research goal until both the causes of—and suitable treat­ ment options for—these diseases are identified. Modeling Parkinson’s disease (PD), in particu­ lar, has been an extraordinarily difficult task. In humans, the disease typically develops over sev­ eral decades. While the hallmark pathology of PD remains the loss of dopamine neurons in the sub­ stantia nigra together with cytoplasmic inclusions known as Lewy bodies in surviving neurons, PD is now known to affect multiple brainstem nuclei and other brain regions, and also to involve sys­ temic pathology (Braak et al., 2004; Forno, 1996; Spillantini et al., 1997). No model to date has been able to recapitulate all of these pathological fea­ tures. Additionally, the etiology of the majority of human PD cases is unknown, with known mono­ genic mutations accounting for <10% of all cases. Because the cause of PD is unknown, a single toxicant exposure or genetic alteration cannot be utilized in the animal to correctly model most cases of PD. While these difficulties suggest that our current models have significant limitations, there is much to be excited about. Decades of modeling with neurotoxins have contributed much to our understanding of human PD—and current models continue to evolve, even as new models are developed. The majority of animal models of PD can be roughly divided into genetic—those utilizing in vivo expression of PD-related mutations, or neuro­ toxic—those using environmental or synthetic neu­ rotoxin administration. Genetic animal models, while recapitulating rare mutations in a-synuclein, Parkin, Pink1, and DJ-1 that are known to elicit human PD, have mostly failed to recapitulate the key neurobehavioral or pathological features of clinical PD (Chandran et al., 2008; Chen et al., 2005; Goldberg et al., 2003, 2005; Itier et al., 2003; Kitada et al., 2007; Manning-Bog et al., 2007; Masliah et al., 2000; Matsuoka et al., 2001; Perez and Palmiter, 2005; Richfield et al., 2002; Von Coelln et al., 2004; Yamaguchi and Shen, 2007).

Neurotoxin animal models have a much longer history and their use remains widespread. There are many fine reviews on neurotoxin animal mod­ els of PD (e.g., Betarbet et al., 2002; Dauer and Przedborski, 2003). The goal of this chapter is to briefly summarize classic PD models and describe important advances and improvements to these longstanding models and also to describe emerging models that have yet to be fully characterized. While in vitro models of PD can provide useful mechanistic data and serve as an initial screening for potential neuroprotective agents, animal mod­ els are best suited to address the complexity exhib­ ited in human PD. Therefore, this chapter is limited to discussion of in vivo findings, except where inclusion of in vitro data is absolutely neces­ sary to provide mechanistic data.

Rationale for neurotoxicant-based models of PD Historically, neurotoxin-induced impairments of the nigrostriatal dopamine system have been the most common system to model PD in animals—a popularity that remains in place to date. There are several key features of these models that have led to their widespread use and continued evolution. First, neurotoxins can be selected based upon a capacity to functionally alter or lesion specific neuronal populations. Such a rationale is particu­ larly useful in PD, where specific neuronal popu­ lations undergo selective loss. This approach was utilized as early as the 1950s when Carlsson uti­ lized reserpine to elicit brain catecholamine deple­ tion (Carlsson et al., 1957). Reserpine was chosen because it was known to deplete serotonin, which shares structural similarities to catecholamines (Carlsson, 2002). In some cases, selectivity is discov­ ered by accident—an animal or human is exposed to a toxin and selective damage is observed—such is the case with several models described in this chap­ ter. Toxins that have been found to lesion specific neuronal populations relevant to a given disease may then be developed into animal disease models. Alternatively, as our understanding of neurobiology

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increases, prediction of toxin selectivity improves. Much epidemiological data also supports the notion that environmental neurotoxin exposure contributes to some cases of sporadic PD (Di Monte, 2001; Fall et al., 1999; Gash et al., 2008; Gorell et al., 1997, 1998; Hageman et al., 1999; Kuhn et al., 1998; Liou et al., 1997; Pezzoli et al., 1996; Semchuk et al., 1993; Tanner et al., 2009). Therefore, the choice to use neurotoxins to model PD is rooted in both the etiological data and the capacity to induce relevant pathogenic features. Classic in vivo neurotoxicant-based models Animal models of PD have now existed for some 50 years. While there have been many attempts and derivations, a few models have persisted through the decades to provide an immense amount of invaluable data that has significantly contributed to our understanding of PD. Early modeling of behavior and dopamine depletion Perhaps the earliest pharmacologic attempt at a PD model resulted from work by Carlsson and Hilarp in the 1950s, in which they depleted brain catecholamines using reserpine (Carlsson et al., 1957). This depletion resulted in akinesia, and sub­ sequent work identified dopamine depletion as the cause of the behavioral phenotype. The phenotype was rescued by administration of L-DOPA, the biochemical precursor to dopamine (Bertler et al., 1958; Carlsson et al., 1958). Ultimately, the work led to the conclusions that (1) dopamine depletion in the basal ganglia was central to the cardinal behavioral features of PD and (2) alleviation of symptoms could be achieved through L-DOPA supplementation (Bertler et al., Carlsson, 1959). These were powerful early observations elucidat­ ing the proximal cause of motor-related PD symp­ toms and the discovery of a treatment regimen that remains the single most effective therapy for

parkinsonian motor impairment. Even with the huge advances that were made using the reserpine model, there were major limitations: other neuro­ transmitter systems are affected by reserpine, neu­ rochemical depletions are temporary, and nigral dopamine cell loss does not occur. Methamphetamine is a psychostimulant that eli­ cits dopamine depletion, and like reserpine, it does not induce loss of nigral dopamine neurons (Fibiger and Mogeer, 1971). The mechanism is thought to be at least partially due to dopamine release through action on the dopamine transporter (Schmidt et al., 1985; Sonsalla et al., 1986, 1989). Following metham­ phetamine administration, motor behavior deficits characteristic of striatal dopamine depletion occur in rodents (Walsh and Wagner, 1992). While metham­ phetamine is useful to study the effects of dopamine depletion, it is an acute model and does not replicate the key pathological features of human PD. By 1919 it was known that rigidity and tremor in parkinsonism were associated with nerve cell loss in the substantia nigra (Tretiakoff, 1919). The later work by Carlsson identified dopamine depletion as the key to the development of motor symptoms, and the subsequent work by Moore related these two findings by demonstrating that the nigrostria­ tal pathway is dopaminergic (Moore et al., 1971). At that point, it became clear that loss of striatal dopamine in PD was due to frank degeneration of this pathway. As such, models that acutely elicit dopamine depletion without the underlying term­ inal or cell body loss ultimately have limited rele­ vance in modeling human PD. Thus, animal models that reproduce dopamine cell loss in the substantia nigra were needed to accurately repli­ cate the pathology observed in PD. 6-Hydroxydopamine History and summary The use of 6-hydroxydopamine (6-OHDA) as an experimental dopaminergic neurotoxin is one of the oldest and most utilized models of PD (for

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excellent reviews see Schwarting and Huston, 1996a, b). Indeed, much of the information on the behavioral, biochemical, and physiological effects of dopamine depletion and nigral dopa­ mine cell loss was derived from this model. 6-OHDA was first isolated in the late 1950s (Senoh et al., 1959; Senoh and Witkop, 1959) and it was initially found to produce denervation of noradrenergic cardiac fibers (Porter et al., 1963, 1965). Ungerstedt pioneered the use of this neu­ rotoxin to lesion the dopaminergic nigrostriatal system in the rat (Ungerstedt, 1968). Use of 6-OHDA as a dopaminergic neurotoxin remains widespread today and the usefulness of this model in PD research endures. The potential relevance of the neurotoxin to clinical PD is underscored by the fact that this molecule has been found in urine samples (Andrew et al., 1993) of patients. Additionally, 6-OHDA has been proposed as a putative endogenous neurotoxin in dopamine neurons because of favorable oxidative conditions (Soto-Otero et al., 2000). The structure of 6-OHDA is very similar to dopamine. However, the presence of an additional hydroxyl group renders the molecule toxic to dopamine neurons. 6-OHDA is able to enter catecholamine neurons through the dopamine transporter, where it is thought to auto-oxidize and lesion cells through oxidative stress (Sachs and Jonsson, 1975). 6-OHDA does not cross the blood–brain barrier and, therefore, must be stereotaxically infused into the parenchyma. By a significant margin, the most common use of 6-OHDA is unilateral infusion into the rat medial forebrain bundle. One of the most attractive fea­ tures of this model is the ability of each animal to serve as its own control, with a lesioned and unle­ sioned hemisphere. This is particularly useful in behavioral analysis, where behavioral deficits can be very difficult to assess in bilateral models. It should be noted, however, that some compensa­ tory changes on the “good” side undoubtedly occur and there are bilateral alterations in physiol­ ogy and function. Dopamine depletion, nigral dopamine cell loss, and neurobehavioral deficits

have all been successfully achieved using variations of 6-OHDA models (Schwarting and Huston, 1996b, 1997). Mice are also sensitive to 6-OHDA (Asanuma et al., 1998; Fung and Uretsky, 1980; Matsuura et al., 1996), although the model is used in mice much less frequently than in rats. The discrepancy in use is likely due to the added diffi­ culty of stereotaxic surgery and the lower amount of tissue obtained in a much smaller animal.

Recent advances The 6-OHDA model has now been around for more than 40 years. While it is most commonly administered in the medial forebrain bundle, pro­ ducing acute and severe nigrostriatal denervation, there have been recent advances that attempt to more closely recapitulate the key features of human PD. Striatal infusion of the toxin produces progressive degeneration resulting in lower levels of dopamine cell loss (Sauer and Oertel, 1994). A progressive lesion allows potentially neuropro­ tective agents to be tested after toxin administra­ tion and nigrostriatal degeneration have been initiated—a temporal scenario that is much more relevant to clinical treatment regimens (Lindholm et al., 2007). Using this partial and progressive regi­ men, multiple groups have now been able to model non-motor behavioral features such as emotional and cognitive alterations in the rat (Branchi et al., 2008; Tadaiesky et al., 2008). Depression is a com­ mon feature of PD and has recently been success­ fully modeled in a rat 6-OHDA PD model (Winter et al., 2007). This is an important aspect of PD to model because a variety of psychiatric disorders are common in PD (Aarsland et al., 1999a, b) and will be important to model in order to develop better therapies. Recently, 6-OHDA use has been expanded to additional animals in which genetic manipulations and high-throughput screening can be conducted. 6-OHDA treatment can elicit oxidative stress and dopamine neuron loss in the zebra fish (Parng et al., 2007). Similarly, 6-OHDA elicits dopamine

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cell death in the round worm (Caenorhabditis elegans) (Nass et al., 2002). Use of 6-OHDA in these systems may be more cost effective and may allow more potentially therapeutic compounds to be screened. Promising compounds could then, in theory, be tested in higher order animals with greater anatomical similarity to humans. Intracerebral infusion of 6-OHDA has long been used to model behavioral, neurochemical, and pathological features associated with severe lesioning of the nigrostriatal dopamine system. Several recent advances have shown that model­ ing of non-motor functional deficits can also be achieved with the model. Gastrointestinal (GI) dysfunction is one of the earliest reported com­ plaints in PD and the functional impairments can become severe (Edwards et al., 1991; Pfeiffer, 2003). A severe unilateral 6-OHDA lesion can elicit decreased propulsion, modeling at least a portion of human PD GI deficits (Blandini et al., 2009). Speech and vocalization abnormalities have also been well documented in human PD and have now been reported in 6-OHDA-lesioned rats (Ciucci et al., 2007, 2009). Much recent research has also focused on the psychiatric aspects of PD. Thus, even though most formulations of the 6­ OHDA model result in severe and acute motor impairment, non-motor behavioral and systemic alterations have been recently modeled. The use of 6-OHDA as a dopaminergic neuro­ toxin has a long history, providing a wealth of data on the pathogenic features of PD and potential neuroprotective regimens. The recent expansion into additional animal species and the modeling of non-motor functional deficits indicate that 6­ OHDA will continue to be an import tool for a long time in PD research.

devastating and previously unknown neurological effects that arose from MPTP intoxication spanned from the creation of one of the most important models of PD to the emergence of experimental neurotoxicology as a major force in neurodegenerative disease research. MPTP-elicited parkinsonism first became widely known after a group of intravenous drug users presented with symptoms similar to PD (Langston et al., 1983). MPTP was produced as accidental side-product during the illicit chemical synthesis of 1-methyl-4-phenyl-4-propionoxypi­ peridine, an opioid analgesic drug. Following this report, one of the most prominent and important animal models of PD was created. Through much insightful mechanistic work, MPTP was found to cross the blood–brain barrier, to be metabolized in astrocytes to its active metabolite (1-methyl-4­ phenyl-dihydropyridine, MPPþ), and to enter catecholamine neurons though the dopamine transporter (Javitch et al., 1985). Inside dopamine neurons, MPPþ concentrates in mitochondria and inhibits complex I; the resulting adenosine-5’­ triphosphate (ATP) depletion and oxidative stress are thought to be the main mechanisms that cause catecholaminergic cell dysfunction and death (Nicklas et al., 1985). The MPTP model is now one of the most widely used models of PD. MPTP is typically adminis­ tered systemically because it is able to cross the blood–brain barrier. Mice and monkeys have been used extensively to model PD (Heikkila et al., 1984; Langston et al., 1984; Pileblad et al., 1984). For unknown reasons, rats are resistant to MPTP (Boyce et al., 1984; Chiueh et al., 1984), so the active metabolite, MPPþ, must be administered stereotaxically to achieve nigral dopamine cell degeneration (Sirinathsinghji et al., 1988).

MPTP Recent advances History and summary The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) story has provided many lessons. The

For all its history, the MPTP model continues to evolve, with several new developments. New routes of exposure are a major development.

22

Repeated nasal delivery produces characteristic behavioral deficits and pathology of nigral dopa­ mine cell degeneration (Rojo et al., 2006). Inter­ estingly, nasal delivery of pesticides that induce experimental PD such as rotenone and paraquat has not been found to induce similar pathology (Rojo et al., 2007). Understanding the mechan­ isms by which nasal MPTP induces experimental PD may be useful in identifying toxins that may pose an inhalation risk. Modeling important functional deficits using the MPTP model has also significantly improved. Separating general malaise or systemic effects on behavior from deficits due to a nigrostriatal lesion is difficult in bilateral rodent models. Several stu­ dies have now identified beam-walking deficits and gait abnormalities that are present in MPTP mice (Fernagut et al., 2002; Kurz et al., 2007; Quinn et al., 2007). Analysis of these functional deficits has proven a powerful endpoint in asses­ sing therapeutic potential (Pothakos et al., 2009). GI defects are a major symptom of multiple neu­ rodegenerative diseases, including PD. Recently, MPTP was found to alter colon motility (Ander­ son et al., 2007). Much has been learned about clinical PD from various versions of the MPTP model. One major obstacle that remains is effective transla­ tion of neuroprotective studies in experimental animals to clinical trials in humans. Even a cur­ sory review of the literature reveals that MPTP parkinsonism has been “cured,” attenuated, or prevented by a variety of compounds, including monoamine oxidase B inhibitors, vitamin E, the mixed lineage kinase inhibitor CEP-1347, the glutamate agonist riluzole, and coenzyme Q10 (Andringa et al., 2003; Beal et al., 1998; Benazzouz et al., 1995; Perry et al., 1985; Saporito et al., 1999); however, for unclear reasons, these findings have not resulted in clinical trial successes (Lang, 2006; Olanow et al., 2006; Parkinson Study Group PRECEPT Investigators 1989, 2007; Storch et al., 2007). Nonetheless, the MPTP model has been and remains an important tool in PD research.

Paraquat History and summary Paraquat is a widely used broad-spectrum herbicide. Inhalation of paraquat can cause severe and even fatal pulmonary toxicity in humans. Numerous other species also exhibit pulmonary toxicity after exposure (oral LD50 22–262 mg/kg, depend­ ing on species) (Clark et al., 1966; Ecobichon, 2001; Smith and Heath, 1976). Paraquat was first identified as a putative neurotoxicant based on its structural similarity to MPPþ, the active metabo­ lite of MPTP. Because of its use as a pesticide, the possibility that this compound could be an envir­ onmental contributor to the etiology of PD has received a great deal of attention. One of the ear­ liest in vivo regimens examining PD-relevant end­ points was conducted in the frog. Here, cumulative paraquat dosing was found to produce many behavioral features characteristic of PD, as well as decreased brain dopamine levels (Barbeau et al., 1985). Furthermore, systemic injection in mice elicits dose-dependent (5–10 mg/kg) decreases in movement and dopamine cell counts in the substantia nigra (Brooks et al., 1999). Addition­ ally, repeated paraquat administration was found to produce selective loss of nigral dopamine neu­ rons (McCormack et al., 2002). Paraquat reportedly enters the brain through a neutral amino acid carrier (McCormack and Di Monte, 2003). Because of its structural similarities to MPPþ, the mechanism by which paraquat elicits dysfunction of dopamine neurons was initially thought to be through selective complex I inhibi­ tion. Accordingly, in vitro mechanistic experi­ ments showed that both compounds produce significant oxidative stress after administration (Chun et al., 2001). However, paraquat has long been known to undergo redox cycling with gen­ eration of reactive oxygen species (ROS) (Fisher et al., 1973; Ilett et al., 1974). Furthermore, it has been directly demonstrated that paraquat does not act by direct inhibition of complex I (Richardson et al., 2005). Interestingly, paraquat has been

23

found to have a 28-day half-life in the mouse brain, with associated ongoing lipid peroxidation after intraperitoneal administration (Prasad et al., 2007). Therefore, it has been a useful compound in modeling the chronic development of nigros­ triatal lesions and associated functional deficits. The paraquat model was also further developed as a “mixture model” through the co-administra­ tion of the fungicide maneb (Thiruchelvam et al., 2000). The paraquat/maneb model was developed based on consideration of the overlapping geogra­ phical use of the two compounds. This model has been used to explore the temporal relationship of exposure to the neurodegenerative phenotype. Developmental exposures were later found to increase sensitivity (Thiruchelvam et al., 2002). Additionally, as with many other toxins, sensitivity increases with age (Thiruchelvam et al., 2003). Indeed, humans are exposed over the course of a lifetime to a myriad of environmental toxins—and cases of environmentally induced PD are unlikely to result from exposure to a single compound. Many epidemiological studies have implicated pesticide and herbicide exposures in the etiology of PD. However, exposure to a specific toxin as a causative agent has yet to be definitively shown. Nevertheless, prior studies have shown a sig­ nificant association between paraquat and PD (Hertzman et al., 1990; Liou et al., 1997). Most epidemiological studies tend to report that signifi­ cantly elevated risk (significant odds ratios) is associated with overall pesticide exposure. How­ ever, when paraquat is specifically examined, the odds ratios are often elevated, but not significantly so (Dhillon et al., 2008; Firestone et al., 2005). Interestingly, in one study, when the contribution of both maneb and paraquat exposures was taken into account, the odds ratio increased and reached significance (Dhillon et al., 2008).

Recent advances In an early characterization of the paraquat model of PD, nigral dopamine cell loss was observed.

However, striatal dopamine depletion was not found and tyrosine hydroxylase activity was ele­ vated 28 days after treatment, indicating that the model recapitulates pathological features of clinical PD, but may have differing neurochemical effects (McCormack et al., 2002). Interestingly, long-term examination suggests that paraquat administration in the rat produces chronic neurodegeneration and may be useful in modeling the “preclinical” stages of PD. After 4 weeks of paraquat treatment, a small but statistically insignificant nigral dopa­ mine neuron loss was found and confirmation of previously reported increases in dopamine neuro­ transmission was observed (Ossowska et al., 2005). However, after 24 weeks, significant dopamine neuron loss and dopamine depletion were observed, indicating development of a chronic neurodegen­ erative process. New additive and synergistic combinations of paraquat with other compounds are also being tested for in vivo production of the PD phenotype. The accumulation of iron in the substantia nigra is a well-known phenomenon in human PD (Dexter et al., 1987). Accordingly, exposure to iron or alterations in homeostasis during different life span time points have been repeatedly hypothe­ sized as risk factors for PD (Bartzokis et al., 2004; Johnson et al., 1999; Powers et al., 2003; Rhodes and Ritz, 2008). The paraquat model has recently been used to test such a phenomenon. Neonatal iron exposure combined with adult paraquat exposure was found to produce age-dependent dopamine cell loss in the nigra (Peng et al., 2007). Therefore, recent research indicates that paraquat and paraquat/maneb models are valu­ able in testing the contribution of developmental, age-dependent, and “multiple hit” mechanisms of dopamine neuron cell loss. Mounting epidemiological data continues to implicate paraquat exposure as an important com­ ponent of environmental risk factors for PD. These newer data further support the continued development and use of the model in animals. Additionally, newer long-term data suggest that the PD phenotype develops chronically and may

24

be more useful in modeling earlier stages of the disease than other models.

Rotenone Rotenone is a naturally occurring compound found in the roots of several plant species and has been extensively used as an insecticide and to kill fish. It is a well-known, selective inhibitor of mitochondrial complex I (Ravanel et al., 1984). Rotenone is highly lipophilic and is able to cross the blood–brain barrier rapidly. Systemic complex I inhibition has been observed in PD patients (Parker et al., 1989; Schapira et al., 1989). Therefore, toxins that produce complex I inhibition have received considerable attention as both potentially causative agents and possible modeling tools. Unlike MPTP, which produces complex I inhibition only in catecholaminergic neurons, rotenone produces systemic complex I inhibition. The first reported attempt to use rotenone to model PD was made through stereotaxic injection into the parenchyma at a concentration 500 000­ fold greater than its IC50 for complex I (Heikkila et al., 1985). In this report, dramatic decreases in striatal dopamine and serotonin were observed. While some later studies have utilized cerebral infusions to produce neurochemical and beha­ vioral deficits (Antkiewicz-Michaluk et al., 2004; Saravanan et al., 2005; Sindhu et al., 2005), the lesions produced by such high doses are not likely specific for dopamine neurons and projections. Indeed, infusion of rotenone into the striatum at similar doses was recently found to elicit liquefac­ tive necrosis surrounded by gliosis and ventricular dilation (Rojas et al., 2009). Thus, it is not clear that stereotaxic injection of rotenone offers any advantage over other toxins, such as 6-OHDA. Human data on systemic complex I deficiency in PD led researchers to test peripheral routes of rotenone administration. Administration of 10–18 mg/kg/day was found to produce “nonspeci­ fic” brain lesions and peripheral toxicity (Ferrante

et al., 1997). However, when rotenone was admi­ nistered chronically at lower doses to achieve com­ plex I inhibition similar to that observed in platelets of PD patients, it produced highly selec­ tive nigrostriatal degeneration (Betarbet et al., 2000). Remarkably, for the first time in an animal model, cytoplasmic a-synuclein-positive inclusions similar to Lewy bodies were observed in surviving dopamine neurons. The rotenone model also pro­ vided the first proof of concept that systemic mito­ chondrial impairment could produce selective nigrostriatal degeneration; it further suggested that nigral dopamine neurons have a unique sen­ sitivity to complex I inhibition. The most common regimen of the model is chronic systemic adminis­ tration in the Lewis rat, which may be more sensi­ tive than other strains (Betarbet et al., 2000).

Recent advances Along with the paraquat model, the rotenone model is still relatively new when compared to the MPTP and 6-OHDA models. Therefore, with expanded use, much information is still being obtained about the model and it is continuing to undergo refinement and expansion into additional species. Every model of PD has major limitations. The most obvious problem for the rotenone model was variability in the percentage of animals that exhibited a clear lesion following systemic administration— typically 30–50% for most batches (Betarbet et al., 2000). While those animals that have a clear lesion provide valuable data on pathogenic processes, variability limited use of the model in neuroprotection and potentiation studies. Chronic systemic infusion using osmotic minipumps has been the most common delivery regimen. Recently, intraperitoneal delivery was reported to elicit consistent behavioral and neurochemical deficits, although mortality was high in these stu­ dies (Alam and Schmidt, 2002, 2004). With mod­ ification of the delivery vehicle and dosing regimen, rotenone produced lesions in all animals

25

(Cannon et al., 2009). In this study, consistent apomorphine-responsive motor deficits, catecho­ lamine depletion, nigral dopamine cell loss, and a-synuclein-positive intracellular aggregates were observed. The development of a regimen that consistently produces lesions is a major advance­ ment for the rotenone model. Moreover, given its ability to reproduce key pathological features of human PD, a consistent rotenone model should be a highly valuable tool to conduct neuroprotection and potentiation experiments. Indeed, experi­ ments testing neuroprotection have already been conducted using this regimen. For example, there is much conflicting data on the efficacy of melatonin as a neuroprotective agent in PD models and there was some concern that melatonin’s apparent neuropro­ tective actions may have been mediated by its docu­ mented effects on dopamine transporters. Using the modified rat rotenone model, it was found that mel­ atonin actually potentiated rotenone-induced stria­ tal dopamine depletion and terminal loss, and nigral dopamine cell loss (Tapias et al. 2010). Rotenone has also been found to effectively and selectively lesion dopamine neurons in several other species that are more amenable to genetic manipulation than the rat. For example, knockout or overexpression of specific genes is much more common in mice than in rats. Some (but not all) laboratories have found rotenone to work in mice, although the delivery parameters typically require substantial modification (Pan-Montojo et al., 2010; Takeuchi et al., 2009). L-Dopa-responsive beha­ vioral deficits and dopamine cell loss are also observed in Drosophila (Coulom and Birman, 2004). Indeed, flies with DJ-1 and Parkin muta­ tions—known PD-related genes—exhibit increased sensitivity to rotenone (Meulener et al., 2005; Wang et al., 2007). Similarly in C. elegans, expres­ sion of PD-related mutations increases sensitivity to rotenone (Saha et al., 2009; Ved et al., 2005). Zebra fish have also been shown to be sensitive to rotenone (Bretaud et al., 2004). Even species such as snails exhibit decreases in movement and neu­ rochemical and pathological alterations after rote­ none (Vehovszky et al., 2007). Thus, the rotenone

model has now been successfully applied to a wide range of species, several of which are amenable to genetic manipulation. Therefore, gene–environ­ ment interactions can easily be investigated using variations of this model. The relevance of the rotenone model to clinical PD continues to increase as additional similari­ ties to human PD are identified. Indeed, recent research has shown the rotenone model to recapitulate apomorphine-responsive behavioral deficits; accumulation and aggregation of a-synu­ clein; a-synuclein- and polyubiquitin-positive Lewy bodies and Lewy neurites; early and sus­ tained activation of microglia; oxidative modifi­ cation and translocation of DJ-1 into mitochondria in vivo; impairment of the nigral ubiquitin-proteasome system; and a-synuclein pathology in enteric neurons and functional def­ icits in GI function, including gastroparesis (Betarbet et al., 2002, 2006; Cannon et al., 2009; Drolet et al., 2009; Greene et al., 2009). Remark­ ably, the rotenone model has also been shown to predict previously unknown features of human PD. For example, the rotenone model was found to produce accumulation of iron in the substantia nigra through a novel mechanism involving transferrin and transferrin receptor 2 (Mastroberardino et al., 2009). In this study, the same alterations were subsequently confirmed in brains of PD patients. Thus, the rotenone model predicted what would be found in human PD. Recent advances suggest that the model will also be highly useful in studying gene–environment interactions, neuroprotection, potentiation, and pathogenesis studies. As such, the rotenone model will likely be an important component of in vivo PD research for many years to come.

Emerging models While there are numerous established PD toxin models, the lack of translation of animal research to clinical trial successes clearly indicates that

26

better animal models are needed (Lang, 2006; Lang and Obeso, 2004). Indeed, several newer models are being developed. While these newer models will require much more time to fully char­ acterize, preliminary results are encouraging. Industrial chemical exposures have long been suspected to play a role in neurodegeneration. Recently, trichloroethylene (TCE) exposure was linked to PD (Gash et al., 2008). Several workers with the highest levels of exposure were diagnosed with PD and those with lower level exposures displayed many PD features including slowing of movement. In the same report, the authors found that chronic TCE exposure in rats produced loss of striatal dopamine and loss of nigral dopamine neurons. Thus, in a single report, the authors identified a potential PD neurotoxin and charac­ terized an animal model that may prove useful in modeling PD. Because human PD is unlikely to result from a single exposure, “multiple hit” models utilizing more than one exposure are gaining popularity. Inflammatory models of PD using lipopolysac­ charide (LPS) are covered in another chapter regarding this issue, but it is worth noting that these inflammatory models are now being combined with established PD models. An LPS þ MPTP regimen was recently found to produce functional deficits in gait as well as dopamine depletion (Byler et al., 2009), but single exposures to either toxin did not cause deficits.

Wish list for the future: what would an ideal model look like? The models reviewed here—both old and new— are continually evolving. However, there is clearly a need for new models. Models that take into account more than one exposure or more than one factor are gaining popularity. Gene–environment interaction models—those that utilize transgenic animals exposed to toxins—are emerging. Animals expressing human mutations known to cause PD are in some cases more sensitive to MPTP (Kim

et al., 2005; Nieto et al., 2006; Rathke-Hartlieb et al., 2001). However, such mutations have not always been found to increase susceptibility in other models (Perez et al., 2005). It is commonly stated that most PD cases likely result from a combination of environmental exposures and genetic susceptibility. However, there is not an adequate system in place to test this hypothesis. Complete knockout of a protein or massive transgene overexpression is often utilized in genetic models. Such model systems do not accurately represent genetic events that occur in humans— and there may be “off-target” effects, including developmental or compensatory changes. On the other hand, animals that more accurately express the genetic mutations and polymorphisms associated with sporadic PD could, in theory, be used to screen putative neurotoxins. Such models would be valu­ able for testing relevant gene–environment interac­ tions, identifying causative agents, and informing individuals with specific genetic backgrounds about the risks of certain classes of compounds. Current models provide excellent reproduction of the striatal dopamine depletion and nigral dopamine neuron loss observed in clinical PD. Numerous agents have been found to be protec­ tive in both the 6-OHDA and MPTP models and have yet to produce positive results in clinical trials. There is currently not enough information to determine whether neuroprotective effects seen in the paraquat and rotenone models will be pre­ dictive in humans. Ultimately, however, we need models of PD with high predictive value, although it may be difficult to know which model is best until at least one neuroprotective agent is found to work in humans. At this point in time, it can be predicted with relative certainty that neuroprotec­ tive agents that are effective when administered before toxin exposure, but which are ineffective when administered later, have little chance being useful in humans. Finally, there is a need for con­ tinued development of chronic models to more accurately mimic human PD—and such models should be used to confirm findings from acute models.

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Summary Modeling PD using toxins has a long history, result­ ing in the discovery of dopamine’s involvement in PD and the identification of the therapeutic poten­ tial of L-DOPA, still the most efficacious treatment for PD. Continual improvement in toxin models has occurred and we now have the ability to replicate the majority of pathogenic features of PD. Toxin models are even beginning to be used to predict what might occur in human PD. However, there is much progress to be made, particularly as regards the development of models that can accurately pre­ dict effective neuroprotective agents in humans. Acknowledgments We thank Maxx Horowitz for a critical reading of this manuscript.

Abbreviations 6-OHDA GI LPS MPP+ MPTP PD ROS

6-hydroxydopamine gastrointestinal lipopolysaccharide 1-methyl-4-phenyl­ dihydropyridine 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine Parkinson’s disease reactive oxygen species

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 3

Behavioral analysis of motor and non-motor symptoms in rodent models of Parkinson’s disease Stephen B. Dunnett
and Mariah Lelos School of Biosciences, Cardiff University, Cardiff, South Wales, UK

Abstract: Alongside the classical motor symptoms, non-motor symptoms are increasingly recognised to play a major role in the disability associated with Parkinson’s disease in humans. Animal models based on experimental depletion of forebrain dopamine have traditionally focussed on the simple and easy to measure motor impairments, and they reproduce well the bradykinesia, rigidity and impairments in the initiation and sequencing of voluntary goal-directed movement. However, a more comprehensive analysis is now urgently required. In this chapter we summarise the predominant unilateral and bilateral dopamine lesion, toxin and genetic models of human parkinsonism, and review the consequences in more complex cognitive, motor learning and psychiatric (‘behavioural’) domains. Theoretical and experimental advances in our understanding of information processing and associative plasticity within the striatum are not only revolutionising our understanding of normal striatal function but also bear directly on our understanding of the processes that underlie non-motor as well as motor disability in human disease, including in Parkinson’s disease. Keywords: Nigrostriatal lesions; Rotation; Locomotor activity; Sensorimotor tests; Motor learning; Reinforcement; Motor impairment; Cognitive impairment; Psychiatric impairment; Animal models

disease may be related to loss of forebrain dopa­ mine activity (Carlsson, 1959), that dopamine was the neurotransmitter of the substantia nigra neu­ rons (Dahlström and Fuxe, 1964), and that dopamine replacement may provide an effective treatment (Carlsson et al., 1957) arose from the studies of experimental animals in the 1950s and 1960s. Since then, animal models have been fun­ damental to our understanding of the functional pathology of the human disease and for the development of all classes of modern therapeutics.

Introduction It has been known since the early twentieth century from postmortem studies that Parkinson’s disease (PD) is associated with Lewy body pathology and degeneration in the substantia nigra (Lewy, 1914) but the realization that Parkinson’s
Corresponding author. Tel.: þ1-44-2920875188; Fax: þ1-44-2920876749;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84003-8

35

36

Indeed, animal models of Parkinson’s disease have provided a protypical exemplar for transla­ tional medicine, not least because the neurochem­ istry, neuropharmacology, and behavioral functions associated with experimental manipula­ tion of forebrain dopamine system have proved to be both tractable and highly sensitive to experi­ mental analysis. A critical component in this multi-disciplinary program has been the contribution of behavioral neuroscience for the experimental analysis of the functional consequences of manipulating central neurotransmitter systems and pathways. One aspect of this contribution has been to provide more sensitive outcome measures in developing novel therapeutics that are valid representations of symptoms of concern in treating the human dis­ ease. Equally, behavioral neuroscience has sought to understand the fundamental functional organi­ zation of the underlying neural systems, which has contributed to our understanding of the disease itself and of its essential components. This chapter reviews the recent advances in the tools and tech­ niques for behavioral analysis of both motor and non-motor functions in animal models of human PD, spanning both the classical focus on dopa­ mine dysfunction and more recent attention to the potential contributions of changes in comple­ mentary non-dopaminergic neural systems.

Animal models of PD Classical pharmacological models Pharmacological depletion of dopamine transmis­ sion, whether by blocking synthesis (with drugs such as a-methyltyrosine), blocking storage (e.g., reserpine, as used in Carlsson’s classic studies), or blocking the dopamine receptors (with neuroleptics such as haloperidol), produces a profound distur­ bance in the motor behavior of the experimental animal, manifested by hypokinesia and a decline in the initiation of all voluntary behaviors. In the extreme case (at high doses), this takes the form of

“catalepsy”, in which the animal maintains a rigid abnormal posture and resists displacement. Many of the early drugs (including reserpine and a-methyltyrosine) affected noradrenaline systems as much as dopamine systems, so that Carlsson had to rely in his early studies on the different dis­ tribution of the two neurochemicals, noting the high concentration of dopamine in basal ganglia struc­ tures known to be involved in motor function, to infer that dopamine depletion was the specific sub­ strate of hypokinesia. This inference has been amply confirmed by subsequent studies using selective dopamine lesions as well as much more specific drugs, including selective dopamine recep­ tor antagonists. Conversely, drugs that increase dopamine activ­ ity, such as the amphetamines, produce marked hyperactivity, the magnitude of which correlates well with the activity of the different isomers of dopamine, rather than of noradrenaline turnover and release. Moreover, drugs that increase dopamine synthesis (e.g., byL-dopa) or dopamine receptor agonists (e.g., apomorphine) have the capacity to reverse the symptoms, providing a restoration of activity in animals rendered akinetic or cataleptic by dopamine depletion, and providing the experi­ mental basis for most contemporary pharma­ cotherapies used in patients. At moderate doses, the activation provided by amphetamine induces increased locomotor activity in experimental animals but at higher doses total locomotion declines to be replaced by intense focused repetitive actions known as “stereotypy”. Detailed analysis of the dose–response profiles of amphetamine action led Lyon and Robbins (1975) to characterize the effects of amphetamine as essen­ tially disinhibitory, to increase the rates of initiation of all prepotent responses, which would compete for expression: at the highest doses only the most sali­ ent and brief movements would achieve expression. Not only the phenomenology of stereotypy but the theoretical analysis accompanying it bears strong relation to our contemporary understanding of the dyskinesias associated with chronic L-dopa therapy in patients (Cenci and Konradi, 2010).

37

Bilateral 6-OHDA lesions

Unilateral 6-OHDA lesions

The ability to model Parkinson’s disease in animals was transformed in the 1960s by the introduction of the catecholamine neurotoxin 6-hydroxydopa­ mine (6-OHDA). Injection of 6-OHDA into the lateral ventricles produces a profound bilateral forebrain depletion of dopamine (and noradrena­ line) in rats and mice. As with pharmacological blockade, this is associated with akinesia and cat­ alepsy, but on a chronic basis, resulting in pro­ found and lasting impairments in eating and drinking (“aphagia” and “adipsia”), and loss of body weight such that the animals will die without intensive care and tube feeding (Ungerstedt, 1971a; Zigmond and Stricker, 1972). Depending on the level of dopamine depletion, the animals may eventually recover the ability to drink and feed themselves, along with a gradual recovery of spontaneous activity and engagement in other voluntary behaviors, such as grooming (Zigmond and Stricker, 1973). As with many catecholaminergic drugs, 6-OHDA is toxic for noradrenaline as well as dopamine neurons, although the dopaminergic selectivity of the lesions can be enhanced by pretreatment with the noradrenaline uptake inhibitor desmethyl imipramine that blocks uptake of the toxin into noradrenergic neurons. The recovery process after extensive and selective dopamine depletion appears in large part to depend on the significant degree of plasticity in residual dopamine neurons spared by the lesion, which can compensate to a large degree for the lost activation by upregulation presynaptically in neu­ ron firing, dopamine synthesis, storage, release, and turnover, and postsynaptically of dopamine receptor sensitivity and postsynaptic signaling (Zigmond et al., 1990). Nevertheless, even recov­ ered animals remain profoundly bradykinetic; they are difficult to motivate for the normal rewards or to train in a variety of learning paradigms, and indeed they provide significant welfare challenges as a practical experimental model for routine laboratory use.

The utility of the 6-OHDA model was trans­ formed with the introduction of the unilateral lesion model, in which the toxin is injected stereo­ tactically into the ascending nigrostriatal bundle. First introduced to aid anatomical analysis of fore­ brain projection pathways (Andén et al., 1966; Ungerstedt, 1971c), the unilateral 6-OHDA lesion was seen to be associated with marked postural bias and turning asymmetry toward the side of the lesion. Notably, if the animal is activated whether by a stressor (e.g., tail-pinch, cold water) or by a stimulant drug (e.g., amphetamine) the postural bias is transformed into an intense circling response, known as “rotation”, lasting for the duration of stimulation. Thus, under amphetamine activation, unilateral lesioned rats exhibit ipsilateral rotation at a rate up to 10–15 turns per minute for the 4–5 h duration of drug action (Ungerstedt, 1971d; Ungerstedt and Arbuthnott, 1970). The utility of the rotation as a behavioral outcome is that it is directly observable, it is easy to measure in an objective manner, and recording can be automated (Dunnett, 1993; Ungerstedt and Arbuthnott, 1970). Moreover, with the nigrostriatal pathway remain­ ing intact on one side of the brain, the animals are able to engage fully in all normal motivated and regulatory behaviors and remain fit and healthy. An intriguing early observation deriving directly from the rotation method is that directly acting dopamine agonists (such as apomorphine) induce intense rotation as do stimulants, but, against initial expectation, the direction of the rotation was seen to be in the opposite direction, contral­ ateral to the side of lesion. Ungerstedt (1971b) hypothesized that this may be attributable to the development of supersensitivity of the postsynaptic receptors following deafferentation, a prediction that was subsequently confirmed in biochemical receptor-binding studies (Creese et al., 1977). As a result of the ease of objective monitoring and recording, rotation has been very widely used as a behavioral tool to study fundamental princi­ ples of organization and plasticity in the nervous

38

system. It has provided a powerful model of dis­ ease in the development of receptor agonists for application in schizophrenia and depression, as well as in modeling symptomatic, neuroprotective, and reparative treatment strategies for Parkinson’s disease. Other neurotoxins Epidemiological studies have associated the inci­ dence of Parkinson’s disease to exposure to a range of environmental, industrial, and agrochem­ ical toxins, in particular pesticides. Ingestion or administration of several of these agents, such as rotenone or paraquat, to rodents can induce neurodegeneration with selective vulnerability of ventral mesencephalic dopamine neurons, and associated behavioral deficits. The compound that has received most attention as an experimental tool is the meperidine analogue 1-methyl-4-phenyl-tetrahydropiridine (MPTP). MPTP was first discovered as the result of the development of a severe parkinsonian syndrome in drug addicts who self-administered the compound after poorly conducted chemical synthesis of a designer drug of abuse. Apart from the acute and severe onset in young patients, their parkinsonian symptoms appeared indistinguishable from idio­ pathic Parkinson’s disease in their symptom profile, profound loss of fluorodopa uptake in the striatum in Positron Emission Tomography (PET) scan, and responsiveness to L-dopa. The severity of syndrome is chronic and exhibited little spontaneous recovery, most likely related to the extent of the initial dopa­ mine depletion, and the patients very rapidly devel­ oped all the dyskinetic and rapid wearing off side effects seen in advanced idiopathic disease. MPTP is equally effective in inducing a profound parkinso­ nian syndrome in experimental primates, and has been widely used for development of novel thera­ peutics in monkey models of human Parkinson’s disease. Typically, a single dose treatment will yield partial lesions and significant recovery, whereas multiple dosing is required to offset acute

compensation to produce a stable model of disabil­ ity. With peripheral injection and bilateral lesions, monkeys suffer the same extreme welfare sequelae as seen with bilateral 6-OHDA lesions in rodents. However, effective unilateral lesions can readily be induced by injection of the toxin into the ascending carotid artery, affecting brainstem distribution and toxicity just on the one side, with attendant welfare benefit of unilateral denervation but without the need for stereotaxic surgery. MPTP is less widely used in rodents. The com­ pound has limited toxicity in rats due to an absence in this species of the particular isoform of monoamine oxidase, MAO-B, that is necessary to convert MPTP into the MPPþ ion that is the active form of the toxin. Conversely, MPTP does produce marked dopamine depletion in mice but its action appears to be attributable, at least in part, to down-regulation of the activity of the tyr­ osine hydroxylase enzyme in the biosynthetic pathway for catecholamines in dopamine neurons, rather than to primary selective neurodegenera­ tion of the neurons themselves. MPTP is also highly toxic to man, imposing significant health and safety issues for its use other than in a high class of safety environment, and for rodent stu­ dies, 6-OHDA still remains the neurotoxic model of choice. Genetic models Although traditionally considered not to be a genetic disease, Parkinson’s disease can run in families. The first familial disease mutation to be identified was in the a-synuclein gene (Polymer­ opoulos et al., 1997), which provided the stimulus for identifying a-synuclein as the principle protein forming the core of Lewy bodies (Spillantini et al., 1997), the globular protein inclusions in neurons both within and beyond the substantia nigra that provides the principal neuropathological hallmark of idiopathic Parkinson’s disease. Subsequently, mutations have been identified in several other genes, including leucine-rich repeat kinase 2

39

(LRRK2), Parkin, DJ-1, and PTEN-induced kinase 1 (PINK1). Collectively now known as the Park family of genes, they exhibit common asso­ ciation with different components of the mito­ chondrial energy chain, suggesting a common pathway for neurodegeneration in all forms of Parkinson’s disease, idiopathic as well as genetic, and driving neuroprotective strategies related to promoting cellular bioenergetics and the control of oxidative stress (Henchcliffe and Beal, 2008). These recent advances in molecular genetics offer the prospect of a new generation of animal models based on transgenic and knock-in gene modification. Following its initial discovery, inser­ tion of additional copies of both the wild-type and the mutant form of a-synuclein in Drosophila resulted in filamentous inclusions in neurons, adult loss of dopamine neurons, and impairments in wall climbing “locomotion” (Feany and Bender, 2000). Similarly, in a-synuclein transgenic mice, neuronal inclusions and dopamine loss have been recorded (Masliah et al., 2000) but the motor symptoms were rather modest. The specific muta­ tion (notably A34T) influences the pattern of cel­ lular pathology, the degree of mitochondrial dysfunction, and the magnitude of behavioral symptoms (Martin et al., 2006). Parallel studies using adeno-associated viral vectors to transfect the brainstem dopamine neurons in normal mice with mutant A34T a-synuclein also demonstrate the development of cellular pathology and neurite aggregations in the dopamine neurons but the mice do not develop Lewy bodies, the specific charac­ teristic subcellular pathology of the human disease (Eslamboli et al., 2007; Kirik et al., 2002). Interest­ ingly, mice in which the a-synuclein gene has been deleted appear to be relatively resistant to the toxicity associated with 6-OHDA in comparison to wild-type mice (Alvarez-Fischer et al., 2008). More recently, transgenic mice have been gener­ ated expressing a range of other familial mutations, including in the DJ-1, LRRK2, and UCH-L1 genes (Chandran et al., 2008; Li et al., 2009; Setsuie et al., 2007), and DJ-1-deficient mice are more suscepti­ ble to the toxicity of MPTP (Manning-Bog et al.,

2007). Although none of the strains exhibit as severe a behavioral syndrome as that associated with overt toxic lesions of the midbrain dopamine neurons, mice carrying the human LRRK2 muta­ tion exhibit perhaps the clearest cellular pathology and behavioral impairments, in particular invol­ ving an akinesia that is responsive to both apomor­ phine and L-dopa treatment (Li et al., 2009).

Motor and sensorimotor tests for rats Methods to assess simple motor impairments in parkinsonian rats and mice have been the subject of many reviews (Cenci and Lundblad, 2005; Dunnett, 2005; Dunnett and Robbins, 1992; Schallert and Tillerson, 2000), and so can be summarized rather briefly here. Locomotor activity Bradykinesia and akinesia, the poverty or loss of voluntary movements, are cardinal symptoms of PD and are readily assessed in experimental rodents by a variety of test measures of spontaneous and druginduced locomotor activity. Rodent activity is typi­ cally recorded by photocell, video, or radiometric monitoring in an automated test arena (Robbins, 1977). Relatively complete bilateral nigrostriatal lesions induce a profound and lasting immobility, so that the animals engage in no voluntary beha­ viors; they do not eat, drink, or groom, and they require intensive care to be kept alive (Marshall et al., 1974). By contrast, if the lesions are incom­ plete, spared dopamine neurons have a marked capacity to compensate via a variety of pre- and postsynaptic mechanisms (Zigmond et al., 1990), so that the animals may eventually recover the abil­ ity to feed, drink, and maintain body weight, although their capacity to regulate physiological status precisely never fully recovers. Such lesions may provide useful models for assessing strategies to promote plasticity and recovery of function but are less than ideal for pharmaceutical studies of

40

symptom alleviation or reconstructive studies of structural repair because of the variability and chan­ ging nature of the symptom baselines and the sig­ nificant welfare issues in maintaining such severely affected experimental animals. Rotation If instead the nigrostriatal lesions are made uni­ laterally, the contralateral dopamine pathways remain intact to maintain the animals in full health. Nevertheless, the animals exhibit a marked spontaneous motor bias toward the ipsilateral side, and if activated by mild stress or pharmaco­ logically with a stimulant drug the animals exhibit a vigorous head-to-tail circling behavior, known as “rotation”. The rate of rotation is easily quantified using an automated “rotometer” test apparatus (Ungerstedt and Arbuthnott, 1970), and the rate of rotation is seen to correlate directly on an ani­ mal-by-animal basis with the degree of loss of midbrain dopamine neurons measured anatomi­ cally and striatal dopamine loss measured bio­ chemically (Hefti et al., 1980; Schmidt et al., 1983). Rotation induced by presynaptic stimulants such as amphetamine or postsynaptic receptor agonists such as apomorphine has been widely adopted as the simplest, most direct measure and most objective behavioral measure of motor impairment in those parkinsonian models that permit unilateral application (in particular ones involving central injection of a toxin) (Dunnett, 2005; Pycock, 1980) but is of course less suitable for models involving symmetrical or bilateral damage, such as the newer genetic models described above. Further issues with use of rotation as a measure of functional outcome relate to (i) the requirement for pharmacological activation to reveal the deficit, (ii) a relative lack of face validity for the human disease, and (iii) the mechanism and underlying neuroanatomical pathway(s) by which an asymmetry of dopamine activation in the striatum translates into an ipsilateral turning bias remains mysterious.

Sensorimotor tests and “neglect” Early formulations of the nigrostriatal impairment highlighted the fact that animals with unilateral lesions failed to respond to stimuli applied in con­ tralateral space or to the contralateral side of the body, and characterized the syndrome as a con­ tralateral “neglect” (Marshall et al., 1971), akin to the neurological syndrome of neglect shown by patients with parietal lobe dysfunction. Further analysis suggests that the impairment is not pri­ marily a sensory failure to detect contralateral stimuli but an impairment in initiating appropriate contralateral response to the eliciting stimuli (Carli et al., 1985). Nevertheless, those early stu­ dies developed a range of neurological tests that still prove useful in characterizing motor asymmetries in experimental animals that may appear normal when observed moving freely in their home cage, and without recourse to drug-induced activation. Thus, when given a choice, animals with unilateral 6-OHDA lesions spontaneously select food pre­ sented from the ipsilateral side, show reduced use of the contralateral paw for stabilization and bal­ ance when rearing, show more foot-slips and falls when traversing narrow beams or grids, are much more likely to turn in the ipsilateral direction when confronting an obstacle, and show impaired step­ ping and placing reflexes when moved into whisker or paw contact with surfaces or edges. Many of these measures have now been formalized into standardized tests such as the cylinder, corridor, and stepping tests widely used in the experimental literature for monitoring functional recovery after experimental treatments (Cenci and Lundblad, 2005; Dunnett, 2005; Schallert and Tillerson, 2000). Skilled reaching In addition to affecting spontaneous and reflexive behaviors, nigrostriatal lesions profoundly impair manual dexterity and animal use of the limbs for goal-directed and skilled movement. Whishaw and colleagues first provided a detailed analysis of rats

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with unilateral nigrostriatal lesions impairing the initiation and accuracy of skilled forelimb move­ ments in a task in which hungry rats were required to reach through the bars of a cage to retrieve food pellets from a tray positioned outside the cage (Dunnett et al., 1987; Whishaw et al., 1986), compar­ able in magnitude to the deficits observed after unilateral motor cortex aspiration or excitotoxic stria­ tal lesion. In that study, performance with the impaired limb was probed by restricting use of the unaffected (ipsilateral) limb by use either of a bracelet or by intramuscular injection of local anaes­ thetic. In subsequent studies, the paw used was constrained by positioning the food pellets offcenter, outside a narrow slot, that could only be reached by the contralateral paw. This was then com­ bined with a form of movement analysis derived from human dance notation in order to describe the detailed components of normal reaching by rats (Whishaw and Pellis, 1990). Moreover, the nigrostria­ tal deficit was characterized as involving a particular difficulty in adjusting postural and base support, as well as in multiple components of the specific reach­ ing movement, which included aim, pronation and supination, and use of the digits to actively grasp and reach food (Miklyaeva et al., 1994). The downside of such an approach is the requirement for intensive qualitative frame-by­ frame analysis of video records of individual reaches, which is both time consuming and requires a highly trained experimenter. An alternative approach has been to develop a simpler objective measure of skilled reaching, the “staircase” test, which involves a test apparatus in which rats or mice reach down either side of a central plinth to retrieve food pellets from the steps of a staircase. The total number of pellets displaced and retrieved provides separate indices of the maximum dis­ tance a rat can reach with each limb, and of the usually lesser distance over which it can coordinate a successful grasp to retrieve the pellet (Abrous and Dunnett, 1994; Baird et al., 2001). Unilateral dopamine lesions markedly impair rats’ abilities of to reach, grasp, and retrieve pellets with the con­ tralateral forelimb in the staircase test (Abrous

et al., 1992; Montoya et al., 1990), an impairment that is of comparable magnitude to that observed after unilateral striatal or cortical lesions (Montoya et al., 1990, 1991). Reaching tests have been of particular interest in studying functional recon­ struction because, in spite of the apparent compar­ ability of the lesion deficits, recovery of function after dopamine-rich nigral grafts in nigrostriatal lesion rats is typically far more limited (Abrous et al., 1993; Dunnett et al., 1987; Montoya et al., 1990; Nikkhah et al., 1995) than that which can be achieved after striatal grafts in striatal lesion rats (Döbrössy and Dunnett, 2003; Dunnett et al., 1988; Montoya et al., 1990), suggesting different mechanisms of repair and recovery in the different models (Dunnett, 1995).

Non-motor symptoms There is a growing recognition that Parkinson’s disease involves non-motor as well as motor symptoms, spanning cognitive, neuropsychiatric, autonomic, and neuro-endocrine domains. As a consequence, many non-motor symptoms—includ­ ing depression and apathy, sleep disturbance, urinary and erectile dysfunction, and frank dementia— are frequently underdiagnosed as not directly associated with the primary Parkinson’s disease, yet impact significantly on the quality of life and efficacy of treatment (Chaudhuri et al., 2006). At the same time, Parkinson’s disease has long been recognized to involve pathology outside of the degeneration of the nigrostriatal neurons and associated loss of forebrain dopamine innerva­ tion, a perspective that is strengthened by the recent emphasis on degeneration in other brain­ stem nuclei as representing an earlier primary pathogenic event that gives rise to the cascade of neurodegenerative changes implicating wide­ spread brain areas including, but not just, the dopamine neurons (Braak et al., 2003). In the light of the classical association of dopamine dys­ function with the motor symptoms in Parkinson’s disease (Marsden, 1992), it is therefore not

42

surprising that non-motor symptoms are often considered to be attributable to the non-dopami­ nergic substrates. Nevertheless, many of the nondopaminergic symptoms of PD can respond well to dopaminergic treatment, so that “non-motor symptoms are not synonymous with a non-dopa­ minergic cause” (Chaudhuri and Schapira, 2009). Good animal models have the potential to pro­ vide an important tool for resolving the extent to which cognitive, psychiatric, and other non-motor functions affected in Parkinson’s disease are under the regulation of dopaminergic neuronal activity both within and beyond the nigrostriatal system. However, outside the cognitive domain (and argu­ ably even within it), such studies are in their infancy. In an interesting recent study, Branchi and colleagues (2008) undertook a first attempt to characterize social and emotional (as well as cog­ nitive) behaviors following bilateral partial dopa­ mine depletion made by intrastriatal 6-OHDA injection in rats. Alongside the expected reduc­ tions in general locomotor activity, they reported an increased duration spent in the open arms of an elevated plus maze test of anxiety-like behavior, shorter latencies to commence floating and increased duration of floating in the Porsolt forced swimming test of depression-like behavior, and reduced offensive behaviors in a social interaction test. In a rather similar study in partial bilateral striatal lesion rats, Tadaiesky and colleagues (2008) also found increased durations of “depres­ sive” floating in the forced swim test, and impair­ ments in social interaction behavior. However, in contrast to the former study, they found both reduced preference for sucrose than control rats and reduced duration spent in the open arms of the elevated plus maze (Tadaiesky et al., 2008), suggesting a reduced sensitivity to reward and increased anxiety-like behaviors, which fits better with hypotheses of PD-like non-motor change predicted from the clinical literature. In view of the recent interest in non-motor symptoms of PD, it is likely that attempts to develop better models of comparable symptoms in rats with partial bilat­ eral dopamine depletion are ripe for development.

“Cognitive” impairment Cognitive change following dopamine depletion in experimental animals has been the subject of more extensive consideration over the past three decades. Nevertheless, designing experiments that are unconfounded by changes in motor beha­ vior and sensitivity to reward has proved extre­ mely challenging. The striatum receives a rich afferent input from frontal association cortex, so that a role in cognitive as well as motor behavior has long been hypothesized (Divac et al., 1967; Öberg and Divac, 1979). Consequently, loss of dopamine regulation has often been predicted to impair the selection and initiation of executive actions initiated at the level of prefrontal cortex. Focal bilateral striatal 6-OHDA lesions do indeed disrupt classical tests of prefrontal cognition, such as delayed alternation, spatial learning, and inhibi­ tion of prepotent responding in operant “DRL” paradigms (Dunnett and Iversen, 1982; Taghzouti et al., 1985). However, there is an immediate issue of speci­ ficity: are the cognitive deficits of PD attributable to a specific dysfunction in the nigrostriatal dopa­ mine projection itself or are changes in other dopaminergic or non-dopaminergic circuits pri­ marily responsible? Although dopamine dysregu­ lation of striatal outflow from the prefrontal cortex may indeed be the locus of impairment, it is equally plausible that disturbance of prefrontal corticolimbic midbrain A10 projections are of more importance, or alternatively expression of Lewy body pathology in the cortex, ventral forebrain, or other brainstem (e.g., noradrenergic) cortical projections. In parallel to the impairment of differential reinforcement of low rates operant responding observed following lateral striatal 6-OHDA lesions (Dunnett and Iversen, 1982), which was selected as a sensitive test of orbito­ frontal-like cognition in the rat (Kolb et al., 1975), 6-OHDA lesions in other dopamine projection areas, including the prefrontal cortex and habe­ nula (Sokolowski and Salamone, 1994; Thornton et al., 1990), and in forebrain projections of the

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dorsal noradrenergic bundle (Salmon et al., 1985), have equally been shown to disrupt DRL timing of performance. Nevertheless, there is growing evidence that dopamine depletion specifically within the dorsal and ventral striatum is indeed associated with impairments in performance on a range of cognitive tasks, including delayed alter­ nation, characteristic of prefrontal function (Braga et al., 2005; Simon, 1981; Taghzouti et al., 1985). A second, more difficult issue of specificity is that the rats also exhibit various degrees of akine­ sia, neglect, and motor biases in all these tasks, which can profoundly influence their ability to execute the responses required to reveal normal cognitive performance. Several studies of spatial navigation in a water maze are instructive in this regard. Rats with unilateral or bilateral depletions of neostriatal dopamine have repeatedly been shown to be impaired in learning the location of the escape platform in the Morris water maze task (Hagan et al., 1983; Mura and Feldon, 2003; Whishaw and Dunnett, 1985). However, probe trials and transfer tests have in each case suggested that the impairment may not be attributable to a fundamental deficit in spatial learning per se but instead relates to the animals’ akinesia and impaired swimming (Hagan et al., 1983), an inabil­ ity to use distal cues for guidance (Whishaw and Dunnett, 1985), or the adoption of inappropriate thigmotaxic swim paths (Mura and Feldon, 2003) in comparison to control rats. Thus, there is the need to develop tasks in which the rats’ ability to detect subtle stimuli and the motor execution of the response has relatively little influence on the primary measure of task performance. A potential strategy to address this issue is to use relatively salient stimuli and simple motor responses in tasks involving choice responses on separate discrete trials, rather than tasks (such as free operant tests) where performance is critically determined from the rate of responding. Thus, for example, we can compare the performance of animals in a simple position habit task in a T maze, a reversal of that trained response, and in an alternation task (which requires the animal to

alternate its choice between the two arms on con­ secutive trials); the controlling stimuli and motor responses are the same in all three tests, only the rule for selecting the correct arm changes. If a dopamine-depleting lesion affects correct choices differentially between the tasks then the reason cannot be due to a change in the sensory or motor demands and we conclude that the impairment is indeed in learning or executing the appropriate rule, i.e., “cognitive”. This has proved extremely difficult to demonstrate, not least because any lesion of sufficient size to reveal clear deficits is associated not only with profound akinesia but also with insensitivity to the controlling rewards. Conversely, a lesion small enough to avoid defi­ cits in motivation is likely to be associated with considerable plasticity and recovery of function insufficient to allow the extensive training and testing required. It is plausible that recent advances in achieving more effective circum­ scribed and stable terminal lesions may allow this problem to be circumvented (Kirik et al., 1998), but the critical experiments have not yet been achieved.

Motor learning, habits, and reinforcement Whereas we can agree that a true cognitive impairment needs to be unconfounded from sim­ ple motor or sensory impairments, it may well be that changes in motivation and reward should be considered as a component of the cognitive pro­ cess itself. Thus, “neglect” of controlling stimuli might be considered either as a fundamental def­ icit in sensorimotor integration or as a component of higher cortical attentional processes that are integral to cognitive processing and executive decision making. Indeed, the involvement of the neostriatum (and by extension its dopamine affer­ ents) in motor learning and habit formation falls squarely at the interface of motor control and cognition, highlighting the degree to which such distinctions are somewhat arbitrary and artificial unless carefully specified.

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Dopamine and reinforcement Dopamine has long been associated with hedonic processes, through the analysis of the mechanisms of drugs of abuse (such as cocaine and the ampheta­ mines) on central dopamine release and action at dopamine receptors, in particular in ventral striatum. The rewarding effects of dopamine activation are readily apparent both in the identification of nigros­ triatal forebrain dopamine pathways as a sufficient substrate for intracranial self-stimulation (ICSS)—a process whereby an animal will readily learn to press a lever in order to self-administer small pulses of electrical stimulation to electrodes implanted into critical “reward pathways” in the forebrain (Hall et al., 1977)—and in the phenomenon of “condi­ tioned place preference”, whereby the animal will repeatedly return to a location that has been asso­ ciated with delivery of a stimulant drug such as amphetamine, cocaine, or heroin (Tzschentke, 1998). More recently, electrophysiological studies by Wolfram Schultz and others have identified distinctive patterns of dopamine neurone firing associated with expectation of reward (Schultz, 2000), leading to the hypothesis that the ascending dopamine pathway provides the substrate for signals of reward converging upon the striatum. Such signals may serve to “stamp in” coincident patterns of synaptic activation in the process for associative learning involved in habit formation within the striatum (Calabresi et al., 2007). Increasingly sophisticated neural network models have been applied to the striatum over the past decade in formulating the principles of information processing within the striatum that are necessary for effective habit learning and motor plasticity (Humphries et al., 2009). These principles may now be utilized to interpret the effects of dopa­ mine manipulations on reward-based learning. Procedural learning Carli and Robbins introduced a choice reaction time task in a novel operant apparatus, the nine-

hole box, which has proved particularly powerful for the analysis of dopaminergic regulation of striatal control in motor learning. The nine-hole box apparatus is an operant chamber in which a horizontal array of response holes, each with its own stimulus light, allows the rat or mouse to respond to choice stimuli, for food or liquid reward delivered to a separate hopper at the back of the box (Robbins et al., 1993). The sim­ plest use of the nine-hole box is in the “5-choice serial reaction time” (5CSRT) in which the rat must detect and respond to a brief stimuli occur­ ring at random in any one of five evenly spaced stimulus holes (the intervening four holes are cov­ ered with blank plates). This provides a simple test of attention, and is sensitive to pharmacological manipulation or lesions of noradrenergic, seroto­ nergic, and cholinergic projections to the neocor­ tex in rats (Carli et al., 1983; Muir et al., 1992; Ruotsalainen et al., 1997) and mice (Fletcher et al., 2007; Humby et al., 1999). A different task, involving a simultaneous choice reaction time in the same apparatus, is particularly sensitive to lesions within striatal circuits. Separate groups of rats were trained to hold their nose in the central stimulus hole, before presentation of a brief eccentric light flash in a lateral hole; the rat is then required to respond rapidly either in the stimulus hole (the “Same” version of the task) or in the symmetrical hole on the opposite side of the cen­ tral hole (the “Opposite” version). Carli and col­ leagues (1985) found that unilateral dopamine lesions profoundly impaired responding on the contralateral side, irrespective of whether the imperative stimulus was presented on the contral­ ateral (“Same” side) or the ipsilateral (“Opposite” side). This study provided the first unequivocal evidence that dopamine depletions produce an essentially motor rather than sensory or sensori­ motor impairment. Moreover, the rats were impaired in particular not only in accuracy in con­ tralateral responding but they also exhibited selec­ tive impairments in “reaction time”, i.e., the latencies to withdraw the nose from the central hole in preparation for a contralateral response,

45

but not in making the identical withdrawal move­ ment in preparation for a correct ipsilateral response, whereas the time taken to execute the lateral movement was unaffected. These data clearly suggest therefore that the dopamine dener­ vation impairment relates to the selection and initiation of a goal-directed movement in contral­ ateral space, rather than an inability to make the contralateral movements per se (Carli et al., 1985). In subsequent studies, Brown and colleagues provided advance signals that prepared rats for the correct response and found that as in PD patients the impairment associated with dopamine depletion is seen particularly in context of choice rather than in simple reaction time versions of the task, an effect they described as “impaired motor readiness but preserved response preparation” (Brown and Robbins, 1991). Motor learning and reinforcement In our own studies, we have utilized the Carli task to probe the efficacy of alternative strategies for striatal repair including application of striatal tis­ sue transplantation to alleviate choice reaction time deficits associated with excitotoxic striatal lesions (Brasted et al., 1999) and use of nigral cell transplantation or neuroprotective gene ther­ apy to reverse the effects of unilateral 6-OHDA lesions of the ascending dopamine pathways (Dowd and Dunnett, 2004; Dowd et al., 2005). In the course of these studies, however, it was noted that the 6-OHDA lesion deficit itself was not apparent immediately; it took several days of further training to emerge, and it remitted after a 3-month gap in testing, following which it then required several days further training to re­ emerge (Dowd and Dunnett, 2004). These results clearly confirmed the original Carli and Robbins hypothesis that the core deficit is not fundamen­ tally motor—an inability to actually execute the required response—and indeed bears all the hall­ marks of an associative impairment, which resem­ bles the phenomenon of “extinction”, in which the

rate of responding to a previously reinforced sti­ mulus wanes when the reinforcer is omitted, and learning of a new action–reward association occurs. This also requires multiple trials for the animal to learn that the previously reinforced con­ tingency no longer applies and exhibits sponta­ neous recovery after a protracted period without testing. We therefore hypothesized, in line with the identification of dopamine pathways providing a substrate for signals of reward during learning to impinge on striatal motor learning circuits (Schultz, 2000), that a unilateral dopamine lesion eliminates the reward-related feedback from hav­ ing access to the striatal circuits that maintain learned responding, weakening the synaptic con­ tacts that provide the circuit substrate for pre­ viously established motor responses, without affecting the animals’ abilities to execute the rele­ vant responses per se (Dowd and Dunnett, 2004, 2007). The apparent differences in the specific profile of results in the two sets of studies may relate in part to differences in specific parameters of the lesions, timing, and task implementation, but primarily relates to our focus on the first days of training and “acquisition” of the impair­ ment, as opposed to the focus by Carli and Rob­ bins on the impairment once stabilized. Striatal dissection of instrumental learning Although the role of the striatum in motor learn­ ing and habit formation has been hypothesized for several decades (Gaffan, 1996; Mishkin et al., 1984), there has been a significant renewed inter­ est recently in the precise functional processes underlying associative learning within this neuroa­ natomical system. For example, elegant electro­ physiological studies reveal the emergence of stable changes in cell firing in discrete ensembles of striatal cells during habit learning, extinction, and reaquisition (Barnes et al., 2005; Jog et al., 1999), and distinct patterns of dopamine firing appears to be able to encode different reward-related and aversion-related outcomes (Morris et al., 2009).

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A classical distinction within the animal learn­ ing literature has been between Pavlovian and instrumental conditioning. In Pavlovian condition­ ing, the animal learns to associate a previously neutral stimulus with a stimulus of physiological value, as in Pavlov’s dog learning that a bell is predictive of food, so that after repeated pairings the bell comes to induce salivation in advance of the actual presentation of the food (“S–S” learn­ ing). In instrumental conditioning, as first formu­ lated in Thorndike’s law of effect, the animal learns that if a particular response is followed by a reinforcing stimulus within a particular stimulus configuration, such as a lever press leads to pre­ sentation of food, the likelihood of increasing that behavior is increased (“S–R” learning).1 The stria­ tum has been particularly associated with the lat­ ter, instrumental learning, and the learning of motor skills and habits outside of consciousness. More recently, animal learning theory has focused on the nature of the associations that are learned within instrumental learning, with a distinction drawn between responding with knowledge about the expected outcome such that the beha­ vior is goal-directed and motivated by the incen­ tive (“A–O”, Action–Outcome learning) as distinct from automated responding to antecedent stimuli (“S–R” learning) in which the behavior becomes established independently of the reinfor­ cing stimuli that originally rewarded the responses. S–R responding is typically a feature of overtraining and represents an automatic or habitual responding irrespective of outcome, which continues when the motivational state is changed or the outcome is devalued and which is slow to extinguish when the reinforcer is omitted (Yin and Knowlton, 2006). Responding under an S–R association corresponds to the classical 1

Note that in classical theory, the food serves simply as a “reinforcer” to stamp in the likelihood of the response being repeated when in the presence of the same antecedent discri­ minative or contextual stimuli. The animal does not learn any­ thing about the food itself nor does the food provide an incentive for the behavior.

concept of habit learning, whereas A–O respond­ ing corresponds to more voluntary or goal-direc­ ted action. So, is the striatum primarily associated with S–R-based habits or A–O-based voluntary goal-directed action? It seems both. The striatum receives a rich topographic projec­ tion from the whole neocortex (Kemp and Powell, 1970; McGeorge and Faull, 1989) and is function­ ally heterogeneous corresponding to the sensory, motor, and associative cortical areas with which each striatal area is principally connected (Divac, 1968; Dunnett and Iversen, 1981). It is within the dorsolateral striatum (DLS), receiving heavy con­ nections from the sensory and motor areas of neocortex, that striatal lesions disrupt habit learn­ ing of the S–R type, whereas A–O actions appear to be dependent upon the dorsomedial striatum (DMS), receiving its dominant inputs from frontal association cortex. Thus, in normal rats trained to lever press for sucrose reward, if the reward is devalued after only a short period of training by pairing of sucrose with the emetic lithium chloride, then responding is reduced when the rats are put back in the lever pressing apparatus; however, if the rats have received extensive training so that responding becomes habitual, then they continue responding when put back in the test cages after reward devaluation. Lesion or NMDA receptor blockade of the DMS blocks the effect of reward devaluation even after a short training (Yin et al., 2005a, b). Conversely, after lesions of the DLS rats continue to show reduced responding after reward devaluation, even after extensive training (Yin et al., 2004, 2006a). Thus, when the habit system is disrupted, control over instrumental per­ formance reverts to the system controlling the performance of goal-direct action (Yin et al., 2004). The operation of dorsal striatum in control of instrumental learning is subject to dopaminer­ gic control, in particular for modulating the striatal plasticity necessary to establishing the A–O cir­ cuits in the early stages of learning, when the system underlying goal-directed action is domi­ nant (Wickens et al., 2007). Indeed, dopamine depletion in the DLS disrupts the transfer of

47

goal-directed actions into habitual responding (Faure et al., 2005). Mice that have been geneti­ cally modified to overexpress dopamine by a knock-down of the selective transporter exhibit reduced sensitivity of stimulus control over condi­ tioned behavior but no change in instrumental learning per se (Yin et al., 2006b), and they exhibit an increased resistance to extinction, again sug­ gesting an increased transfer to habitual respond­ ing (Hironaka et al., 2004). New genetic and optical tools open the way for identifying the molecular mechanisms for dopaminergic modula­ tion of striatal plasticity in instrumental learning at the receptor and subcellular levels (Surmeier et al., 2009), which is likely to be a topic for major attention during the next decade.

Summary and conclusions Animal models of PD have traditionally focused on motor symptoms; they are easier to observe, easier to measure, and have obvious face validity in their similarity to the cardinal defining symp­ toms of the human disease. However, in recent years, clinical studies have highlighted the impor­ tance of a range of parallel non-motor symptoms, not only in the cognitive domain (“subcortical dementia” and impairments in frontal-type execu­ tive function) but also disturbances of mood and emotion (such as depression, anxiety, and “anhe­ donia”) and in a range of physical and physiologi­ cal functions. Animal studies have been extended, typically with some delay, to consider each of these non-motor symptom domains, with some success. In particular, recent studies both on phy­ siological plasticity within the striatal circuits and on the functional associative processes that under­ lie goal-directed and habitual learning are revolu­ tionizing our understanding of fundamental striatal processes mediating instrumental learning, which may be expected to open up new under­ standing of the fundamental functional processes underlying human PD. Such theoretical advances enhance, in turn, the prospect of being able to

develop more potent strategies for modification and treatment of the full complex of non-motor as well as motor symptoms in improved animal models, and are directly relevant to the develop­ ment of better treatments for the full spectrum of core problems confronted by patients.

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 4

Genetic mouse models of Parkinson’s disease: The state of the art Iddo Magen and Marie-Françoise Chesselet
Department of Neurology, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA

Abstract: The identification of several mutations causing familial forms of Parkinson’s disease (PD) has led to the creation of multiple lines of mice expressing similar genetic alterations. These models present a unique opportunity for understanding pathophysiological mechanisms leading to PD in a mammalian brain and provide models that are suitable for the preclinical testing of new therapies. Different lines of mice recapitulate the symptoms and pathological features of PD to various extents. This chapter examines their respective advantages and highlights some of the key findings that have already emerged from the analysis of these new models of PD. Keywords: alpha-syn; mice models; Parkison's disease; cell loss; pathology; motor deficits; non-motor deficits; substantia nigra

characteristics of PD: the age-dependent progressive neuronal loss, the presence of Lewy bodies, and extensive non-motor symptoms. More importantly, these models are not based on mechanisms known to cause PD in humans (Pandya et al., 2008). Beyond the reproduction of neuropathological and clinical manifestations of PD, which cannot be expected to occur fully in a mouse, the most important criterion a useful model should fulfill is mechanistic rele­ vance. This is essential to gain meaningful informa­ tion on the pathophysiology of the disorder and for its usefulness in testing potential therapies. Unfor­ tunately, much work and controversies in the field of PD models have focused on the ability of models

Introduction Traditionally, rodent models of Parkinson’s disease (PD) have been generated by using neurotoxins that kill, more or less specifically, the nigrostriatal dopaminergic neurons. This approach fulfills one criterion expected of a PD model: to reproduce the canonical loss of dopaminergic neurons that leads to the characteristic motor symptoms of the disorder. These models, however, lack other key


Corresponding author. Tel.: +1-310-267-1781; Fax: +1-310-267-1786;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84004-X

53

54

to reproduce characteristics of the human disease rather than to mimic mechanisms that may lead to different manifestations in different species while sharing molecular similarities. The role of genetic factors in PD has long been overlooked because the large majority of cases occur sporadically, i.e., without clear familial ante­ cedents. Familial forms, however, do exist and in 1997, the first mutation leading to PD was identi­ fied in the gene encoding the vesicular protein alpha-synuclein (a-syn), a missense mutation from alanine to threonine in position 53 (A53T) (Polymeropoulos et al., 1997) followed by the dis­ covery of another missense mutation, alanine 30 to proline (A30P) (Krüger et al., 1998). The importance of this finding was immediately highlighted by the discovery that a-syn, although not mutated in the very large majority of PD cases, does accumulate in the brain of patients as a component of the key pathological feature of the disease, the Lewy bodies (Spillantini et al., 1998). Furthermore, duplication or triplications of the a-syn gene also lead to PD, with a clear relation­ ship between level of a-syn expression and age of onset of the disease (Chartier-Harlin et al., 2004; Ibáñez et al., 2004). The role of increased levels of a-syn in PD is further supported by studies show­ ing an association between polymorphisms in the a-syn gene promoter and age of onset, and recent data from genome-wide association studies (GWAS) indicating a close association between the a-syn gene and PD risk (Satake et al., 2009). Together, these data strongly indicate an involve­ ment of a-syn in sporadic PD and suggest that mice overexpressing this protein (mimicking the familial cases of gene multiplication) should pro­ vide useful models of the disease. Indeed, numer­ ous lines of a-syn-overexpressing mice have been generated, using a variety of promoters and transgenes (Fernagut and Chesselet, 2004). Most reca­ pitulate some, although not all, aspects of PD. This chapter will focus primarily on these models. Other genes have subsequently been identified in familial forms of PD as reviewed in detail else­ where in this volume. Recessive mutations in

parkin, PINK1, and DJ-1 are usually modeled by knockout strategies, leading to the absence of the corresponding protein. Although most published models of these mutations do exhibit deficits, these are usually modest and much fewer work has been done in these lines. Very recently, two models of mutations in LRRK2, which in humans, cause dominant forms of the disease, have been published and will be briefly described. Mice overexpressing human a-syn Two major variables have to be considered when reviewing the various lines of mice overexpressing a-syn: the promoter used to drive the transgene and whether the transgene expresses or not the mutations that cause familial forms of PD in humans. Other significant variations in the models are linked to the background strain, the presence or absence of endogenous mouse a-syn, and whether the full-length or a truncated form of the protein is expressed (Table 1). Overexpression of a-syn under the tyrosine hydroxylase promoter A primary criterion of success for an animal model of a neurodegenerative disorder is the reproduc­ tion of the pattern of characteristic cell death observed in patients with the disease. Many early lines of a-syn-overexpressing mice used the tyro­ sine hydroxylase (TH) promoter to drive the transgene. TH is the limiting enzyme of catecho­ lamine synthesis and is selectively expressed in dopaminergic, noradrenergic, and adrenergic neu­ rons. Accordingly, the use of this promoter was motivated by the prominent death of several groups of catecholaminergic neurons in PD, pri­ marily the nigrostriatal dopaminergic neurons, but also the noradrenergic neurons of the locus coer­ uleus (Baloyannis et al., 2006; Zarow et al., 2003). The hope was to reproduce the canonical cell death of these neuronal populations in the mice.

Table 1. Overview of a-syn transgenic mice References

Human a-syn

Promoter

Genetic background

Expression levels

Neuropathology

Behavioral phenotype

Masliah et al. (2000)

WT, A53T (behavioral deficits were found only in mice expressing WT protein)

PDGF-b

C57BL/6×DBA2

10–80% of a-syn in humans

WT I in Cx, H, OB, and TH+ cells in SN, ubiquitin+, glia+ in B, C, H, M, Th; # TH+ terminals and TH levels and activity in STR, # DA levels in STR;

WT Middle age onset; mild # in rotarod, " in thigmotaxis

Rockenstein et al. (2002)

2–4× endogenous expression

Hashimoto et al. (2003)

# Neurogenesis and " apoptosis in DG (young age), # neurogenesis and " apoptosis in OB (young and old age) with intact proliferation

Winner et al. (2004, 2008) Yacoubian et al. (2008)

Alteration of expression of about 200 genes related to transcription in the SN

Koob et al. (2010)

Lovastatin # a-Syn aggregation and neurodegeneration in temporal Cx A53T # Proliferation and " apoptosis in SVZ, # neurogenesis (including that of TH+ cells) and " apoptosis in OB (all changes in aged but not young mice) Sharon et al. (2003)

WT

PDGF-b

C57BL/6

NA

Regulation of a-Syn oligomerization by fatty acids

NA

Liu et al. (2010)

WT

PDGF-b

C57BL/6

NA

Upregulation of GRK5

NA

van der Putten et al. (2000)

A53T

Mu Thy­ 1

C57BL/6

NA

Motor neurons degeneration; I: B, C, SC, T; ubiquitin+

Early onset, severe (rotarod)

(Continued)

Table 1 (Continued ) References

Human a-syn

Promoter

Genetic background

Expression levels

Neuropathology

Behavioral phenotype

Kahle et al. (2000)

WT, A30P, A53T (alterations were found only in mice expressing A30P variant)

Mu Thy1

C57BL/6

Two-fold endogenous a-syn; higher in symptomatic than in presymptomatic mice

A30P I: A, B, Cx, H, SC, SN, STR;

A30P Young age onset: " swim speed

Neumann et al. (2002) Frasier et al. (2005) Poon et al. (2005)

B: LB-like structures and " Tau phosphorylation, GFAP+ staining and TS amyloid staining; hyperphosphorylation of a-syn in cytosol in SC and phospho-Ser129 staining in A and Cx; GFAP+ in SC; # Serotonin in STR of males only and " serotonin in frontal Cx of both genders; # 5-HIAA in frontal Cx of males only, # 5­ HIAA in STR of males and " 5­ HIAA in STR of females

Freichel et al. (2007) Schell et al. (2009)

Middle age onset: # rotarod in females; paralysis, weakening of the extremities, abnormal tail posture, unsteady gait; " locomotion and stereotypy, # fear response (freezing and active avoidance), deficit in probe trial in Morris water maze Old age onset: # rotarod/OR no motor phenotype

Oxidation of mitochondrial associated metabolic proteins— Car2, Eno1, and Ldh2 Rockenstein et al. (2002) Song et al. (2004) Fleming et al. (2004, 2006, 2008a, b) Fernagut et al. (2007) Wang et al. (2008) Watson et al. (2009) Wu et al. (2009)

WT, A30P (behavioral deficits were found only in mice expressing WT protein)

Mu Thy­ 1

C57BL/6 × DBA2

10-fold human levels 10× endogenous expression Higher expression of WT a-syn than A30P a-syn

WT Young mice: I: OB, SN, LC other brain regions. Increased size with paraquat and age. Old mice: a-Syn aggregation and neurodegeneration in temporal Cx (reduced by lovastatin); no TH+ cell loss in SN or LC up to 18 m, Decreased cortical NE 7 m; decreased striatal TH and DA 14 m Increased MPTP-induced mitochondrial alterations, filamentous neuritic aggregations, axonal degeneration, and formation of electron denseperinuclear cytoplasmic inclusions in the SN

WT (male) Young age onset: 2 m: progressive increased errors on grid and decreased fine motor skills, deficit in pole test, # hindlimb steps in cylinder, no change in gait Abnormal motor response to L-DOPA, amphetamine and apomorphine. 3 m: Olfactory deficits in buried pellet, block and habituation/dishabituation tests Abnormal circadian rhythm, cognition, fear Middle age onset: Slower colonic transit but increased fecal response to novelty stress and CRF Abnormal cardiovascular function,

Koob et al. (2010)

altered corticostriatal plasticity; abnormal corticostriatal transmission

Zhou et al. (2008)

Y39C

Mu Thy­ 1

FVB/N

2.5-fold of expression in WT

Cx: I, phospho-Ser129 staining, ubiquitin+, apoptosis. No TH+ cell loss in SN

Old age onset: # rotarod, # Morris water maze, no change in locomotor activity

Ikeda et al. (2009)

A30P + A53T (DM)

Human Thy-1

C57BL/6 × DBA2

NA

I: B, DN, SN; atrophy in C and Cx, nitration of a-syn, ubiquitin + in B and DN, dystrophy in DN, possible TH+ cell loss in SN 12 m (no stereology), astrocytosis in C, "Phospho­ a-syn

Young age onset: # rotarod and pole test (progressive). Reversal by PBA

Ono et al. (2009)

#DA in STR and hypothalamus, #serotonin in hypothalamus, #ACh in STR, #TH levels in SN (most deficits reversed by PBA) Lee et al. (2002) Von Coelln et al. (2006) Martin et al. (2006) Unger et al. (2006) Miller et al. (2007)

WT, A30P, A53T (alterations were found only in mice expressing A53T variant)

Mouse prion

C3H/HeJ × C57BL/6J backcrossed into C57BL/6J, Parkin KO

4–15 times > non-Tg

A53T

A53T

I: B, C, M, SC; GFAP+ in B, C, M, SC; ubiquitin+, Fluoro Jade B+ in B; Axonal swelling and degeneration, somal chromatolytic changes and nuclear condensation in B and SC, Lewy body-like inclusions in the cytoplasm of Cx neurons and SC motoneurons, cytoplasmic inclusions in dendrites and neurons, degenerating mitochondria. 75% loss of motor neurons

Middle age onset: hyperactivity (reversed by D1 antagonist), hyperactivity and # startle response (unaffected by parkin absence) Middle/old age onset: reduced ambulation, ataxia, dystonia (fatal) with no effect of parkin absence

" D1 receptor in SN, # DAT in NAc and STR, # DA uptake in STR Altered profile of gene expression related to inflammation in B (Continued)

Table 1. (Continued) References

Human a-syn

Promoter

Genetic background

Expression levels

Neuropathology

Giasson et al. (2002)

WT, A53T

Mouse prion

C57BL/C3H

2.5–30-fold end. a-syn, higher expression of the A53T than the WT a-syn 2–4-fold " with paraquat/maneb in B, C, and Cx compared to expression of A53T a-syn in salinetreated Tg mice

WT

WT

" Susceptibility to LPS-induced neuroinflammation, which caused TH+ cell loss and I in SN (no loss in Tg alone)

Young age onset:# anxiety

Norris et al. (2007)

Gao et al. (2008) Sotiriou et al. (2009) Graham and Sidhu (2010) George et al. (2008)

Gispert et al. (2003) Cabin et al. (2005)

A53T I: B, C, SC, STR, Th; #TH+ terminals in SC and #TH levels in OB and SC; GFAP+ and gliosis in SC; altered neuronal morphology, diffuse ccumulation of a-syn, Wallerian degeneration in ventral root of SC, axonal degeneration of sciatic nerve.

Behavioral phenotype

A53T Young age onset: # rotarod and altered anxiety (" at 2 months and # at 4 months) Middle age onset: hyperactivity, " rotarod Old age onset: hunched back, freezing, paralysis (fatal)

" Susceptibility to LPS-induced neuroinflammation, which caused cell loss and I in SN, and to paraquat/maneb, which caused I in C, Cx, H, and mitochondrial dgeneration (no loss in Tg alone) #DAT in STR, #NE in OB, SC, STR WT, A53T (alterations were found only in mice expressing A53T variant)

Mouse prion

FVB/N, FVB × 129, a-syn KO

5–20-fold end. a-syn, higher expression in A53T than in WT mice

A53T Altered neuronal morphology, diffuse accumulation of a-syn, axonal degeneration of sciatic nerve is greater in KO mice

A53T Young/middle age onset: # vertical activity Middle age onset: # stride length, grip strength; # rotarod Old age onset: lethal motor phenotype is more severe in a-syn KO mice

Yavich et al. (2005, 2006) Gureviciene et al. (2007, 2009)

WT, A30P (alterations were found only in mice expressing A30P variant)

Mouse prion

C57BL/ 6J × DBA2 backcrossed into C57BL/6J

Aged A30P compared to adult A30P: 1.5-fold increase of mutant gene

A30P a-Syn accumulation in STR, C and H, abnormal NE mobilization in H, higher increase of dopamine overflow by L-DOPA, LTD in the DG in aged mice

Oksman et al. (2009)

A30P Middle age onset: # rearing and " stereotypy Middle/old age onset: # locomotion, " sensitivity to effects of L-DOPA on locomotion, rearing, stereotypy and dopamine overflow No change in rotarod

Gomez-Isla et al. (2003) Nieto et al. (2006)

Matsuoka et al. (2001) Manning-Bog˘ et al. (2003) Yu et al. (2008)

WT, A30P, A53T (alterations were found only in mice expressing A30P variant)

Hamster prion

C57BL/6J × SJL

5–15-fold end. a-syn. Expression is highest in A30P

A30P GFAP+ in Cx and H, Tg mice are more susceptible to MPTP, which # TH+ cells in SN and #DA in STR only in Tg mice (no effect of Tg alone)

A30P

Young/middle age onset: #

rotarod, tremor, dystonia, (fatal)

WT, A30P, A53T

Rat TH

Swiss Webster × C57BL/DBA

A53T Striatum, OB > cerebellum

WT Tg mice are resistant against paraquat-induced neurodegeneration

NA

A53T Ubiquitin Tg mice are resistant against paraquat-induced neurodegeneration but are more vulnerable to MPTP, which # DA in OB and " DA metabolism in CN and OB and " nitrotyrosine in OB only in Tg mice (no effect of Tg alone) (Continued)

Table 1 (Continued ) References

Human a-syn

Promoter

Genetic background

Expression levels

Neuropathology

Behavioral phenotype

Richfield et al. (2002)

WT, A30P+, A53T (DM)

Rat TH

C57BL/6J

30–50% of mouse a­ syn

WT Altered profile of gene expression related to dopaminergic phenotype in SN, microglial activation in SN and TNF-a release

WT Young age onset: " sensitivity to effects of amphetamine (" locomotion) and MPTP (# locomotion) Old age onset: effect of high dose apomorphine on horizontal activity is diminished

Thiruchelvam et al. (2004) Chen et al. (2006) Miller et al. (2007) Su et al. (2008, 2009)

DM " DAT density in STR, # DA, DOPAC, HVA in STR (only at old age), # TH+ neurons and Lewy bodylike I in SN, " TH+ cell loss in SN and " dopaminergic alterations in STR following paraquat/maneb (compared to paraquat/maneb-treated non-Tg mice) Impairment in ubiquitin­ proteasome system in frontal Cx, M and STR Altered profile of gene expression related to dopaminergic phenotype in SN Microglial activation in SN, "proinflammatory molecules in SN and STR and "TH mRNA in SN (young age) and #TH mRNA in STR (middle age)

DM Young age onset: # sensitivity to effects of amphetamine (" locomotion) but " sensitivity to effects of MPTP (# locomotion); "coordination " decline of horizontal locomotor activity and coordination with age Middle age onset: " sensitivity to locomotor effects (#) of paraquat/ maneb Old age onset: direction of effect of high dose apomorphine on horizontal activity is reversed (" and not #)

Tofaris et al. (2006)

Truncated (1–120)

Rat TH

C57BL/6J × CBA/ ca backcrossed into C57BL/6J, asyn KO

NA

I: SN, OB; microglial activation in SN; # DA and HVA in STR

Old age onset: # spontaneous activity with no treatment and higher " of spontaneous activity in response to amphetamine

Wakamatsu et al. (2008a, b)

Truncated (1–130)

Rat TH

C57BL/6J

NA

Non-progressive (developmental) TH+ cell loss in SN but not VTA, axonal damage to STR, # TH, DA and HVA in STR, # expression of genes related to DA system in M

Young age onset: # spontaneous activity and reversal by L-DOPA and DA agonists

Maskri et al. (2004)

A30P + A53T (DM)

Chicken beta actin, mu TH, mouse prion

C57BL/6

Four times higher in msprp-Syn than in chb-actin-Syn and TH-Syn

NA

NA

Nuber et al. (2008)

WT, A30P, A53T

CaM-tTA (tet-off)

C57BL/6 (WT and A30P), C57BL/ C3H (WT and A53T)

WT Less (£90%) than human control; nonsignificant increase (112%) only in Cx of CaM-aSyn mice. Highest expression in the forebrain—OB, Cx, and BG; 2–3-fold of end. mouse a-syn

WT Trend to # TH+ neurons in SN; # DA in OB (reversed by ceasing gene expression); # neurogenesis in DG (reversed by ceasing gene expression), loss of postmitotic neurons in DG due to apoptosis (reversed by ceasing gene expression), synapse loss in H and OB

WT Young age onset: # rotarod (progressive); Progression halted by ceasing gene expression

A30P Expressed only in OB

A30P # Neurogenesis (including that of TH+ cells) and " apoptosis in OB (reversed by ceasing gene expression)

Marxreiter et al. (2009) Lim et al. (2010)

A53T 2–5-fold of end. mouse a-syn

Old age onset: # retention in the Morris water maze

A53T Loss of postmitotic neurons in DG due to apoptosis (reversed by ceasing gene expression), synapse loss in H and OB (Continued)

Table 1 (Continued ) References

Human a-syn

Promoter

Genetic background

Expression levels

Neuropathology

Behavioral phenotype

Plaas et al. (2008)

A30P

KI in endogenous a-syn

C57BL/6

NA

# DA, DOPAC in STR; # DA in mesolimbic area

Old age onset: # beam walk test, # stride length, # locomotor activity, catalepsy. Diminished reserpine-induced enhancement of amphetamine effect on horizontal activity

Daher et al. (2009)

Truncated (1–119), A53T

ROSA26

C57BL/6J

NA

# DA, DOPAC, HVA in STR, " NE in STR, " 5-HT in Cx

NA

Kuo et al. (2010)

WT, A30P, A53T

Endogenous a-syn (using BAC)

FVB/N × 129S6// SvEvTac

mRNA: colon > brain, WT > A30P > A53T, protein: 1.3–2-fold of end. a-syn in brain, 100–1000-fold of end. a-syn in colon

A53T Proteinase-K-resistant aggregates in ENS neurons

A53T Young age onset:# colonic motility in males Middle age onset: # rotarod, # locomotor activity (progressive)

Data are grouped by different models of a-syn using different promoters to induce transgene expression; the data in each model are further subgrouped by different lines of mice generated by different research groups, with the first reference referring to the originator of the line and followed by other groups using the same line in chronological order. The different models are ordered chronologically (by the year of the first report of the model), and so are the different lines within each model. 0–6 Months of age are considered “young age”, 6–12 months “middle age”, and over than 12 months “old age”. In cases where two age ranges appear separated by a slash (e.g., young/middle), the onset either was in the borderline between two ranges or was reported in different age ranges by different groups using the same mice. Abbreviations shown in the table: A, amygdala; B, brainstem; BAC, bacterial artificial chromosome; BG, basal ganglia; C, cerebellum; CaM, calmodulin; CN, caudate nucleus; Cx, cortex; DA, dopamine; DAT, dopamine transporter; DG, dentate gyrus; DM, doubly mutated; DN, dentate nucleus; end. a-syn, endogenous levels of a-syn; ENS, enteric nervous system; GFAP, glial fibrillary acidic protein; H, hippocampus; HVA, homovanillic acid; I, inclusions; LC, locus coeruleus; LTD, long-term depression; M, midbrain; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NA, not available; NAc, nucleus accumbens; NE, norepinephrine; OB, olfactory bulb; PBA, sodium 4-phenylbutyric acid; PrP, prion protein; SC, spinal cord; SN, substantia nigra; STR, striatum; SVZ, subventricular zone; T, telencephalon; Tg, transgenic; Th, thalamus; TH, tyrosine hydroxylase; TNF, tumor necrosis factor; VTA, ventral tegmental area; WT, wild-type

63

Dopaminergic deficits and neuropathology in mice overexpressing a-syn under the control of the TH promoter and their relation to behavior The TH promoter can be expected to cause mainly dopaminergic and noradrenergic deficits when it drives the expression of a-syn, as TH is selectively expressed in dopaminergic and nora­ drenergic cells. However, only in two studies using TH as promoter was there a selective loss of nigrostriatal dopaminergic neurons—in mice expressing doubly mutated a-syn, a situation not encountered in humans (Thiruchelvam et al., 2004), and in mice expressing a truncated form of the protein with the amino acids 1–130 (Wakamatsu et al., 2008a). The former study also reported a greater decline of horizontal activity with age in the transgenic mice and Lewy bodylike inclusions in the substantia nigra. The cell loss was progressive and increased from 8.5 to 19 months and was aggravated by a combined treat­ ment of the pesticides paraquat and maneb, which also decreased locomotor activity (Thiruchelvam et al., 2004). The latter study reported an impair­ ment of axon terminals in the striatum and concomitant decrease in striatal dopamine and homovanillic acid (HVA) content and TH expres­ sion at 8 and 52 weeks. Behaviorally, spontaneous locomotor activities of Syn130m were reduced but were improved by treatment with L-DOPA. The loss of nigral dopaminergic neurons was not pro­ gressive as it was apparent at 8, 26, and 52 weeks to the same extent and seemed to occur during embryogenesis along with the onset of expression of the transgene because TH expression decreased throughout the embryonic period. The expression of key genes of the dopaminergic system was reduced (Wakamatsu et al., 2008a). Another study by the same group demonstrated a dosedependent effect of L-DOPA and an effect of dopaminergic agonists on reduced exploratory behavior at young/middle age, thus reproducing the results of the previous study and supporting the involvement of decreased striatal dopamine content in the behavioral deficits in this line of

mice, as the level of dopamine receptors did not decrease (Wakamatsu et al., 2008b). A decrease in spontaneous activity at 18 months (Tofaris et al., 2006) was found in another line of mice expressing the truncated form of a-syn containing the first 120 amino acids (1–120) along with a higher increase of spontaneous activity by amphetamine. The decreased spontaneous activity was asso­ ciated with inclusions of a-syn co-localized with TH staining and microglial activation in the sub­ stantia nigra and decreased dopamine and HVA in the striatum at different time points. a-Syn inclusions were also present in the olfactory bulb and were co-localized with TH staining (Tofaris et al., 2006). The results of the above-mentioned three studies indicated that truncated human a­ syn is deleterious to the development and/or sur­ vival of nigral dopaminergic neurons, as no neu­ ropathology was found in mice expressing the wild-type (WT) protein. Indeed, truncated forms of a-syn 1–120 and 1–130 were found in PD brain extracts and seemed to aggregate faster (Crowther et al., 1998; Serpell et al., 2000) and to be more susceptible to oxidative stress than the full-length protein (Kanda et al., 2000). In stark contrast to these studies, transgenic mice expressing WT, A30P, or A53T a-syn under the control of the rat TH promoter, thus achieving increased levels of expression in the substantia nigra, did not display dopaminergic def­ icits despite the accumulation of a-syn in dopami­ nergic neurons (Matsuoka et al., 2001). No ubiquitin immunoreactivity was detected and the number of dopaminergic neurons and dopamine levels were not different from control mice, sug­ gesting that a-syn overexpression is not sufficient to produce dopaminergic deficits in these mice. A major factor linking a-syn overexpression and pathology may be the level of expression of the protein, with high levels being required to produce deficits in the short life span of the mouse. As long-term exposure to the pesticide para­ quat constitutes possible risk factors for sporadic PD (Andersen, 2003), paraquat was adminis­ tered to mice overexpressing human WT a-syn

64

under the control of the rat TH promoter. Those mice expressing a-syn, whether WT or mutant, were protected against paraquat-induced neuro­ degeneration even though there were protein aggregates in their brains, whereas their control littermates were not resistant to paraquat. This resistance was attributed to an increased level of HSP70, a chaperone protein known to protect against paraquat neurotoxicity (Manning-Bog˘ et al., 2003). In contrast, A53T a-syn worsened the neuropathology caused by combined administration of paraquat and maneb (Thiruchelvam et al., 2004). Other alterations in TH-aSyn mice Microglial activation was found as early as 1 month of age in the substantia nigra of mice overexpressing the WT a-syn (Su et al., 2008) and its mutated form A53T (Su et al., 2009) under the control of the rat TH promoter. In both studies, release of proinflammatory molecules was found in the substantia nigra and striatum, while in the latter, a release of proin­ flammatory molecules and a decrease in TH mRNA and a-syn protein were also found well after microglial activation was resolved (Su et al., 2009). These data suggest that microglial activa­ tion may be an early hallmark of PD, which may precede cell death of dopaminergic neurons. Other studies have found only moderate dopa­ minergic deficits at the neurochemical level. For example, mice expressing WT a-syn or a-syn with a double A30P + A53T mutation under the control of the rat TH promoter had an increased density of the dopamine transporter (DAT) and a subsequent increased sensitivity to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Richfield et al., 2002). Reduced striatal levels of dopamine and its metabolites were only observed in the line carrying the double mutation, together with a reduced locomotor activity and coordination at middle age (Richfield et al., 2002). Yu et al. have also found an increased sensitivity to MPTP in mice carrying the A53T mutation, as

MPTP decreased dopamine in the olfactory bulb and increased dopamine metabolism in the caudate nucleus and the olfactory bulb only in the trans­ genic mice but not the WT mice. It also increased a-syn expression in the striatum and tyrosine nitra­ tion in the olfactory bulb (Yu et al., 2008). More subtle changes were found in the molecular level. Analysis of gene expression pattern in mice overexpressing the WT and doubly mutated A53T + A30P human a-syn under the rat TH promoter revealed gene dosage-dependent dysre­ gulation of several genes important for the dopa­ minergic phenotype in the substantia nigra at time points preceding neuronal cell death, further sup­ porting the idea of subtle changes that precede cell death and may cause mild motor deficits (Miller et al., 2007). Another subtle effects of the expres­ sion of doubly mutated gene under the control of the TH promoter were impairments in the ubiqui­ tin proteasome system (UPS) in the frontal cortex, midbrain, and striatum (Chen et al., 2006), abnorm­ alities of which based on postmortem tissue studies have been associated with cases thought to repre­ sent idiopathic Parkinson’s disease (iPD) (Mcnaught et al., 2002). Although the finding of dopaminergic cell death in the substantia nigra (Thiruchelvam et al., 2004; Wakamatsu et al., 2008a) validated the TH model as relevant to PD, a major drawback of the restricted expression of a-syn when driven by the TH promoter is that it does not reproduce the extent of pathology seen in patients. Indeed, Lewy bodies and a-syn expression in neurites (Lewy neurites) are widely distributed in the cen­ tral and peripheral nervous system of patients with PD (Braak et al., 2003; Halliday et al., 2005). Pathological studies even suggest that extra-nigral pathology may precede dopaminergic cell loss in the substantia nigra and proceeds in a caudo­ rostral gradient, with the exception of the olfac­ tory bulb, which is affected early in the course of the disease. Behaviorally, most of the studies reported only moderate motor deficits, which may reflect the restricted distribution of a-syn pathology.

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Neuropathology and behavioral deficits in mice overexpressing a-syn under the control of the PDGF-b promoter To better mimic the broad distribution of a-syn pathology seen in humans, other studies used the platelet-derived growth factor (PDGF)-b, the prion, or the Thy-1 promoters. The first human a-syn-overexpressing mice generated expressed WT a-syn driven by the PDGF-b promoter (Masliah et al., 2000). These mice displayed intraneuronal inclusions immunoreactive for a-syn and sometimes ubiquitin in regions typically affected in synucleinopathies such as the neocortex, olfactory bulb, and midbrain. However, these inclusions lacked the characteristic fibrillar components of Lewy bodies. Interestingly, TH-positive terminals in the striatum, as well as striatal TH levels and activity, were reduced in the line with the highest expression of the transgene, although no changes were detected in the substantia nigra. These neu­ rochemical deficits, which were accompanied by a reduced motor performance on the rotarod, were only detectable at 12 months. Further examination of this line also revealed a 25–50% decrease in striatal dopamine at 12 months of age and increased thigmotaxis (Hashimoto et al., 2003). These results suggest that the induction of func­ tional deficits in the nigrostriatal system may require a certain level and duration of accumula­ tion of a-syn. The a-syn inclusions found in the olfactory bulb (Masliah et al., 2000) may account for impaired neurogenesis found in this region in two studies by the same group (Winner et al., 2004, 2008). The first study has found impaired neurogenesis in the hippocampus and olfactory bulb, and induced neuroapoptosis in the hippo­ campus of mice overexpressing the human WT a-syn (Winner et al., 2004). The second one repro­ duced these results, showing that the neurogenesis of TH+ neurons in the olfactory bulb of aged mice was affected more than that of other cells in this region. In mice expressing the WT a-syn it was attributed to neuroapoptosis while in mice expres­ sing the mutant protein A53T the decreased

neurogenesis was attributed to decreased prolifera­ tion. In addition, proliferation of cells in the subventricular zone, where most of the newly born neurons are generated and where a-syn was widely expressed, decreased in both adult and aged mice expressing the A53T mutation (Winner et al., 2008). This suggests that WT and mutant a-syn mediate pathology through different mechanisms and highlights the role played by age­ ing in the development of a-syn pathology under the control of the PDGF-b promoter, similar to the previously reported results (Masliah et al., 2000). Despite the great importance of these stu­ dies in revealing a mechanism, which may account for olfactory and perhaps cognitive deficits in PD, their findings were not correlated to functional deficits in these studies. Molecular alterations in mice overexpressing a-syn under the control of the PDGF-b promoter All of the later studies on mice overexpressing a-syn under the control of the PDGF-b promoter focused on molecular and morphological hallmarks of this model but none of them on behavioral alterations. Mice expressing the human WT pro­ tein had increased a-syn oligomer levels following exposure of living mesencephalic neurons to polyunsaturated fatty acids (PUFAs), whereas saturated FAs decreased them. PUFAs directly promoted oligomerization of recombinant a-syn, and transgenic mice accumulated soluble oligomers with age (Sharon et al., 2003). It was concluded that a-syn interacts with PUFAs in vivo to pro­ mote the formation of highly soluble oligomers that precede the insoluble a-syn aggregates asso­ ciated with neurodegeneration. Another lipid that was suggested to interact with a-syn and thus accelerate its aggregation is cholesterol (Bieschke et al., 2006; Bosco et al., 2006). A recent study found aggregates of a-syn in the temporal cortex as well as a decreased neuronal density in this region and an increase in plasma levels of

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cholesterol and its oxidized metabolites in mice overexpressing human WT a-syn under the PDGF-b promoter. These alterations were corre­ lated with 2–4-fold increase in the expression of a­ syn compared to the WT mice and were reversed by lovastatin, a cholesterol synthesis inhibitor (Koob et al., 2010). These studies suggest that different lipid molecules can promote a-syn aggre­ gation and the resulting neuropathology following a-syn overexpression and that agents regulating their levels or synthesis may be beneficial in the treatment of PD, as suggested by epidemiological studies of statin use in PD patients. More moder­ ate changes in the PDGF-aSyn mice were an alteration in the expression of some 200 genes related to transcription in the substantia nigra in mice overexpressing the WT protein, at 3 months of age when nigral neuropathology is not apparent (Yacoubian et al., 2008), implying that subtle changes in this region may precede cell death. Finally, Liu et al. (2009) have found that overexpression of WT human a-syn upregulated the nuclear and cytoplasmic expression of G proteincoupled receptor kinase 5 (GRK5), which was found to inhibit bcl-2 transcription and expression, thus pointing to a role for nuclear GRK5 in the regulation of apoptosis-related genes and perhaps in the mechanism of PD. Interestingly, a-syn phosphorylation at Ser 129, one of the pathologies characteristic to PD, did not increase despite the increase in GRK5 suggesting that other kinases may phosphorylate a-syn as well. Overall, it seems that the few overt changes caused by overexpression of a-syn under the PDGF-b promoter are mainly restricted to the olfactory bulb and the hippocampus and that the most severe deficits, whether they are functional or anatomical, occur relatively late. No overt pathology was found in the nigrostriatal system, which is particularly vulnerable in PD. The major changes to this system seem to be on the molecu­ lar (alteration in gene expression in the substantia nigra), biochemical (decreased dopamine and TH level and activity in the striatum), and, moder­ ately, anatomical levels (decreased TH terminals

in the striatum and decreased neurogenesis). A possible reason for these limitations may be the relatively low level of the transgene, which was found to be only 10–80% of control levels of expression. Therefore, this model does not seem to effectively mimic the major behavioral hallmark of PD—both motor and non-motor dysfunction. Mouse prion protein promoter Mice expressing the doubly mutated (A30P and A53T) protein under three different promoters, the chicken beta-actin (chbetaactin), the mouse tyrosine hydroxylase 9.6 kb (msTH), and the mouse prion protein (msprp), all displayed expres­ sion of the transgene. Although all promoters directed the expression into the brain and specific neuron types, they differed from each other with respect to the expression pattern in peripheral organs, the number and the regional distribution of expressing cells in the brain, and the level of the transgene expression. The msprp promoter was found to give the highest level of transgene expression and also targeted the expression exclu­ sively to neurons, including those in regions where no expression of the transgene was found under the control of the other promoters, especially the substantia nigra (Maskri et al., 2004). This sug­ gested that the pattern of expression under the control of this promoter may reliably mimic the neuropathology observed in PD. This potential advantage, however, was mitigated by the high level of transgene expression in motor neurons, which conferred behavioral deficits not character­ istic of PD as described in the following section. Behavioral and neuropathological alterations in mice overexpressing the A53T variant under the control of the mouse PrP promoter (PrPSynA53T) Mice expressing WT or A53T a-syn under the control of the mouse prion promoter developed

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a dramatic and fatal motor impairment by 8 months of age (Giasson et al., 2002). Neuropatho­ logical assessment revealed a wide distribution of a-syn-positive inclusions in the spinal cord, brain­ stem, cerebellum, striatum, and thalamus with a relative sparing of the motor cortex and a total sparing of the substantia nigra. Although the pathological changes were moderate in the basal ganglia and related cortical area, the distribution of inclusions in brainstem and medullar structures was similar to that of PD (Braak et al., 2003). Interestingly, there was no overt neuronal loss in the spinal cord or in the basal ganglia despite a severe and rapidly fatal motor phenotype. Similar to the study by van der Putten et al. (2000), the high expression of the transgene in the spinal cord may account for the severity of the motor symptoms. Another line of mice expressing human A53T a-syn driven by the murine prion promoter also displayed a marked motor phenotype including a reduced ambulation with ataxia and dystonia developing around 10–15 months of age and leading to death in a few weeks (Lee et al., 2002). Abnormal a-syn accumulation was present in the midbrain, cerebellum, brainstem, and spinal cord, and glial fibrillary acidic protein (GFAP) immunoreactivity was evident in these affected areas. Interestingly, motor deterioration occurred early in the lines with a high level of transgene expression. Similarly, pathological changes were more noticeable in the lines with the highest expression and in symptomatic com­ pared to asymptomatic mice, suggesting a “dose– effect” relationship between a-syn expression, pathological aggregation, and motor outcome (Lee et al., 2002; Masliah et al., 2000). None of the dopaminergic markers investigated in these A53T mice was different from controls (Lee et al., 2002). In the same study, mice overexpres­ sing the WT or A30P variant of the protein did not develop detectable behavioral or neuro­ pathological modifications despite the fact that the levels of expression of the transgene were comparable to those of the A53T line. Another

attempt to use the mouse prion promoter to drive WT or A53T a-syn resulted in a widespread dis­ tribution of the protein in the case of mutated a­ syn, while the WT protein was more restricted (Gispert et al., 2003). Mice expressing A53T a­ syn showed altered neuronal morphology, parti­ cularly in the olfactory bulb, and developed a reduction in vertical activity, stride length, grip strength, and rotarod performance. Similar to pre­ vious studies using the mouse prion promoter (Giasson et al., 2002; Lee et al., 2002), the A53T mutation was associated with more pronounced neuropathological and behavioral alterations. In all three studies, the magnitude of alterations was correlated with the levels of expression (Gias­ son et al., 2002; Gispert et al., 2003; Lee et al., 2002). In accordance with the previously reported studies, mice expressing A53T mutant a-syn developed neuronal degeneration and cell death in brainstem and spinal cord, and they exhibited large axonal swellings, somal chromatolytic changes, nuclear condensation, and spheroid eosi­ nophilic Lewy body-like inclusions that contained human a-syn and nitrated synuclein in the cyto­ plasm of cortical neurons and spinal motor neu­ rons. Approximately 75% of motor neurons were depleted. Electron microscopy revealed cytoplas­ mic inclusions in dendrites and axons, some of which were degenerating mitochondria and were positive for human a-syn. Mitochondrial changes included reduction of complex IV activity in spinal cord and terminal deoxynucleotidyl transferasemediated biotinylated UTP nick end labeling-posi­ tive matrices and p53 at the outer membrane. Subsets of neurons in neocortex, brainstem, and spinal cord ventral horn were positive for terminal deoxynucleotidyl transferase-mediated biotiny­ lated UTP nick end labeling, cleaved caspase-3, and p53, an indication of apoptosis (Martin et al., 2006). Similar to the previous studies, this study also showed no effect of the A30P mutation. Finally, mice expressing A53T a-syn stained posi­ tively for the anti-a-syn and anti-ubiquitin anti­ body, an evidence of Lewy body-like inclusions of a-syn, in the brainstem, cerebellum, spinal

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cord, and cortex at old age. GFAP+ staining in the brainstem, cerebellum, midbrain, and spinal cord and positive staining for Fluoro Jade B in the brainstem, an evidence of neurodegeneration, were all found in these mice, which also displayed hyperactivity and decrease in startle acoustic response at 9 months and showed a severe motor phenotype leading to death. The changes were not affected by the absence of Parkin, a ubiquitin E3 ligase that was suggested to be involved in a-syn degradation (Kitada et al., 1998; Zhang et al., 2000), suggesting that PD caused by a-syn and parkin mutations may occur via independent mechanisms (Von Coelln et al., 2006). Although the last two studies reproduced the behavioral findings and some of the neuropathological find­ ings in the spinal cord and brainstem and showed no dopaminergic deficits or nigral cell death, they did show neurodegeneration in the former regions, which was not found in earlier studies and may account for the abnormal startle response in the latter study (Von Coelln et al., 2006). Another non-motor symptom found in young PrPSynA53T mice was decreased anxiety. A tendency to decrease locomotion was also observed in this study, but neuropathology was not documented (George et al., 2008). Mice expressing the human mutant protein A53T on a a-syn null background showed a marked expres­ sion of the transgene (37-fold of endogenous a­ syn) in the spinal cord but not in the brain (0.35 of endogenous a-syn). The expression was found throughout the spinal cord including the ventral horn, ventral white matter, neuronal cell bodies and axons in the dorsal and ventral roots, and also in the sciatic nerve (Cabin et al., 2005). Wallerian degeneration was found in the ventral roots and sciatic nerve axons were damaged. In accordance with the axonal damage, astrogliosis was found in the spinal cord and the mice developed a neuro­ pathy characterized by limb weakness and paraly­ sis with onset beginning at 16 months of age, and gait measurement showed shorter stride length at 17–18 months, but rotarod performance was not affected. Intriguingly, in mice expressing the WT

mouse a-syn protein, Wallerian degeneration and GFAP+ staining were not evident and axonal damage to the sciatic nerve was much less marked, which may account for the longer life span observed in these mice (Cabin et al., 2005). A possible explanation for the protective effect of endogenous a-syn was the presence of six amino acids in the murine protein differing from the human protein, in addition to the A53T mutation, which could help the mice tolerate the mutation. Interaction between external insults and a-syn expression under the control of the mouse PrP promoter A53T mice treated chronically with a combination of the pesticides paraquat and maneb (but not with either pesticide alone) showed drastic increase in neuronal a-syn pathology throughout the central nervous system including the hippocampus, cere­ bellum, and sensory and auditory cortices at 8 months of age. a-Syn-associated mitochondrial degeneration was observed in these mice but not in WT a-syn transgenic mice or the A53T mice treated with saline. The expression of a-syn increased by 2–3-fold compared to the expression in mutant mice untreated with the pesticides in the cerebellum, cortex, and brainstem, and filamentous a-syn aggregates were found in axon terminals. Because a-syn inclusions accumulated in pesti­ cide-exposed transgenic mice without a motor phenotype, it was concluded that a-syn aggregate formation precedes disease onset. This study sup­ ported the notion that environmental factors can promote a-syn pathologies and work in concert with genetic vulnerability to promote PD progres­ sion (Norris et al., 2007). a-Syn also seems to confer sensitivity to inflammation as mice displayed higher susceptibility to lipopolysaccharide (LPS)-induced neuroinflammation following LPS injection to the substantia nigra, which caused cell loss selec­ tive to TH+ cells in mice expressing WT a-syn on an a-syn-KO background but non-selective loss in those expressing A53T a-syn on an a-syn-KO

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background, at 12 months, suggesting perhaps that TH+ cells are more sensitive and that the A53T is more detrimental than the WT protein when combined with inflammation. This cell loss was mediated by oxidative and nitrative species. Interestingly, non-transgenic mice and mice expres­ sing the WT and A53T all displayed lower TH immunoreactivity in the substantia nigra while the TH+ neurons in the a-syn-KO mice were spared, suggesting that a-syn promotes inflammationinduced cell death. Dendrite length and dopamine uptake decreased in midbrain TH+ neurons. The differential outcomes of LPS treatment could not be attributable to different inflammatory responses as these were similar in all lines. Cyto­ plasmic inclusions of a-syn were found only in the substantia nigra of the LPS-treated mice expressing WT and A53T a-syn but not their control litter­ mates (Gao et al., 2008). The last two studies high­ light the importance of a-syn overexpression as a risk factor for development of PD, when combined with external insults. Genetically, the PrPSynA53T mice may indeed be more susceptible than nontransgenic mice, as increased alterations in gene expression profiles were found in the brainstem of these mice at a time point when neuronal death and behavioral phenotype are apparent. Unsurpris­ ingly, one of the functional categories that was sig­ nificantly and mostly changed in PrPsynA53T mice was immune/inflammation (49 genes, 16.3% of all genes) (Miller et al., 2007). Alterations in catecholamine systems in mice expressing A53T a-syn under control of the mouse prion promoter Alterations in the dopaminergic and noradrener­ gic systems were also reported in the A53T line; higher expression of the dopamine D1 receptor in the substantia nigra, lower expression of DAT in the nucleus accumbens and the striatum, and reduced uptake of dopamine in the striatum were related to elevated daytime and nighttime activity in these mice at 7 months of age and increased

locomotor activity at 9 and 19 months. In agree­ ment with the neurochemical findings, the hyperac­ tivity was dependent on D1 dopaminergic receptor but not on 5-HT1B serotonergic receptor (Unger et al., 2006). In this study too, A30P-expressing mice did not demonstrate any alterations com­ pared to WT mice. Likewise, a very recent study showed a decrease in the striatal DAT at 8 months in PrPSynA53T mice along with hyperactivity at 12 months. An increased anxiety was found at 2 months but reduced anxiety was found at 12 months, and similarly, rotarod performance was impaired at 2 months in the transgenic mice but was better at 12 months (Graham and Sidhu, 2010). This suggests that decreased levels of the DAT that may increase dopamine levels may cause hyperactivity and perhaps reduced anxiety, although the interpretation of these results is hampered by the presence of the hyperactivity. Mice expressing the A53T mutation displayed decreases in norepinephrine but not dopamine levels in the spinal cord, olfactory bulb, and stria­ tum at 15 months. These were associated with a decrease in TH density in the spinal cord and in TH levels in the spinal cord and olfactory bulb, indicating a noradrenergic terminal loss in the spinal cord (Sotiriou et al., 2009). TH enzyme levels and density did not decrease in the striatum despite the decrease in norepinephrine, suggesting perhaps a decreased enzyme activity in noradre­ nergic but not dopaminergic terminals in this region. TH+ cell counts in the substantia nigra and locus coeruleus did not change, in line with previous findings (Giasson et al., 2002; Von Coelln et al., 2006), though a-syn accumulated in these regions (Sotiriou et al., 2009). Thus, it is possible that noradrenergic terminal loss in the projection areas of the locus coeruleus preceded cell loss in this region and caused the selective decrease in norepinephrine as opposed to unchanged dopa­ mine levels. Indeed, synaptic loss was found in the locus coeruleus of PD patients and a-syn pathology occurs in the locus ceoruleus prior to the substantia nigra in PD (Baloyannis et al., 2006; Braak et al., 2003).

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Alterations in mice overexpressing the A30P variant under the control of the mouse prion promoter Even though the studies comparing expression of the WT, A30P, and A53T forms of the gene did not detect any behavioral or neuropatholo­ gical effect of A30P expression, a few studies did find behavioral and other deficits, though they were more moderate and had later onset. Mice with the A30P mutation displayed 1.5-fold increase of a-syn compared to a-syn knockout mice, and aged mice bearing the mutation expressed it 1.5 times higher than adult A30P mice. a-Syn accumulated especially in the stria­ tum, cerebellum, and hippocampus at 21 months but no Lewy body-like inclusions were observed. Compared to WT mice, decreased locomotion had its onset in old age, while decreased rearing had its onset at middle age and was progressive. Evoked dopamine release in the striatum decreased in A53T mice (Yavich et al., 2005). Similarly, at 6 and 11 months, locomotion and rearing decreased in this line of mice and they showed increased sensitivity to the effects of L-DOPA on stimulated dopamine release in the nucleus accumbens shell, and on locomotor activity, rearing, and stereotypy (Oksman et al., 2009). Mice from the C57BL/6J background expressing WT and A30P a-syn under the control of the mouse prion promoter showed modest synaptic changes at 4–5 months— paired pulse facilitation decreased in the two trans­ genic lines and in a-syn knockout mice, whereas LTP and frequency facilitation in response to highfrequency stimulus decreased only in a-syn knock­ out mice. WT a-syn and A30P a-syn accumulated to the same extent in the mossy fibers of the den­ tate gyrus (DG) (Gureviciene et al., 2007). This suggests that a-syn is crucial to normal synaptic plasticity, in accordance with what is known about the interactions of a-syn with synaptic proteins (Chandra et al., 2005). Long-term depression in response to stimulation of the DG (instead of the normal response of long-term potentiation) was found at 24–25 months and was associated with

impaired exploratory activity (Gureviciene et al., 2009). Other changes that were found were more subtle, such as abnormal mobilization of norepi­ nephrine in the hippocampus of mice expressing the A30P mutation (Yavich et al., 2006). Hamster prion protein The use of the hamster prion promoter to express WT, A30P, or A53T a-syn resulted in a predominant localization of the transgene in the hippocampus, cortex, substantia nigra, and cerebellum. Although dopaminergic markers were not affected and neu­ rodegeneration was not observed, astrogliosis in the cortex and hippocampus, as well as a severe motor syndrome including tremor and dystonic posture occurred in the line with the highest expression level (Gomez-Isla et al., 2003). In another study, mice expressing the A30P variant were more vulnerable to the effects of 150 mg/kg of the neurotoxin MPTP, which decreased the number of TH-positive cells in the substantia nigra at 6–8 months and the level of dopamine in the striatum at 3–4 months (Nieto et al., 2006). In a conditional mouse model of a-syn expression, a relatively weak expression of the human WT a-syn was found under the control of the hamster prion protein promoter, in comparison to the CaM promoter, in the basal ganglia and the cor­ tex. Only in the olfactory bulb was there a strong expression of the protein under the control of the hamster prion promoter (Nuber et al., 2008). In general, the hamster prion protein promoter does not seem to induce substantial expression of the transgene or overt pathology as compared with the mouse prion promoter. In conclusion, although the expression of a-syn in this line appeared less widespread than when the mouse prion promoter was used, many of the lines in which mutant a-syn was driven by a prion promoter developed a severe motor disorder related to the level of expression of the transgene (Cabin et al., 2005; Giasson et al., 2002; Lee et al., 2002), which may be accounted for by the

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marked expression of the transgene in the spinal cord. In addition, hyperactivity was also found (Unger et al., 2006; Von Coelln et al., 2006), which may be related to upregulation of the dopaminergic system (Unger et al., 2006) and precede the severe and, in many cases, lethal behavioral phenotype. The substantia nigra seems to be spared in these models (Giasson et al., 2002; Sotiriou et al., 2009). In general, mice expressing the A53T mutation seem to be affected much more severely and earlier than both WT mice and mice bearing the A30P muta­ tion under the control of the prion promoter, both neuropathologically and behaviorally. This is in accordance with the finding that aggregation of the A53T protein was much faster than that of the A30P (Li et al., 2002). Overexpression of a-syn under the control of the Thy-1 promoter The murine Thy-1 promoter turned out to be particularly useful to drive high levels of expres­ sion of the human WT a-syn in neurons. The advantages of using this promoter include very high levels of expression and lack of restriction to catecholaminergic neurons (Kahle et al., 2000; Rockenstein et al., 2002). Motor phenotype following a-syn overexpression under control of Thy-1 promoter (Thy1-aSyn) and its relation to pathology Van der Putten et al. (2000) were the first to report neuropathology and early-onset motor def­ icits in the rotarod in mice overexpressing the human gene with the A53T mutation under the Thy-1 promoter (van der Putten et al., 2000). aSyn inclusions were found in the telencephalon, brainstem, cerebellum, and spinal cord. Neurons stained positive for ubiquitin, indicating a Lewy body-like pathology (van der Putten et al., 2000). Although the neuropathological assessment

revealed the presence of a-syn and ubiquitin-posi­ tive inclusions in structures classically affected in synucleinopathies, most of the pathological changes were observed in the spinal cord with a prominent degeneration of motor neurons but without any modification in the nigrostriatal sys­ tem (Sommer et al., 2000; van der Putten et al., 2000). The lack of nigral pathology may be due to a lack of expression of the transgene in the nigros­ triatal system or weaker levels of expression than those reached in the spinal cord in this particular line of mice. Other studies indicated that this pro­ blem was not a general characteristic of the use of the Thy-1 promoter. Indeed, a comparative study of PDGF-b a-syn and another line of Thy-1 mice revealed that the Thy-1 promoter induced a higher and more widespread expression of the transgene than the PDGF-b, although the spinal cord was spared (Rockenstein et al., 2002). On the other hand, the PDGF-b promoter was able to induce expression of a-syn in glial cells, which was not the case of Thy-1 (Rockenstein et al., 2002). In a separate line of mice, a-syn inclusions in the brainstem and spinal cord of mice expres­ sing the A30P mutation, astrogliosis in the spinal cord, hyperphosphorylation of a-syn in the spinal cord, and alterations in the levels of serotonin and its metabolite 5-HIAA in the frontal cortex and striatum were associated with motor impair­ ments after the first 12 months of life. The bio­ chemical and behavioral changes occurred mostly in female mice (Neumann et al., 2002). In contrast, the same promoter did not cause any particular motor phenotype in mice expressing either WT or A30P a-syn even with the observation of accumu­ lation of the protein in the cerebellum, substantia nigra, neocortex, and brainstem (Kahle et al., 2000). Thus, the overall phenotype of mice overexpressing a-syn under the Thy-1 promoter criti­ cally depends on background and transgene insertion as well as the overall level of overexpres­ sion achieved, and each line must be considered separately for its ability to reproduce PD-like phenotypes without confounding spinal cord pathology.

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The motor phenotype of mice from the mixed background C57BL/DBA2 overexpressing human WT a-syn under the Thy-1 promoter (Thy1-aSyn) generated by the Masliah’s group (Rockenstein et al., 2002) was extensively characterized for the presence of motor and non-motor deficits, pathology, and dopamine alterations. Because the transgene is inserted in the X chromosome in these mice, all studies included male mice exclusively. These mice pre­ sent broadly distributed proteinase-K-resistant a-syn aggregates throughout the brain and accu­ mulation of a-syn in peripheral neurons (Ferna­ gut et al., 2007; Rockenstein et al., 2002). Dopamine levels are reduced by approximately 40% in the striatum of these mice at 14 but not 8 months of age, whereas a progressive loss of nor­ adrenaline in the cortex can be detected from 8 months (N. Maidment and M.-F. Chesselet, unpublished observations). Nevertheless, these mice displayed significant motor and coordination impairments and a reduction in spontaneous activity and in body weight as early as 2 months of age. Motor performance and coordination impairments progressively worsened with age and sensorimotor deficits appeared at 6 months, whereas fine motor skills were altered at 4 months and worsened at 8 months (Fleming et al., 2004), suggesting that overexpression of a-syn induced an early and progressive behavioral phenotype. In contrast to WT mice, Thy1-aSyn mice did not show amphetamine-induced stereotypies (Flem­ ing et al., 2006) without decreases in the ability of amphetamine to increase dopamine release, suggesting that chronic overexpression of a-syn led to abnormal pharmacological responses. As long-term exposure to the pesticide para­ quat constitutes possible risk factors for sporadic PD (Andersen, 2003), paraquat was administered to mice overexpressing human WT a-syn under the control of Thy-1 promoter. Even though para­ quat intoxication increased the number of protei­ nase-K-resistant aggregates in the substantia nigra, it did not worsen behavioral impairments and neither was the paraquat-induced TH+ cell

loss in the substantia nigra affected by a-syn, as opposed to a previous study on a different model of a-syn expression (Fernagut et al., 2007; Manning-Bog˘ et al., 2003). Thus, aggregates of a-syn in the substantia nigra are not directly cor­ related with the magnitude of motor deficits but may signal the presence of neuronal dysfunction long preceding neuronal death. Another line of mice expresses the doubly mutated gene (A30P + A53T) under the control of the human Thy-1 promoter. Motor deficits were observed at 3 months and were progressive. Striatal dopamine and hypothalamic serotonin decreased at 10 and 17 months of age, and hypothalamic dopamine and striatal acetylcho­ line decreased at 17 months of age. Eosinophilic inclusions stained positive for a-syn were found in the dentate nucleus and substantia nigra, and atrophy was found in the cerebellum and cortex. Intracellular inclusions were also found in the brainstem. These inclusions lacked the halo structure of classical Lewy bodies. Nitration of a-syn and ubiquitin-positive cells were found in the brainstem and TH staining was weaker in the locus coeruleus of transgenic mice (Ikeda et al., 2009). Nigrostriatal deficits may account for the motor deficits as inclusions were found in the substantia nigra and dopamine decreased in the striatum at time points when motor deficits were evident (7 and 10 months, respectively), though nigral cell death was not reported. Another study showed impaired performance in the rotarod and pole tests, which began at 3 months of age and progressed up to 12 months. At 12 months, TH+ cells were reduced in the substantia nigra, staining for phosphorylated a­ syn was enhanced, and dopamine decreased in the striatum; sodium 4-phenylbutyric acid (PBA), a chemical chaperone that supposedly interacts with a-syn, reversed the neuropathol­ ogy as well as the neurochemical and behavioral deficits (Ono et al., 2009), presumably by decreasing the phosphorylation of a-syn, known as one of the pathological hallmarks of PD (Fujiwara et al., 2002).

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Transgenic mice overexpressing the human a-syn with the mutation Y39C, which enhances neurotoxicity and protein aggregation, under the control of the mouse Thy-1 promoter, widely expressed the mutant protein in the brain, includ­ ing the cortex, hippocampus, striatum, thalamus, and substantia nigra, and resulting in 250% expression relative to wild types. At 24 months, transgenic mice developed neuropathology, such as Lewy body-like a-syn inclusions and ubiquitin­ positive inclusions, phosphorylation at Ser(129) of human a-syn, and increased apoptotic cell death in the cortex. At age 9–12 months, transgenic mice began to display motor dysfunction in rotarod testing. Older animals aged 15–18 months showed progressive accumulation of human a-syn oligo­ mers, associated with worse motor function and cognitive impairment in the Morris water maze. By age 21–24 months, a-syn aggregates were further increased, accompanied by even more severe behavioral deficits. However, TH+ cell loss was not found in the midbrain up to 21–24 months. The results suggest that Y39C human a-syn transgenic mice show age-dependent, progressive neuronal degeneration with progressive motor and cognitive deficits. The time course of a-syn oligomer accumulation coincided with behavioral and pathological changes, with an increase from 9–12 months to 15–18 months to 21–24 months, indicating that these oligomers may initiate protein aggregation, disrupt cellular function, and even­ tually lead to neuronal death (Zhou et al., 2008). Non-motor phenotype of Thy1-aSyn and its relation to neurochemical and pathological alterations In addition to motor deficits found in Thy1-aSyn mice expressing human WT a-syn (Fleming et al., 2004, 2006), these mice also displayed a wide range of non-motor alterations reminiscent of non-motor symptoms of PD. Thy1-aSyn mice had decreased colonic function but an increased fecal response to novelty stress and corticotropin releasing factor

injected intraperitoneally (Wang et al., 2008). They also had olfactory deficits at 3 and 9 months in the buried pellet, block, and habituation/dish­ abituation olfactory tests, and these deficits were associated with proteinase-K-resistant a-syn inclu­ sions throughout the olfactory bulb (Fleming et al., 2008b). Apparent decreased anxiety was found in 3–6-month-old a-syn-overexpressing mice (Mulligan et al., 2008). As noradrenaline decreases in the prefrontal cortex of Thy1-aSyn mice (Maidment, unpublished observation) and given the role of noradrenaline in modulating aspects of cognition (Aston-Jones et al., 1999) and the difficulty in strategy switching displayed by PD patients, Thy1-aSyn and WT mice were tested in a reversal learning task that assesses cognitive flexibility. Male Thy1-aSyn mice at 4–5 months of age learned a simple operant strategy as well as controls but showed greater difficulty than WT littermates in their ability to switch their responses at reversal, although they were able to eventually achieve criteria and learn the reversed contingency (Fleming et al., 2008a). Both low- and high-dose L-DOPA had a positive effect of rever­ sal accuracy in the Thy1-aSyn mice. In addition to its effects on the dopaminergic system, L-DOPA is also known to increase noradrenaline, so it is pos­ sible that it improved cognition, in part, by stimu­ lation of noradrenaline receptors. Agonists of the alpha-2 noradrenergic receptor also had a positive effect on reversal accuracy (Fleming et al., 2008a), supporting the role of noradrenergic deficits in the cognitive deficits in these mice. Deficits in the probe trial of the Morris water maze were found in 12-month-old mice expressing the a-syn gene with the A30P mutation under the Thy-1 promoter but they were not related to decreased swimming speed. Decreased freezing time and active avoidance in response to fearful stimuli were also found at 12 months as well as impaired rotarod performance. However, locomo­ tor activity and stereotypy were increased at that time point. Inclusions of a-syn and neuronal dys­ trophy were found in the neocortex and amygdala, the latter region being related to fear responses

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and thus may explain the decreased freezing and active avoidance. In addition, the level of a-syn expression in the brainstem appeared to be related to the degree of motor impairment: presymptomatic mice had no detectable a-syn signal whereas the expression of a-syn was slightly higher in mice with severe motor impairments than in those with mild impairments (Freichel et al., 2007). Similarly, overexpression of A30P caused age-dependent impairments in fear-condi­ tioning behavior at 16–19 months and distinct staining patterns of phosphorylated serine residue (phospho-serine-129 (PSer129)), which is one of the features of neuropathological lesions in PD patients (Fujiwara et al., 2002; Neumann et al., 2002). Somal and nuclear PSer129 immunoreac­ tivity increased with age in hippocampal and cortical areas as well as the lateral/basolateral amygdalar nuclei and was also present in young, presymptomatic mice but not WT controls. These mice further developed age-dependent, specific neuritic/terminal a-syn pathology in the medial parts of the central amygdalar nucleus and one of its projection areas, the lateral hypothalamus. This suggests that a-syn becomes phosphorylated in distinct parts of the brain in this a-synucleino­ pathy mouse model, showing age-dependent increases of nuclear PSer129 in cortical brain areas and the formation of neuritic/terminal PSer129 neu­ ropathology with variable amyloid quality within the fear-conditioning circuitry (Schell et al., 2009).

GFAP+ staining was found in the brainstem of transgenic mice but not control littermates. Stain­ ing was higher in symptomatic than in asympto­ matic mice, suggesting a role for phospho-tau in the symptoms of PD (Frasier et al., 2005). Mice overexpressing this mutation also displayed an increase in the oxidized levels of three mitochon­ drial-associated proteins, carbonic anhydrase, alpha-enolase, and lactate dehydrogenase 2, and reduced activities of these proteins, in brain (Poon et al., 2005). Another study that focused on mito­ chondrial alterations showed that mice expressing WT protein under the Thy-1 promoter were more susceptible to the neurotoxin MPTP than their WT littermates: increased mitochondrial size and neuritic aggregations, axonal degeneration, and inclusions in the substantia nigra were only found in transgenic mice, but not WT, following MPTP administration (Song et al., 2004). These data point to a mitochondrial dysfunction in Thy1­ aSyn, mice that may reflect compromised mito­ chondrial function in PD (Li et al., 2009). Koob et al. (2010) have found, using the Thy-1 promoter to drive a-syn expression, similar results to those found with the PDGF-b promoter (see above), and lovastatin, a cholesterol synthesis inhibitor, affected a-syn in both lines of mice equally. A difference, however, was a much lower level of a-syn in insoluble fraction from brains of Thy1-a Syn mice than in PDGFb-aSyn mice, contrasting with the abundance of proteinase-K-resistant a-syn aggregates in the Thy1-aSyn mice (Fernagut et al., 2007). The reasons for these results are unclear.

Other neuropathologies in Thy1-aSyn mice Phosphorylation of tau protein and of a-syn, as well as the expression of several kinases like JNK and GSK-3b that are induced by cellular stress and are known to phosphorylate tau, increased in the brainstem of 12-month-old mice expressing the A30P mutation under the control of Thy-1 promoter. The phosphorylated kinases co-loca­ lized with phosphorylated a-syn aggregates and phosphorylated tau protein, indicating that they may be responsible for the phosphorylation.

Time course of deficits in various lines of Thy1-a Syn mice A distinct advantage of the Thy1-aSyn mice pro­ duced by the Masliah’s lab (Koob et al., 2010; Rockenstein et al., 2002; Song et al., 2004) is that they present both motor and non-motor deficits at a very young age, i.e., starting at 2 months (Ferna­ gut et al., 2007; Fleming et al., 2004, 2006, 2008b). Accordingly, these Thy1-aSyn mice provide a

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useful tool to study the preclinical stages of PD, prior to the appearance of severe motor symp­ toms, and assess novel drug therapy in genetic models of synucleinopathies. These data indicate that overexpression of a-syn under the Thy-1 pro­ moter is sufficient to cause early behavioral defi­ cits in mice equivalent to those observed in patients with PD prior to the clinical diagnosis, as these deficits were evident at a time point when no overt cell loss in the substantia nigra (Fernagut et al., 2007) or dopaminergic terminal loss in the striatum (N. Maidment and M.-F. Chesselet, unpublished) were found. Potential mechanisms that can explain these deficits are alterations in the corticostriatal pathway that were found at a young age (Watson et al., 2009; Wu et al., 2009), alterations in striatal dopamine synapses, which include a chronic increase in basal levels of extracellular dopamine in the striatum (Maidment et al., 2006), and abnormal electrophy­ siological responses of dopamine receptors to dopaminergic agonists and antagonists in striatal slices (Wu et al., 2005). Compared to other studies that used the Thy-1 promoter and demonstrated motor dysfunction at a much older age (Freichel et al., 2007; Neumann et al., 2002; Zhou et al., 2008), no motor dysfunction at all (Kahle et al., 2000), or late-onset cognitive dysfunction (Frei­ chel et al., 2007; Schell et al., 2009; Zhou et al., 2008), the earlier onset of these deficits in these mice may be due to the high level of expression of the protein (10× levels of endogenous mouse a­ syn). Alternatively, classical motor as well as cog­ nitive tests such as Morris water maze and fear conditioning that were used by other groups may not be sensitive enough to detect subtle motor and cognitive deficits that are present at a young age. Calcium/calmodulin-dependent protein kinase II promoter-controlled tetracycline transactivator (CaMKII-tTA) Few studies investigated the effects of overexpres­ sion of a-syn under the control of the CaM

promoter, which is expressed abundantly in fore­ brain regions, especially the olfactory bulb. The rationale behind using the CaM was to investigate neurogenesis, which occurs postnatally mainly in the olfactory bulb and the hippocampus where the promoter is most active. A conditional overex­ pression under this promoter is made possible due to the addition of tTA domain, which sup­ presses the transgene expression in response to doxycyline administration, and allows for the analysis of the progression of a-syn-dependent neuropathology and motor symptoms and of their possible reversal when the expression of the transgene is halted at certain ages and for defined periods of time. Nuber et al. (2008) have found high expression of WT a-syn expression under the CaM promoter in the cortex, basal ganglia, and olfactory bulb. Reduced retention in the Morris water maze 7 days after a single probe trial was found at the age of 52 weeks and deterioration in rotarod performance was found starting from 30 weeks of age. Repressing the expression of a-syn starting from 58 weeks of age halted the deteriora­ tion of motor function but did not restore it, and neither did it affect body weight, but it restored dopamine level in the olfactory bulb while it was reduced when the gene was expressed. These data suggest that a-syn-induced pathology and the resulting deterioration of motor function are reversible when treated early enough at the development of PD. CaM-aSyn mice in which the transgene expression was repressed starting from 16 weeks of age for 8 weeks showed restored neurogenesis in the hippocampus compared to untreated transgenic mice in which neurogenesis was reduced. There was also a strong trend for reduction of TH+ cells in the substantia nigra. (Nuber et al., 2008). This study suggested a possi­ ble link between decreased neurogenesis, a-syn pathology, and cognitive deficits. In another study, mice overexpressing the A30P variant under the control of the CaM promoter showed decreased dopaminergic neurogenesis in the olfactory bulb, which was restored by suppres­ sing the transgene expression starting from 2

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months of age for 2 months, in association with total elimination of a-syn overexpression (Marxreiter et al., 2009). Finally, in one line of WT and A53T mutant a-syn overexpression in the forebrain under CaM promoter, massive loss of postmitotic neurons in the hippocampal DG was observed during postnatal development, with hippocampal synapse loss as evidenced by reduced levels of pre- and postsynaptic markers. This degeneration was attributed to cell death rather than decreased proliferation because mar­ kers of cell proliferation were not changed. It was shown to be restricted to the developmental per­ iod, as no hippocampal neuron loss was observed when mutant and WT a-syn expression was repressed until the Tg mice were mature postna­ tally and then induced for several months. These data implied that developing neurons are more vulnerable to degenerate than mature neurons as a consequence of forebrain WT and mutant a-syn overexpression (Lim et al., 2010). It seems that overexpression of a-syn, be it WT, A30P, or A53T, under the control of the CaM promoter greatly affects neurogenesis in regions in which new neurons are constantly generated such as the DG and olfactory bulb, either by affecting the proliferation of new neurons or by affecting the fate of newly generated neurons shortly after birth. The relationship of these observations to PD, however, remains unclear as similar changes have not been reported in the brain of patients. Other promoters Several other promoters were used occasionally to drive a-syn expression, including the endogenous mouse a-syn promoter in mice with a knock-in A30P mutation. These mice developed a signifi­ cant deficit in motor performance tests related to nigrostriatal function—the beam walk test and the ink test, at 13 and 17 months of age and 16 months of age, respectively. They also displayed dimin­ ished enhancement of the locomotor effect of amphetamine induced by pretreatment with the

vesicular monoamine transporter 2 (VMAT2) inhibitor reserpine, possibly reflecting a functional dopamine deficiency. Dopamine and its metabo­ lite DOPAC were indeed reduced in the striatum and dopamine was reduced in the mesolimbic sys­ tem at 15 months of age, thus perhaps supporting the latter hypothesis (Plaas et al., 2008). The mutant a-syn may interfere with the correct func­ tioning of VMAT2 and induce deficits in the nigrostriatal system. Indeed, VMAT2 has a pro­ tective effect against the development of PD (Glatt et al., 2006). In another study, A53T-aSyn and the C-terminally truncated synuclein with 119 amino acids (aSyn119) were expressed in the ROSA26 locus. Since disease-associated muta­ tions in a-syn promote the accumulation of these truncated filaments compared to the WT protein and C-terminally truncated species can enhance the aggregation of WT a-syn at low substoichio­ metric ratios (Li et al., 2005; Liu et al., 2005; Murray et al., 2003), the hope was that C-terminal truncations of a-syn could potentially advance dis­ ease progression or propagation through promot­ ing the pathological aggregation and accumulation of a-syn selectively in TH-abundant neuronal populations like the substantia nigra. Therefore, a construct with the Cre-loxP-based transgene cas­ sette was made, which is regulatable and can turn on and off the expression of the transgene either in catecholaminergic or in all neurons with TH or nestin, respectively, and thus express the transgene in a region-dependent manner. Expression of human aSyn119 expression was confirmed in brain regions containing TH-positive neurons or nerve terminals including the olfactory bulb, stria­ tum, cerebral cortex, and ventral midbrain. At 10 months of age, aSyn119 expression, however, was less than 10% of the endogenous a-syn in the regions where it was determined. Possibly as a result of this low expression, no nigral cell death of neurons in general or TH+ neurons in particu­ lar were observed at 10–12 months of age. Never­ theless, striatal dopamine and its metabolites were reduced whereas striatal norepinephrine and 5­ HT in the prefrontal cortex increased in aSyn119

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mice at 12–13 months of age (Daher et al., 2009). This strengthens the idea that subtle changes in the dopaminergic system can occur in the absence of overt cell death. However, as behavioral phe­ notypes were not characterized in these mice, it is not known whether these changes are related to motor or non-motor symptoms. Finally, a very recent work (Kuo et al., 2010) demonstrated beha­ vioral and gastrointestinal abnormalities in mice expressing A53T a-syn under the P1 artificial chromosome (PAC) on an a-syn-null background. These mice had an early onset of impaired rotarod performance and decrease in locomotor activity, which was progressive, even though a-syn inclu­ sions, TH+ cell loss in the substantia nigra, or dopamine loss in the striatum were not found even at old age, suggesting that deficits in systems other than the dopaminergic may play a role. These mice also had decreased colonic motility that may result from proteinase-K-resistant a-syn aggregates that were found in the nuclear and perinuclear cytoplasm of neurons of the enteric nervous system (ENS) but olfactory function and cardiac autonomic innervation remained intact (Kuo et al., 2010) in contrast to the reported olfac­ tory deficits in the Thy1-aSyn model (Fleming et al., 2008b). Abnormal gastrointestinal function was already found in Thy1-aSyn mice at older age (Wang et al., 2008) but this is the first report to show neuropathology in the ENS following a­ syn overexpression. Thus, this model serves as a good model to investigate therapies for the gastro­ intestinal dysfunction in the early, preclinical stages of PD. Summary—a-syn models of PD To conclude, the models of a-syn overexpression in mice developed for PD recapitulate to variable extents the neuropathology and neurochemical and behavioral deficits found in PD patients, depending primarily on the promoter used to drive the expression of the transgene, whether the transgene codes for the WT or mutated

protein, and the genetic background of the mice. The mouse prion protein promoter drives transgene expression at high levels in motor neurons of the spinal cord and brainstem and tends to cause severe motor deficits leading to death. However, its main drawbacks include a lack of cell loss in the substantia nigra and locus coeruleus, both of which have cell loss in PD, the inability to detect subtle motor deficits at preclinical stages, and the presence of disabling but non-PD-related motor neuron dysfunction. These mice are primarily use­ ful to assess molecular mechanisms associated with a-syn-related pathology. In contrast, the use of the murine Thy-1 promoter causes widespread expression of the transgene without motor neuron loss when used in C57Bl6 mice. Nigral DA cell loss was only observed in a few instances but mice often develop loss of DA in the striatum, and a range of motor and non-motor anomalies that appear at different ages, depending on levels of overexpression and the sensitivity of the tests used. The use of the rat TH promoter led to TH + cell loss in a few cases, but did not replicate the broad a-syn neuropathology observed in PD patients, in contrast to the Thy-1 promoter. Mice based on the PDGF-b promoter showed relatively mild changes at the molecular level and also in neurogenesis. With regard to the transgene expressed, the A53T a-syn seems to be more effective in inducing neuropathology and beha­ vioral deficits than the WT protein, probably due to a faster rate aggregation (Li et al., 2002). Regardless of the promoter used to drive a-syn expression, most studies did not demonstrate dopaminergic cell loss at the ages and in the rear­ ing conditions examined. However, they recapi­ tulate at least partially the susceptibility of certain neuronal populations to develop a-syn aggregates, and can help to identify factors responsible for this vulnerability. Moreover, in some of the models, alterations of the nigrostria­ tal dopaminergic projection (decreased striatal levels of TH and/or dopamine) and behavioral impairments indicate that the accumulation of a-syn can significantly alter the functioning of

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dopaminergic neurons even without overt cell death. This suggests that a-syn could cause dopa­ minergic dysfunction in the early phase of the pathogenic process, possibly promoting a subse­ quent cell death. In light of many studies reviewed here, which demonstrated increased susceptibility to certain external insults such as inflammagens (Gao et al., 2008), neurotoxins (Song et al., 2004; Yu et al., 2008), and pesticides (Fernagut et al., 2007; Norris et al., 2007), the a­ syn models can also be used to test the hypothesis that increased levels of expression of a-syn pre­ dispose to develop the disease when individuals are exposed to specific environmental factors. Other studies may suggest that the profile of expression of genes related to the dopaminergic system, such as VMAT or DAT, in individuals with a-syn genetic abnormalities, can account for this susceptibility (Miller et al., 2007). Therefore, the genetic profile of PD patients should be explored more thoroughly with respect to other genes than a-syn or other genes that are impli­ cated in familial PD. The emergence of new con­ ditional transgenic models such as those using the CaM-tTA promoter (Lim et al., 2010; Marxreiter et al., 2009; Nuber et al., 2008) enables to deeply investigate the reversibility of the effects of a-syn pathology and thus focus on designing therapeu­ tic strategies aimed at regulation of a-syn expres­ sion levels at the early stages of the disease. A new generation of models using BAC trans­ genics methods to overexpress a-syn under its normal promoter should bring new information, as should models that mimic posttranslational mod­ ifications of the protein that may influence its pathological potential, such as phosphorylation and nitrosylation.

Genetic mouse models of recessive mutations causing PD The most frequent mutations causing early onset, recessive familial forms of PD occur in the parkin gene (Cookson et al., 2008). Parkin is an E3

ubiquitin ligase, thus suggesting an involvement of the proteasome pathway in PD. Several lines of parkin knockout (KO) mice have been generated by deleting either exon 3 or 7 in the parkin gene. Parkin KO mice show mild, progressive motor def­ icits, anxiety behaviors, alterations in dopamine release, abnormal responses to amphetamine, synaptic anomalies, mitochondrial damage, and increased oxidative stress; none of the available models, however, show a progressive loss of nigros­ triatal dopaminergic neurons (Goldberg et al., 2003; Itier et al., 2003; Martella et al., 2009; Palacino et al., 2004; Periquet et al., 2005; Rodr´ıgez-Navarro et al., 2007; Stichel et al., 2007; Von Coelln et al., 2004; Zhu et al., 2007). This was even the case in a triple transgenic model lacking all three genes (parkin, PINK1, and DJ-1) that cause recessive familial forms of PD (Kitada et al., 2009). One parkin KO line (Von Coelln et al., 2004) shows a loss of neu­ rons in the locus coeruleus but this phenotype is developmentally induced rather than progressive, as expected in a model of PD. Of concern, a model based on careful consideration of the genetic makeup of the KO mice failed to reveal any beha­ vioral deficits (Perez and Palmiter, 2005). How­ ever, a recent study shows that it is possible to induce dopaminergic cell loss in parkin KO mice by exposing them to LPS, an inflammatory stressor (Frank-Cannon et al., 2008). This clearly illustrates that this recessive mutation may make dopaminer­ gic neurons vulnerable to other insults, which can occur in vivo in patients but are usually absent when mice are raised under controlled laboratory conditions. Furthermore, a mice lacking parkin and overexpressing the Pael-receptor, a suspected parkin substrate, develops loss of dopaminergic neurons accompanied by marked endoplasmic reticulum (ER) stress (Wang et al., 2008). More recently, attention was drawn to the pos­ sibility that heterozygote carriers of parkin muta­ tions may be at increased risk to develop PD implying that partial loss of function or a gain of function in parkin may contribute to the disease (Pavese et al., 2009). One mouse model was gen­ erated to explore the possibility that a Q311X

79

mutation in parkin could exert dominant toxicity on nigrostriatal dopaminergic neurons. This hypothesis was supported by data in Drosophila showing a progressive loss of dopaminergic neu­ rons in flies expressing Q311X parkin (Sang et al., 2007). Mice expressing the mutation selectively in dopaminergic neurons by way of a DAT-BAC do show progressive loss of nigrostriatal dopaminer­ gic neuron and motor deficits, as well as a-syn pathology (Lu et al., 2009). These mice provide a novel model to study the mechanisms triggered by parkin mutations that lead to DA neuron death. Like parkin KO mice, PINK1 and DJ-1 KO mice failed to reproduce the canonical deficits in DA neurons observed in PD without additional insult. Unexpectedly for a model of PD, one line of DJ-1 KO mice shows loss of DA neurons in the ventral tegmental area accompanied by nonmotor behavioral deficits (Pham et al., 2009). Most PINK-1 and DJ-1 KO mice, however, show primarily hypoactivity, increased oxidative stress, and increased vulnerability to mitochondrial tox­ ins, without spontaneous loss of nigrostriatal dopaminergic neurons (Chen et al., 2005; Gautier et al., 2008; Gispert et al., 2009; Goldberg et al., 2005; Kim et al., 2005). In agreement with a role of DJ-1 in protecting dopaminergic neurons from oxidative stress, nigrostriatal dopaminergic cell loss can be induced in DJ-1 KO mice by feeding them a diet poor in the antioxidant selenium (Mortazavi et al., 2009). Overall, these data indi­ cate that the KO models of recessive PD-causing mutations are particularly useful to identify addi­ tional factors that may play a role in the patho­ physiology of PD in humans.

Genetic mouse models of LRRK2 mutations causing PD Mutations in LRRK2 in familial forms of PD have been identified relatively recently but have gener­ ated a lot of interests because heterozygote car­ riers of these dominant mutations can develop PD at a late age and with all the characteristics of the

sporadic forms of the disease, including the pre­ sence of a-syn pathology in brain (Cookson et al., 2008). Furthermore, LRRK2 being a kinase, the discovery of its involvement in PD raised the hope of finding a druggable target for neuroprotective therapies. The substrate(s) of LRRK2, however, remain elusive and the exact molecular mechan­ isms triggered by the mutations have not been fully identified. Several mutations have been iden­ tified in LRRK2, the most frequent being the G2019S mutation. However, only mice expressing one of two mutations at R1441 have been pub­ lished so far. Mice with the R1441G mutation develop a strong behavioral phenotype and defi­ cits in dopamine release (Li et al., 2009). Similar dopaminergic deficits, but not the prominent motor defects, were observed in a mouse with the R1441S mutation (Tong et al., 2009). Much work remains to be done in these and other LRRK2 models to realize their full potential for developing new PD treatments. Furthermore, a recent study (Lin et al., 2009) established a tanta­ lizing relationship between LRRK2 and a-syn in the pathophysiology of PD.

Other genetic mouse models of PD A number of other genetic mutations have been identified that cause a loss of dopaminergic neu­ rons and have been considered as useful models of PD. Notably, a lack of the transcription factor Pitx3 leads not only to a small eye and blindness (aphakia mice) but also to a complete loss of nigrostriatal dopaminergic neurons in early post­ natal development. This can hardly be considered a model of PD since it does not reproduce the late, progressive loss of dopaminergic neurons or the other pathological features of the disorder but it is useful to validate behavioral tests indicative of dopaminergic deficits (Hwang et al., 2005). In addition, some of the Pitx3 mice show interesting cognitive deficits, implicating nigrostriatal dopa­ minergic neurons in these functions (Ardayfio et al., 2008). Furthermore, the mice may mimic a

80

relevant pathophysiological mechanism because polymorphisms in Pitx3 have been associated to an increased risk of PD (Haubenberger et al., 2009) and this transcription factor may directly regulate VMAT and DAT expression (Hwang et al., 2009), two genes that have been associated with PD risk as well (Ritz et al., 2009). Genetic models have also been generated based on a mechanism thought to be involved in PD rather than a mutation known to cause the disease in humans. This is the case of the “mitomice”, a model that lacks mitochondrial transcription factor A (TFAM). These mice show a progressive loss of nigrostriatal dopaminergic neurons and related behavioral deficits, providing a model to assess new neuroprotective molecules (Harvey et al., 2008). A number of other genetic risk factors for PD are emerging from GWAS and other genetic stu­ dies. Recent GWAS consistently identified a-syn and the microtubule associate protein tau as most strongly associated with PD (Pankratz et al., 2009). This clearly raises the possibility that introducing mutations in tau may synergize with a-syn overexpression and other PD-causing mutations to more completely reproduce PD phenotypes in mice. Further analysis of the wealth of data generated by the GWAS will likely point in new directions for improving mouse models of PD. Other genetic risk factors are emerging from candidate gene approach and provide particularly interesting insights in new ways to model PD. Of note, some of these genetic risk factors have already been shown to modify the phenotype of established mouse models of PD. For example, polymorphisms in inflammatory genes are asso­ ciated with an increased risk of PD in humans and, as indicated early, chronic inflammation wor­ sen the phenotype of parkin KO mice (FrankCannon et al., 2008; Wahner et al., 2007). Of note, polymorphism in the DAT markedly increase the risk of PD in young onset patients who have been exposed to the agricultural pesti­ cides paraquat and maneb, compared to exposure alone (Ritz et al., 2009). The emerging evidence

for gene–environment interactions as risk factors for PD will generate exciting new avenues for modeling PD in genetic mouse models. In conclusion, although often decried as “imper­ fect”, the available genetic mouse models of PD have already created exciting new opportunities for studying the disorder in a mammalian brain. In order to choose wisely among existing models it is important to consider the question being explored. Whereas most models do not exhibit frank progres­ sive neuronal degeneration of nigrostriatal DA neurons, they often exhibit marked decreases in striatal DA, sometimes with loss of TH-positive terminals. Furthermore, many models exhibit increased sensitivity to other insults, then resulting in the canonical loss of nigrostriatal DA neurons. This may reproduce more faithfully the multiple hits affecting DA neurons during a lifetime in humans compared to the pristine environmental conditions to which laboratory mice are exposed. Importantly, models may provide new insights into PD pathophysiology, whether or not nigrostriatal cell loss occurs during the lifetime of the animal. The distinct advantage of genetic mouse models is that they are based on mechanisms known to cause or increase the risk of PD in humans. This makes them a lot more valuable to identify and test neuro­ protective strategies than the classical models based on the use of neurotoxins that may or may not reproduce steps involved in the pathophysiology of the human disease. The relevance of genetic models based on extremely rare mutations involved in familial rather than sporadic forms of PD remains an open question. Clearly, evidence for an involve­ ment of a-syn both in familial and in sporadic forms of PD is overwhelming. Models mimicking the accu­ mulation of this protein in neurons are particularly useful when they reproduce the broad distribution of pathology observed in human, thus leading to the expression of the non-motor symptoms that have traditionally been difficult to study in animal models. The extent of the phenotype in a-syn-overexpres­ sing mice, however, seems to depend on the level of overexpression of the protein, perhaps explaining why only models with high levels of expression

81

have robust and reproducible alterations in a wide range of behavior, as well as loss of nigrostriatal DA. New models mimicking posttranslational modifica­ tions in a-syn (truncation, phosphorylation) are likely to provide additional insights into the role of these modifications in the future. Finally, increasing knowledge of synergies between environmental and genetic risk factors in PD is emerging as a fruitful source of inspiration for a new generation of increas­ ingly relevant genetic mouse models of PD.

Abbreviations PD a-syn TH PrP PDGF DA LRRK DAT GWAS VMAT LPS

Parkinson’s disease alpha-synuclein tyrosine hydroxylase prion protein platelet-derived growth factor dopamine leucine repeat rich kinase dopamine transporter genome wide association studies vesicular monoamine transporter lipopolysaccharide

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 5

Viral vector-mediated overexpression of a-synuclein as a progressive model of Parkinson’s disease Ayse Ulusoy†, Mickael Decressac‡, Deniz Kirik† and Anders Björklund‡,
†

Brain Repair and Imaging in Neural Systems, Department of Experimental Medical Science, Lund University, Lund,

Sweden

‡ Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, Lund, Sweden

Abstract: The discovery of the role of a-synuclein in the pathogenesis of Parkinson’s disease (PD) has opened new possibilities for the development of more authentic models of Parkinson’s disease. Recombinant adeno-associated virus (AAV) and lentivirus (LV) vectors are efficient tools for expression of genes locally in subsets of neurons in the brain and can be used to express human wildtype or mutated a-synuclein selectively in midbrain dopamine neurons. Using this approach, it is possible to trigger extensive PD-like cellular and axonal pathologies in the nigrostriatal projection, involving abnormal protein aggregation, neuronal dysfunction, and cell death that develop progressively over time. Targeted overexpression of human a-synuclein in midbrain dopamine neurons, using AAV vectors, reproduces many of the characteristic features of the human disease and provides, for the first time, a model of progressive PD that can be applied to both rodents and primates. Keywords: Synuclein; Dopamine; Nigrostriatal system; Motor impairment; Adeno-associated virus; Lentivirus; Viral vectors; Animal models

(MPTP), or rotenone. Although highly useful, these models have a limitation in that they address only one element of the underlying neuropatholo­ gical changes, i.e., free radical damage and the associated mitochondrial dysfunction. Moreover, the neurotoxin models are essentially non-progres­ sive and do not replicate all aspects of the disease. There is, therefore, a strong need to develop new animal models that more faithfully reproduce

the progressive neuropathological changes seen in

Introduction The commonly used animal models of Parkinson’s disease (PD) are based on systemic or local administration of neurotoxins, such as 6-hydroxydopamine (6-OHDA), 1-methyl-1,2,3,4-tetrahydropyridine
Corresponding author.

Tel.: þ46-46-2220540; Fax: þ46-46-2220559; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84005-1

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idiopathic PD. The discovery of a-synuclein (a-syn), in 1997, as a key player in the pathogenesis of familial PD (Polymeropoulos et al., 1997), and as a major component of the characteristic protein inclusions—Lewy bodies and dystrophic neur­ ites—that develop over time in the brains of PD patients (Spillantini et al., 1997), has opened new interesting possibilities to replicate PD-like changes in animals. The new models that have been developed over the last years are based on overexpression of human wild-type or mutated a-syn using either transgenic techniques (trans­ genic flies and mice; reviewed in the chapter by Magen and Chesselet in this volume), or local intracerebral injections of viral vectors carrying the a-syn gene. A large number of transgenic mouse lines overexpressing a-syn have been generated during the last decade. Although very interesting as models of more generalized synucleinopathy, none of the transgenic mouse lines have so far reproduced the prominent, progressive neurodegenerative changes that are the hallmarks of human PD. In this regard, overexpression of a-syn by viral vector delivery offers a valuable alternative approach. Two vector systems have been explored for this purpose: recombinant adeno-associated virus (AAV) and recombinant lentivirus (LV). These vectors transduce neurons in the adult brain with high efficiency, and the expression of the inserted genes is long-lasting, over many years. These tools are also attractive in that they can be used for gene delivery in a broad range of animal species, including mice, rats, pigs, and monkeys. The viral vector approach was initially explored in rats using injections of AAV or LV vectors encoding either wild-type or mutant human a-syn unilaterally into the substantia nigra (SN) (Kirik et al., 2002; Klein et al., 2002; Lo Bianco et al., 2002). In these studies a-syn was efficiently encoding in the nigral dopamine (DA) neurons, accompanied by cellular and axonal pathologies and DA neuron cell loss that developed progres­ sively over time. Subsequently, the AAV vectors, in particular, have been used with similar success

also in mice (Theodore et al., 2008; St Martin et al., 2007) and marmosets (Eslamboli et al., 2007; Kirik et al., 2003). These studies show that targeted overexpression of human a-syn in mid­ brain DA neurons, using AAV vectors, reproduces many of the characteristic features of the human disease and provides for the first time a model of progressive PD that can be applied to both rodents and primates. In this chapter we provide an overview of the results obtained so far using this disease modeling approach, and the mechan­ isms most likely to be involved in the development of neuronal dysfunction and neurodegeneration in the AAV-a-syn model; in addition, we discuss some of the key technical aspects and pitfalls in the use of AAV vectors for this purpose.

Induction of PD-like neuropathological changes by AAV-mediated overexpression of a-syn AAV vectors are particularly useful for targeted gene delivery in the rodent brain. They have a high tropism for midbrain DA neurons, and since they can be produced in high titers, efficient trans­ duction can also be obtained with small injection volumes. In rats, a single 2–3 ml injection of vectors of the AAV2 serotype has been shown to transduce the nigral tyrosine hydroxylase (TH)­ positive neurons with 80–90% efficiency within the transduced area (Kirik et al., 2002; Yamada et al., 2004). Other AAV serotypes, notably AAV2/5 and AAV2/6 (i.e., generated with a transfer plasmid carrying AAV2-inverted term­ inal repeats packaged in an AAV5 or 6 capsid), have been used with similar success (Azeredo da Silveira et al., 2009; Gorbatyuk et al., 2008; Sanchez-Guajardo et al., 2010; own unpublished observations). The transgene expression in mid­ brain DA neurons achieved with LV vectors is clearly lower, usually no more than 50%. The extent of DA neuron cell loss obtained LVmediated overexpression of wild-type or mutated a-syn is also less pronounced, typically between 25 and 35% (Lauwers et al., 2003, 2007; Lo Bianco

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et al., 2002), as compared an average of to 50–60% seen in rats treated with AAV vectors (see below). Cell death In the rat AAV-�-syn model the average loss of TH-positive neurons, as reported in several stu­ dies from independent laboratories using various serotypes of AAV, is relatively consistent and ranges between 50 and 60% reduction in the TH-positive cell numbers in the SN (Chung et al., 2009; Gorbatyuk et al., 2008; Kirik et al., 2002, 2003; Maingay et al., 2006; Yamada et al., 2004, 2005), accompanied by a similar level of THpositive innervation in the striatal target areas (Fig. 1). The time course of cell loss, however, has been variable: from 8 weeks in the Kirik et al. (2002) and Gorbatyuk et al. (2008) studies, to 3–4 months in the Yamada et al. (2004) and Chung et al. (2009) studies. This difference may, at least in part, be due to the level of a-syn expres­ sion obtained with the different vector constructs (the strong expression from the synthetic chicken b-actin (CBA) promoter in the first two studies, as compared with the cytomegalovirus (CMV) and synapsin promoters in the two latter studies). Gor­ batyuk et al. (2008) have reported that the level of expression of human wild-type or mutated a-syn in the SN was about fourfold higher than the endogenous a-syn in rats injected with the CBAdriven vector construct. Similar data are not avail­ able for the alternative vector constructs, but the expression levels obtained is likely to be lower with the CMV and synapsin promoters. In rats, the cellular pathological and neurode­ generative changes induced by intranigral AAVa-syn are associated with the development of motor impairments (Eslamboli et al., 2007; Kirik et al., 2002; Maingay et al., 2006; Yamada et al., 2005). As predicted from the highly variable cell loss (30–80% at 2 months and beyond), overt behavioral impairments were seen only in a subset of the AAV-a-syn-treated animals (~25% in the experiments in rats carried out in our center)

(Fig. 2). Interestingly, the impact of a-syn overexpression seems to be different between the two major DA neuron subtypes, leading to prominent cell loss in the A9 cells of the SN, while the A10 cells in the adjacent ventral tegmental area (VTA) survive, despite similar levels of a-syn expression (Maingay et al., 2006). Signs of a-syn toxicity, including the formation of a-syn-positive inclu­ sions and dystrophic neurites, also developed in the transduced VTA neurons, but this did not seem to affect the survival of the cells in the same way as in the nigral cells. In mice, the nigral cell loss obtained with AAVa-syn vectors is clearly less pronounced. In the studies performed to date, the loss of TH-positive neurons in the SN, as observed at 2–3 months after injection, is in the order of 20–25% (St Martin et al., 2007). This is in line with our own unpub­ lished data. Consistent with this level of cell loss, the treated mice have in our hands shown no or only subtle behavioral impairments. Also in mice, the VTA neurons seem to be less affected.

Axonal pathology AAV-mediated overexpression of human a-syn provides an interesting model for progressive PD-like cellular pathology, including the forma­ tion of a-syn-positive cytoplasmic inclusions and prominent axonal pathology (Fig.1e). The axonal changes develop early after vector injection, pre­ cede DA neuron cell loss and persist in the stria­ tum over many months (Chung et al., 2009; Eslamboli et al., 2007; Kirik et al., 2002, 2003; Klein et al., 2002; Maingay et al., 2006; St Martin et al., 2007; Yamada et al., 2004, 2005). In surviv­ ing nigral neurons that express the human a-syn transgene, the denritic projections in the SN pars reticulata are truncated with distorted morphol­ ogy, and in the projection areas in the striatum the pre-terminal axons display swollen and dis­ torted profiles (Fig. 1g), filled with a-syn-positive aggregates (Fig. 1e). In addition there is a notable

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A

B E

D

G

F

Str

TH C

SN

NSP

cx

Str

α-syn

VTA

S SN

NAc

OT

SN

D

Striatum

TH

VTA E

α-syn Striatum

TH

F

Nigra

G

Nigra

SNc

SNr

TH

TH

Fig. 1. Overexpression of human wild-type a-syn in the rat midbrain leads to PD-like neurodegeneration in the nigrostriatal pathway (NSP), 8 weeks after injection of a single deposit of an AAV6-a-syn vector into the right substantia nigra (SN). Immunohistochemical staining against TH shows reduced DAergic innervation in the striatum in the injected side (a, e) compared to the intact side (a, d). Intra-cytoplasmic inclusions immunoreactive for TH are present in DAeric dystrophic terminals (see arrow heads in panel e), and a substantial loss of TH-positive neurons in the SN on the injected side (a, g) compared to the contralateral side (d), while the A10 neurons in the ventral tegmental area (VTA) are largely spared. Immunohistochemical staining against a-syn (using an antibody specific for human a-syn) shows overexpression of a-syn in the nigrostriatal system; in the midbrain DA neurons, along the NSP and in the axonal terminals in the striatum (Str) (b, c). Asterisk in panel b marks the injection site. Cx, cortex; NAc, nucleus accumbens; OT, olfactory tubercle; SNc, SN pars compacta; SNr, SN pars reticulata.

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Net ipsilateral turns

15

B

5 0

60 50 40 30 20 10

–5

2 4–5 Months after vector injection

C

70

GFP α-syn

10

Cylinder test

No. of steps

Amphetamine-induced rotation

Left paw use (%)

A

0

2 4–5 Months after vector injection

Stepping test 18 16 14 12 10 8 6 4 2 0

2 4–5 Months after vector injection

Fig. 2. Changes in motor behavior in rats given unilateral intranigral injections of either AAV6-a-syn or AAV6-GFP (n = 8 in each group). Although the mean performance of the AAV6-a-syn-injected animals did not differ from the AAV6-GFP-injected ones in either the amphetamine-induced rotation test (a), the cylinder test (b), and the stepping test (c), clear impairments were observed in a subset of a-syn-overexpressing animals. This effect was most pronounced in forelimb use in the cylinder test, where half of the animals showed impairment in the 4–5-month test. Dotted lines in b and c indicate level of performance in normal controls.

loss of fine-caliber TH-positive terminals in the target regions of the affected nigra neurons (Fig. 1a–c). The morphology of these a-syn-containing dys­ trophic axons, which is remarkably similar to those observed in brains from PD patients (Braak and Braak, 2000; Galvin et al., 1999), seems compatible with intra-axonal buildup due to impaired axonal transport (Saha et al., 2004). Indeed, in a recent study, Chung et al. (2009) have shown that the appearance of dystrophic axonal swellings along the nigrostriatal projection is accompanied by altered levels of proteins involved in axonal transport and vesicle exocyto­ sis, pointing to an early and persistent impairment of both axonal transport and synaptic function.

Immune and inflammatory changes AAV-mediated overexpression of either wildtype or A53T mutant a-syn in midbrain DA neu­ rons induces an early and persistent immune/ inflammatory reaction similar to what has been reported in PD patients (Chung et al., 2009; Sanchez-Guajardo et al., 2010; Theodore et al.,

2008). These changes are characterized by an early activation of microglia, expression of neu­ roinflammatory markers and infiltration of lym­ phocytes at a time when a-syn is fully expressed (4 weeks), but before any significant cell loss has occurred. Sanchez-Guajardo et al. (2010) have described two distict patterns of inflammatory/ immune changes depending on the extent of cell death: (1) an early, transient change in the number of activated microglia and long-lasting major histo­ compatibility complex (MHC) II expression associated with persistent a-syn-induced neuro­ pathology, but no cell death; and (2) a second, more protracted microglia response correlated with long-lasting CD68 expression and infiltration of CD4þ and CD8þ lymphocytes, which is seen in cases where a-syn has induced both pathological changes and cell death. At the level of the striatum, the development of prominent a-syn-induced axo­ nal pathology is associated with the appearance of activated microglia, increased MHC II expression, and elevated levels of pro-inflammatory cytokines (interleukin 1b, interferone g and tumor necrosis factor a) (Chung et al., 2009; Sanchez-Guajardo et al., 2010) (Fig. 3). These data show that overexpression of human wild-type or mutant a-syn, in

94 Microglia

Oxidative stress α-syn Aggregation Pro-inflammatory factors (IL1, IL6, TNFα, Chemokines) Toxic intermediates

DA

Astrocytes

Fig. 3. A growing body of evidence suggests that the presence of ongoing inflammation may contribute and hasten the progression of PD. This is supported by evidence of activated microglia, accumulation of cytokines, oxidative damage in post-mortem PD brains as well as the increased expression of genes encoding pro-inflammatory cytokines in the SN. In parallel, post-mortem analysis has shown the presence of a-syn inclusions in astrocytes in the striatum of PD patients. Mice overexpressing human a-syn display increased microglial burden and higher levels of inflammatory cytokines prior to cell loss. This inflammatory environment represents a source of oxidative stress to which DA neurons are particularly susceptible. In addition, recent findings suggest a direct transfer of a-syn from DA neurons to astrocytes, leading to the production of pro-inflammatory mediators. In this model, astrocytes and microglia act as key players in the induction of oxidative stress and the production of toxic a-syn intermediates in the a-syn-overexpressing DAergic neurons, probably acting on both the cell body and the axon terminal level.

the absence of overt neuronal cell death, is sufficient to trigger a sustained neuroinflammatory response, including both microglial activation and adaptive immunity, similar to that seen in progressive PD in patients. In humans the presence of ongoing inflammation may contribute to neurodegeneration and drive the progression of PD. This is supported by evidence of activated microglia and accumulation of cytokines, in post-mortem PD

brains (McGeer and McGeer, 2008), and by experi­ ments in transgenic mice showing that the induc­ tion of a neuroinflammatory response (by local injection of lipopolysaccharide (LPS)) is sufficient to induce a-syn aggegation, cytoplasmic inclusions, and cell death in nigral DA neurons (Gao et al., 2008). In addition, a microarray study has revealed increased expression of genes encoding pro-inflam­ matory cytokines in the SN (Duke et al., 2007) and

95

positron emission tomography (PET) imaging studies have demonstrated increased microglial activation in various brain regions, including the striatum, of patients with idiopathic PD (Gerhard et al., 2006). Enhancement of a-syn toxicity by posttranslational modifications As illustrated schematically in Fig. 4, the damage caused by a-syn overexpression is likely to depend

on the formation of toxic intermediates (oligomeres or protofibrils) that may overwhelm or bypass the ubiquitin-proteasome system/lysosomal degrada­ tion system. The formation of these toxic a-syn intermediates is promoted by oxidative damage and disease causing genetic mutations. In addition, post-translational modifications of a-syn have been implicated in this process. AAVmediated overexpression of post-translationally modified forms of a-syn has been used to generate improved models of PD and to further investigate

Healthy cell

Affected cell

α-Synuclein

UPS/Lysosomal degradation

Dopamine

Oxidative damage Dopamine Post-translational modifications

Toxic intermediates (oligomers/proto-fibrils)

?

Non-toxic fragments

Insoluble inclusions

Dopamine neuron death

Fig. 4. Cellular mechanisms of a-syn-mediated DAergic cell death. In a healthy cell where the levels are within physiological range, a-syn can be degraded via the intracellular degradation pathways (i.e., chaperone-mediated autophagy, macroautophagy or the ubiquitin-proteasome system (UPS)). a-Syn protein is prone to aggregate and form intermediate species under stress conditions (i.e., oxidative and nitrosative stress), or by post-translational modifications. Moreover, a-syn is known to interact with DA or its toxic metabolites leading to the formation of oligomeres or protofibrils. These intermediates are suggested to be the toxic to the cell, unless they are degraded by the endogenous pathways or neutralized by forming insoluble fibrillar inclusions. A widely accepted view postulates that the formation of insoluble aggregates is part of the survival mechanism rather than the cause of neuronal death. In the AAV-a-syn model, where the levels of a-syn in nigral DA neurons are elevated to levels severalfold above normal, increased formation of toxic intermediates is proposed to be a key player in the induction of the inflammatory response (as illustrated in Fig. 3), the formation of inclusions and aggregates (as shown in Fig. 1), and cell death.

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the mechanisms underlying a-syn-mediated neuro­ degeneration. Most of the a-syn located in Lewy bodies in PD brains and related synucleinopathies, and in the aggregates formed in transgenic models of PD, is phosphorylated at serine residue at posi­ tion 129 (S129) (Anderson et al., 2006; Fujiwara et al., 2002; Hasegawa et al., 2002; Kahle et al., 2000; Neumann et al., 2002; Takahashi et al., 2003), and this is the case also in the a-syn-positive aggregates formed in the AAV-a-syn model (Yamada et al., 2004). Although the presence of phosphorylated a-syn in pathological accumula­ tions suggests that S129-phosphorylated a-syn is closely linked to PD pathology, the exact mechan­ ism by which phosphorylation at S129 modulates a-syn aggregation and toxicity in vivo is not clear. Studies in Drosophila have shown that blocking the phosphorylation at S129 residue of the a-syn pro­ tein, using S129A mutant a-syn that cannot be phosphorylated, suppresses DAergic neurodegen­ eration. Whereas when the phosphorylation mimic S129D mutation was introduced, the neurotoxicity was enhanced (Chen and Feany, 2005). In order to gain a better understanding of the role of phosphorylation in a-syn toxicity and aggregation in rodents, several groups have used AAV vectors to overexpress S129D mutant forms of a-syn in the rat midbrain DAergic neurons. Interestingly, how­ ever, AAV-mediated expression of S129D phosphomimic a-syn in rats led to similar or lower levels of toxicity in the SN as compared to the AAVmediated S129A a-syn expression (Azeredo da Silveira et al., 2009; Gorbatyuk et al., 2008; McFarland et al., 2009). At this point, as these site-specific mutations do not replicate the biolo­ gical consequences of the actual phosphorylation event on the natural serine residue, the role of S129 phosphorylation in the induction of a-syn aggregation and neurotoxicity remains controver­ sial. Recently, a-syn phosphorylation at S87 has been documented in post-mortem analysis of synucleinopathy patient brains, as well as in brains of a-syn overexpressing transgenic mice (Mbefo et al., 2010). Interestingly, when micelle-bound a-syn is phosphorylated at S87, it underwent a

conformational change and represented a lower affinity to lipid vesicles, suggesting that phosphor­ ylation of this site might have a role in interactions with other molecules rather than the aggregation process. Further studies are warranted to clarify the effects of phosphorylation of a-syn in the mechanism of neurodegeneration in vivo. Besides phosphorylation, many other post-trans­ lational modifications, such as oxidation, C-term­ inal truncation, ubiquitination, and nitration, have been implicated in the mediation of a-syn aggrega­ tion and toxicity. Using transgenic mouse models several groups have shown that truncated a-syn can lead to increased DAergic dysfunction and pathology (Daher et al., 2009; Tofaris et al., 2006; Wakamatsu et al., 2008), and evidence from in vitro studies suggests that the C-terminal-truncated a-syn is more prone to aggregate and that it can promote aggregation of full-length a-syn (Crowther et al., 1998; Liu et al., 2005). In line with these observations, we have shown that AAV-mediated co-expression of wild-type human full-length a-syn and C-terminal-truncated a-syn in the rat SN promotes the accumulation of patholo­ gical full-length a-syn protein and leads to increased DAergic cell death, suggesting that changes in truncation of a-syn can be responsible from the pathogenesis and progression of PD (Ulusoy et al., 2010). Mechanisms of a-syn toxicity in DA neurons The results obtained in the AAV-a-syn model show that overexpression of human a-syn can induce a-syn-positive cytoplasmic and axonal inclusions and progressive neurodegenerative pathological changes in midbrain DAergic neu­ rons. Our own observations suggest that these degenerative changes are most prominently expressed in the nigral DA neurons and were not seen in any of the non-DAergic neuron sys­ tems that were efficiently transduced by the same AAV-a-syn vector (Kirik et al., 2002). The impact of a-syn overexpression in midbrain DA neurons

97

Dysfunctional terminal

Tyrosine α-syn α-syn

PP2A

DOPA

TH

14-3-3 TH inactive

VMAT2

DA

α-syn

AADC

α-syn α-syn

DAT α-syn

Fig. 5. a-Syn interacts with enzymes involved in DA synthesis, including TH, 14-3-3, PP2A, and AADC, thus taking part in the regulation of the production of the neurotransmitter. DA storage, release and uptake are also influenced by a-syn. Based on this model, overexpression of a-syn in midbrain DA neurons, as seen at early time points in the AAV-a-syn model, will lead to a state of impaired DA neurotransmission due to reduced synthesis and storage of the transmitter and impaired synaptic DA release. The subsequent formation of a-syn aggregates, on the other hand, may lead to an impairment of normal a-syn function in the cell, which in turn may result in the synthesis of larger amount of DA and a reduction in the storage capacity and a leakage of DA from the synaptic vesicles. In addition, reduced bioavailability of a-syn at the synapse could promote the presence of the DAT at the membrane and increase its efficacy for DA reuptake. Taken together, this may induce a dysfunctional state, leading to an increased concentration of free cytosolic DA and a source of detrimental oxidative stress.

is twofold: a suppression of DA synthesis and storage (as reflected in reduced TH enzymcl activity and striatal DA levels) at a stage when a-syn has a diffuse cytoplasmic distribution, followed by the appearance of cytoplasmic inclusions, dystrophic neuritis, and cell death. These data point to interesting interactions between a-syn and DA, in the maintenance of DA neurotransmission, in the regulation of DA homeostasis at the synapse level, and in the formation of toxic a-syn derivatives and cell death. In the AAVa-syn model these three mechanisms, in combination with the a-syn-induced inflammatory response (see above), may interact to induce the progressive pathological changes that develop over time in the transduced DAergic neurons. Effects on DA synthesis In DAergic neurons, the regulation of DA synthesis, storage, release, and re-uptake has been shown to depend, at least in part, on the function of a-syn (Fig. 5). There is considerable evidence that the

activity of the TH enzyme is modulated by a-syn, in particular by regulation of its phosphorylation state. In this line, a reciprocal interplay between a-syn and 14-3-3 proteins has been suggested for the regulation of TH activity (Xu et al., 2002). The chaperone 14-3-3 protein binds to the active phos­ phorylated form of TH and is required for maximal phosphorylation of the enzyme (Ichimura et al., 1987, 1988). Interaction between 14-3-3 protein and TH maintains the latter in its active form by protecting it from dephosphorylation and increases its half-life in DA neurons (Toska et al., 2002). Conversely, it has been demonstrated that a-syn co-localizes with and binds directly to TH with the functional consequence of a reduced enzymatic activity and a decrease in DA synthesis. Indeed, a-syn has the property to bind to TH in its dephosphorylated form and thereby maintains the enzyme in its inactive form (Alerte et al., 2008; Perez et al., 2002). In addition, it was shown that a-syn might also regulate TH activity indirectly by acting on protein phosphatase 2A (PP2A). Overexpression of a-syn in DA cells leads to unaltered expression of PP2A, while the activity of the

98

phosphatase is increased in parallel with a-syn expression (Peng et al., 2005; Perez et al., 2002). This suggests that the inactivation of TH seen at early time points in AAV-a-syn-transduced DA neurons (Kirik et al., 2002) may be due to depho­ sphorylation of the TH enzyme induced by PP2A. Effects on DA storage and release Different studies support that a-syn is involved in synaptic vesicle recycling, DA storage and release in the presynaptic terminal by maintaining a reserve pool of synaptic vesicles. This function is suggested by the observation that the synaptic response to paired stimulus depression of DA release, which is capable of depleting the release pool of vesicles, was impaired in a-syn knockout mice, and the time for replenishing docked vesi­ cles from reserve pool was slower in these trans­ genic animals (Abeliovich et al., 2000). Further evidence in support of a role for a-syn in vesicular recycling and transmitter release comes from in vitro studies of hippocampal neurons (Cabin et al., 2002) and PC12 and chromaffin cells (Larsen et al., 2006), possibly mediated via an interaction with phospholipase D2, which is an important regulator of synaptic vesicle recycling (Payton et al., 2004). In support, Nemani and colleagues (2010) have reported that even modest non-toxic overexpression of a-syn, similar to what was found in PD patients with a triplication of the a-syn gene locus (Miller et al., 2004), has an inhi­ bitory effect on neurotransmitter release. a-Syn has been implicated also in the regulation of DA storage into synaptic vesicles. This process is critical as the low pH environment within the vesi­ cle stabilizes DA and therefore prevents its oxida­ tion and the formation of toxic reactive intermediates in the cytoplasm. Volles and collea­ gues have shown that a-syn aggregates can per­ meabilize synaptic vesicles and postulated that this caused cytosolic leakage of DA (Volles and Lansbury, 2002; Volles et al., 2001). In addition, an in vitro study has provided evidence that the

A53T mutant, but not wild-type a-syn, can modu­ late the vesicular monoamine transporter-2 (VMAT2) (Lotharius and Brundin, 2002; Lotharius et al., 2002). LV-induced overexpression of A53T mutant a-syn in an immortalized human mesence­ phalic cell line (MESC2.10) resulted in downregulation of VMAT2, decreased potassiuminduced and increased amphetamine-induced DA release, enhanced cytoplasmic DA and increased intracellular levels of superoxide. This finding is in line with a reduction of VMAT2 that was observed by PET imaging in the brain of PD patients (Frey et al., 1996). Effects on DA re-uptake The DA transporter (DAT) plays a major role in the regulation of the synaptic content of DA and therefore is a key component of DA neurotrans­ mission. Thus, enhanced activity or expression of DAT on the plasma membrane will result in an increase in intracellular levels of DA and an increased risk for oxidative damage to DA neurons. In vitro studies have shown that DA uptake is modulated by a-syn expression. Under basal con­ ditions, a-syn appears to be a negative regulator of the DAT function due to a decrease in DA uptake velocity, while no changes in the affinity for the neurotransmitter or the level of expression were observed (Adamczyk et al., 2006; Wersinger et al., 2003). Co-immunoprecipitation studies revealed that this effect results from a direct inter­ action between the non-amyloid component domain of a-syn and the last 22 amino acids of the C-terminal tail of the DAT (Lee et al., 2001; Wersinger et al., 2003). Due to its ability to bind to membrane lipids a­ syn can also affect the trafficking of DAT by sequestrating the transporter away from the plasma membrane, limiting the re-uptake of released DA (Lee et al., 2001; Wersinger and Sidhu, 2003). Wersinger and Sidhu (2005) have observed that disruption of the interaction

99

between a-syn and microtubules, using microtu­ bule-destabilizing agents, leads to an enhanced cell surface recruitment of the DAT, suggesting that the regulation of DAT activity by a-syn is due, at least in part, to its ability to tether the transporter to the microtubular network. Together, these studies suggest a model of a-syn-mediated pathogenesis linked to aberrant regulation of DAT function, leading to increased cytoplasmic levels of DA. In support of this idea Wersinger et al. (2003) have observed that the attenuation of DAT function by a-syn has a pro­ tective effect on DA-mediated oxidative stress and cell death. Dopamine-dependent toxicity Aggregation of the a-syn protein is a characteris­ tic feature of PD, but the consequence of this process on cell survival and function remains unclear. In vitro studies have shown that, at high concentration, monomers of a-syn are prone to self-aggregate into insoluble fibrils via an oligo­ meric state (Volles and Lansbury, 2002), and that the disease-related mutant variants (A53T and A30P) of a-syn are prone to aggregate faster than the wild-type form (Conway et al., 2000). Although monomers of a-syn can self-aggregate, various intracellular factors, among them the interaction with DA, oxidative stress, and post­ translational modifications, have been shown to speed up this process (Fig. 4). In support, Xu and colleagues showed that accumulation of a-syn in cultured human DA neurons led to cell death that required DA synthesis and was driven by reactive oxygen species (Xu et al., 2001). Overexpression of a-syn in non-DAergic human cortical neurons, by contrast, had no toxic effect, but rather exhib­ ited neuroprotective property. Oxidative stress has been shown to play an important role in the facilitation of a-syn aggrega­ tion. DA neurons may be particularly susceptible since the free radical production is high in these types of cells, due to their content of both DA and

iron. Interestingly, a-syn protein is itself a target for oxidative damage. Nitration of the protein by reactive oxygen and nitrogen species has been shown to produce toxic intermediates that are prone to aggregate (Giasson et al., 2000). Indeed, in the presence of compounds generating free radicals, cells overexpressing a-syn produce intra-cytoplasmic inclusions of ubiquitinated a-syn (Hashimoto and Masliah, 1999; OstrerovaGolts et al., 2000), and administration of the mito­ chondrial inhibitor rotenone (which causes the generation of reactive oxygen species) has been reported to lead to the formation of a-syn aggre­ gates and the loss of DA neurons in rats (Sherer et al., 2003). In the AAV-a-syn overexpression model it is likely that cytoplasmic DA plays a role in the induction of toxic damage. Free cytosolic DA can interact with a-syn in the generation of toxic species and thus contribute to the susceptibility of a-syn to aggregate in vivo. As described above, this may be further aggravated by the neuroin­ flammatory response induced by toxic a-syn monomers (see Fig. 3), leading to microglial acti­ vation and increased production of reactive oxy­ gen species, such as microglia-derived nitric oxide and superoxide (Gao et al., 2008). It has been shown that DA binds to two different parts of the a-syn protein, including the non-amyloid com­ ponent region (Herrera et al., 2008). Depending on the state of aggregation, this results either in the inhibition of a-syn fibrillation or in the disag­ gregation of fibrils into oligomers that are likely to be the most toxic species (Cappai et al., 2005; Conway et al., 2001; Herrera et al., 2008; Li et al., 2004; Mazzulli et al., 2006; Norris et al., 2005).

Studies on neuroprotection in the AAV-a-syn model Mutations in the parkin gene has been linked to one of the most common causes of familial Par­ kinson’s disease, and it has been suggested that

100

this protein, which is a ubiquitin-proteasome E3 ligase enzyme, may play a role in the defense of the cell against a-syn toxicity. Based on this idea several studies have been performed to explore the possible neuroprotective properties of parkin in the a-syn overexpression model (Lo Bianco et al., 2004a; Yamada et al., 2005; Yasuda et al., 2007). According to Lo Bianco and colleagues LV-mediated overexpression of parkin reduces nigral TH neuron loss, induced by a-syn overex­ pression, from 31 to 9% when cotransfected in the midbrain DAergic neurons in rats (Lo Bianco et al., 2004b). In a similar study, performed with AAV vectors to co-express a-syn and parkin, Yamada et al. (2005) demonstrated protection from a-syn-induced DAergic cell loss, from about 50 to 20%, and prevention of the develop­ ment of motor impairments (Yamada et al., 2005). When tested in the 6-OHDA model of PD, on the other hand, AAV-mediated overexpression of parkin in rat SN led to improvement of motor functions in the 6-OHDA-lesioned rats, but with­ out detectable neuroprotection, as observed at 12 weeks after lesion (Manfredsson et al., 2007). In another study, Verkammen et al. (2006) reported that LV-mediated gene transfer of parkin in the rat 6-OHDA lesion model resulted in a significant (~80%) neuroprotection at 3 weeks after the lesion, although the protective effect of parkin diminished to about 5–10% at later time points. Glial cell-line-derived neurotrophic factor, GDNF, and its functional analogue neurturin, have been shown to provide functional recovery and nigral DA neuron protection in rodent and primate toxin models of PD (Eslamboli et al., 2005; Georgievska et al., 2002; Hoffer et al., 1994; Horger et al., 1998; Kearns et al., 1997; Kirik et al., 2001; Kordower et al., 2000, 2006; Rosenblad et al., 1999, 2003; Tomac et al., 1995). Several clinical studies, performed either by direct delivery of recombinant GDNF protein into the brain or by AAV-mediated overexpression of neurturin into the putamen, have so far failed to show convincing evidence of neuroprotection in PD patients (Ceregene, 2009; Gill et al., 2003; Manning-Bog

et al., 2006; Nutt et al., 2003). Although there is solid evidence that GDNF can act as an efficient neuroprotective agent in the 6-OHDA or MPTP lesion models, the two studies that have been per­ formed in the rat AAV-a-syn overexpression model have so far given negative results (Lo Bianco et al., 2004a; own unpublished observa­ tions). This discrepancy of results obtained in the toxin and AAV-a-syn models is a concern and raises critical questions regarding the predictive value of the toxin models currently used for testing of novel neuroprotective strategies. Use of AAV vectors for overexpression of a-syn in non-dopaminergic systems A particular advantage of AAV vector delivery is that it makes it possible to target a-syn overex­ pression to selected brain structures, also outside the SN. The non-motor symptoms of PD, such as cognitive decline, mood disturbances, olfactory deficits, and balance and sleep disturbances, are likely to reflect, at least in part, the involvement of brain structures outside the midbrain DAergic system. Neuro-pathological studies of the distribu­ tion of a-syn-positive Lewy bodies and Lewy neurites in PD patients point to a progressive involvement of structures outside the nigrostriatal system, including serotonin neurons of the raphe nuclei, noradrenergic neurons of the locus coeru­ leus, cholinergic neurons of the basal forebrain, and ultimately also multiple forebrain, olfactory and cortical areas (Braak et al., 2003). In the attempts to model PD made so far, the AAVa-syn vector injections have been targeted to the midbrain DA neurons alone. In future studies, however, a combination of nigral AAV-a-syn injections and overexpression of a-syn in selected non-DAeric structures, such as the serotonergic, noradrenergic and cholinergic systems, may be used to model a wider range of PD-related patho­ logical changes. In particular, it will be interesting to explore the impact of a-syn pathology in corti­ cal and/or basal forebrain cholinergic neurons on

101

AAV6-α-syn

A

B

AAV6-α-syn

Cx

cc Str

α-syn B C

AAV6-α-syn

C α-syn D

AAV5-GFP Sp

Thal

α-syn

GFP Fig. 6. Overexpression of human wild-type a-syn in neocortex (a–c) and septum (d). Unilateral injection of AAV6-a-syn in sensorimotor cortex results in a good spread of the vector, as illustrated by the strong expression of the protein in the cortical region around the injection site, and transport of a-syn along the corticostriatal and corticothalamic pathways (a). a-Syn­ immunopositive fibers (visualized with an antibody specific for human a-syn) are found in the contralateral cortex (a), the corpus callosum (b), the ipsi- and contralateral striatum (a, c), and the ipsi- and contralateral thalamus (a). Panel d shows the efficient transduction of neurons in the septum-diagonal band area obtained with an AAV-GFP vector of serotype 5. Efficient transport of the GFP protein was observed along the septo-hippocampal pathway and its terminals in the hippocampus (not shown). Inset in panel d shows transduced medial septal neurons in higher magnification. CC, corpus callosum; Cx, cortex; Sp, Septum; Thal, thalamus.

cognitive functions and the effect of a-syn overexpression in locus coeruleus and raphe nuclei on non-motor aspects of behavior, such as mood and sleep. In preliminary experiments (in part unpublished) we have seen that a-syn can be very

effectively expressed in cortical projection neu­ rons by intracortical injection of an AAV6-a-syn (Fig. 6a–c). Similarly, we have found that neurons located in the medial septum-diagonal band region or striatum can be efficiently transduced with AAV5 vectors (Fig. 6d–f), illustrating the

102

versatility of AAV vector delivery for modeling multiple aspects of neurodegenerative diseases in vivo. It should be kept in mind, however, that dif­ ferent serotypes of AAV vectors have different propensity to transduce neurons in different brain regions. Our own observations indicate that the AAV5 serotype is considerably more efficient in transducing rat cortical and hippocampal pyramidal neurons than the AAV2 serotype (Kirik and Bjork­ lund, 2003), and comparisons made after vector injection in the rat and monkey striatum indicate that the AAV2 vector is less efficient in transducing striatal neurons than either the AAV1, AAV5, or AAV8 serotypes (Davidson et al., 2000; Dodiya et al., 2010; Reimsnider et al., 2007). The volume of tissue transduced by single deposits of the AAV2 vector is also much less than that for the other serotypes tested due to more limited diffusion of AAV2-type vectors. The experience gained from studies in adult rats indicate that AAV2 vectors are quite efficient for use in SN, but for gene transfer to neurons in other brain regions other serotypes, such as 1, 5, 6 or 8, are preferred.

to obtain transduction of a major part of the SN­ VTA region with a single 2–3 ml deposit, provided that the vector is of high titer. With low-titer vec­ tor preparations it is difficult to obtain transduc­ tion of more than a part of the SN, leading to partial effects. On the other hand, it is critical that the purification procedures in the production process are efficient in eliminating contamina­ tions, such as excess empty capsid particles and other proteins that may cause non-specific toxic effects or immune reactions. In addition, the salt content of the concentrated vector solution should be within the physiological range, since high con­ centration of salts or other chemicals may impact the outcome. Since production, purification, and titration methods vary between laboratories, it is often difficult to compare results between centers, and even between different vector preparations generated at different occasions within the same production unit.

Technical aspects of AAV-mediated a-syn delivery

Improvements in the techniques for AAV produc­ tion and purification have made it possible to gen­ erate high-titer AAV vector stocks of high purity. Direct administration of such undiluted vector stocks in the brain may result in excessively high expression of the transgenic protein product, which might have unwanted consequences. This deserves careful consideration as optimal working dilution of vectors encoding for functional proteins (e.g., cytoplasmic enzymes or secreted factors), nonmammalian proteins, typically used as markers, or RNA interference constructs may well be different from each other. Even the most commonly used control constructs expressing the green fluorescent protein (GFP) marker protein may become toxic at high concentration. For example, we have recently reported a dose-dependent toxicity following AAV-mediated GFP expression, showing that the very high levels of GFP expressed after injection of very high-titer AAV vectors may induce significant

Successful application of AAV vectors for disease modeling depends on a number of factors related to the quality, purity, and titers of the vector pre­ parations, as well as on the correct handling and use of the vectors in the stereotaxic surgery. The technical details of producing high-quality AAV vectors have been thoroughly reviewed earlier (Grimm et al., 1998; Ulusoy et al., 2008; Zolotukhin et al., 1999, 2002). In this section we will focus on a number of practical issues related to the application of AAV vectors in the brain. Induction of PD-like pathology and DA neuron cell death by AAV-a-syn vector delivery depends on achieving a sufficient level of a-syn expression, in the absence of any non-specific toxicity. The expression level is primarily determined by the titer of the vector. In rats and mice, it is possible

Selection of working titers and non-specific toxicity

103 A

AAV2

B

AAV5

C

AAV6

α-syn Fig. 7. Transduction of AAV-a-syn vectors of different capsid serotypes in the midbrain. The figure illustrates the expression of human wild-type a-syn in the midbrain (lower panels) and striatum (upper panels) delivered by AAV2 (a), AAV5 (b) and AAV6 (c) vectors injected to SN. Note that a-syn expression mediated by AAV2 and AAV6 vectors have distinct specificity for the cells located in SN and ventral tegmental area, whereas AAV5-mediated transgene expression has a diffuse pattern in the midbrain without any apparent specificity. All serotypes illustrated in this figure result in efficient transduction of DAergic neurons as assessed by the expression of a-syn in the striatal terminals. Asterisks denote the approximate site of vector injection.

non-specific damage and cell death (Ulusoy et al., 2009). In this experiment, injection of AAV5-GFP vectors at titers between 3 × 1012 and 3 × 1013 gen­ ome copies/ml in the SN resulted in >30% reduc­ tion in TH-positive cell numbers, which was associated with pronounced microglial activiation. Both the cell death and the microglial activity dis­ appeared after diluting the vector stock to between 2 × 1010 and 3 × 1012 genome copies/ml while GFP expression remained robust and covered the whole nigra (Ulusoy et al., 2009). In light of these obser­ vations, we recommend that an optimal titer range for each vector construct is defined in order to achieve an expression of the transduced protein at sufficient levels, without causing non-specific toxi­ city. For each new batch we suggest that prior to use in actual experiments the vector is tested in vivo at three different dilutions (e.g., undiluted, 3× diluted and 10× diluted) in order to assess the optimal working titer for use in subsequent studies. In case of AAV-a-syn it is important to make sure that the vector is used at the right dilution, i.e., at a titer that allows wide-spread transduction in the SN and VTA without excessive spread to areas outside

the SN-VTA region, as illustrated in Fig. 7 for three representative cases, given single intranigral depos­ its of AAV-a-syn of serotypes 2, 5, and 6. In most experiments an AAV-GFP vector is used to control for non-specific damage. Since GFP is potentially toxic to the cells, many investigators in the field agree that GFP protein does not provide an opti­ mal control in viral vector experiments; however, a consensus on the use of a better alternative has not been reached yet.

Immune reactions Although initial studies suggested that intracereb­ ral injections of AAV vectors do not trigger any significant immune reactions, more recent work showed that a humoral immune response is acti­ vated against the AAV capsid proteins following injections in the central nervous system (CNS), and that re-administration of the same vector leads to a robust inflammatory response (Brockstedt et al., 1999; Chirmule et al., 2000; Manning et al., 1997;

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Peden et al., 2004, 2009). As a consequence, the expression of the second vector injection is signifi­ cantly reduced. The immune response directed toward the second vector deposit, however, is ser­ otype specific and can be avoided by using a differ­ ent AAV serotype for the second injection. In experiments where repeated injections of vectors are needed, therefore, one should select a vector of one serotype for the first injection (e.g., AAV2) and a different serotype vector for the second injec­ tion (e.g., AAV5). Depending on the dose used, a transient glial reaction may be observed at the injection site even after the first administration, but if it occurs, it will subside over time. Nevertheless, the experimental design should include appropriate controls to rule out the possibility that this vector-dependent glial response can affect the results (Peden et al., 2004; Ulusoy et al., 2009). Empty viral particles should, in principle, be possible to use as a control for the immune response elicited by the capsid proteins. However, the detection of transduction efficiency of empty viral particles in vivo is problematic, and the use of empty virus does not make it possible to control for non-specific effects related to the expression of a protein in the target cell. Assessment of levels of AAV-mediated protein expression In most studies using AAV vectors, the outcome of the experiment requires comparison of differ­ ent vector injection groups. For example, we usually compare our experimental groups with control groups at different time points in cohorts of animals that have received injections of the same vector batches. At the same time, we expect that the results obtained in one set of experiments should be possible to compare with the results obtained with the same vector constructs in other experiments performed either in our own labora­ tories or by investigators at other centers. This raises the important issue of how to compare the efficiency of AAV-mediated a-syn overexpression

between different batches of vector. Since the level of AAV-mediated transgene expression is directly related to the number of viable AAV vector particles injected, the method used to assess AAV vector titers becomes important. Infectious AAV vector particles can be quantified by several means, such as determination of total particle numbers and amount of capsid protein, or by assessment of the number of functional genome copies in the vector batch. Currently, the most common method to quantify AAV vectors is to measure the quantity of genome copies per volume using quantitative polymerase chain reac­ tion (qPCR). The values obtained with this tech­ nique are quite standardized and comparable. However, the vector preparations contain not only infective particles but also inactive (e.g., defective) viral particles and their relative propor­ tion can differ significantly from one production site/method to another. Therefore, the genome copy titration method does not provide accurate information on the amount of the infectious AAV particles in a vector batch. Biological assays, such as the replication center assay (Shabram and Aguilar-Cordova, 2000; Yakobson et al., 1987) or the qPCR-based infectious titration assay (Rohr et al., 2005), provide a more meaningful infectious titer estimation, but on the other hand, they are poor predictors of in vivo efficacy and have an intrinsic variability due to experimental conditions such as the cell types used for the assay, cell confluency, and variability in in vitro infection efficiency. A second possibility for defining the efficacy of a viral vector batch is the level of gene or protein expression in vivo. This can be done either by measuring the level of mRNA expressed by the vector or by determining the level of protein expression in vivo. Although there are good examples where the level of transgene expression is very well matched to the injected AAV genome titers (see, e.g., Bjorklund et al., 2009), it is clear that the correlation between the AAV vector titer (genome copies determined by qPCR) and the protein expression levels may not always be

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linear. We have noted that intracerebral injections of vectors that differ two- to threefold in titer may not result in readily measureable differences in the protein product in vivo. Conversely, exact titermatched vectors (by qPCR) may not lead to precisely the same expression levels in the brain. As the half-life of proteins can be very different, and especially when the expression is expected to induce neurotoxicity, leading to cell death and loss of the transgene product (as is the case for a-syn), one cannot rely only on the level of protein expression for assessment of the in vivo efficacy of the vector used.

serotypes have been utilized. Figure 7 illustrates the expression of human wild-type a-syn obtained in the rat ventral midbrain after injection of three different AAV serotypes: AAV2 (Fig. 7a), AAV5 (Fig. 7b), and AAV6 (Fig. 7c). Note also that the projections in the striatal target areas are filled with the transgene product, showing that the a-syn protein is efficiently transported along the nigrostriatal axons. A major variability factor in AAV-mediated gene delivery is the targeting accuracy in the sur­ gical procedure. The delivery of the vector to the appropriate site using a vector batch with suitable titer should result in transduction of the whole SN and in a-syn expression throughout the striatal axonal terminal network. Although, in our hands, the success rate for correct targeting of the SN-VTA area in rats is high (>90%), the situation is different in mice. Targeting the ventral midbrain in the mouse can be difficult both due to the fact that the brain size is smaller than the rat and the head positioning in the stereotaxic frame is less accurate. These factors lead to a higher variability (relative to the size of the nucleus) in the position of the tip of the needle or the glass

Factors related to efficiency of targeting midbrain DA neurons Since the transduction efficacy of different AAV vector serotypes differs between regions and between different animal species for the same target, it is important to carry out in vivo tests to identify most suited AAV serotype for a given in vivo application/target nucleus in the brain. In the rat brain, AAV vectors based on different capsid A

B correctly targeted

Mistargeted

AAV2

SNr

α-syn

ML

AAV5

SNr

ML

Fig. 8. AAV-mediated expression of human a-syn and targeting in mouse SN. Accurate targeting of the vector injection in the SN is critical, and also more challenging, when applied in mice. The figure gives examples of nigral AAV-a-syn injections in two different mice. The injection in panel a is correctly placed, resulting in a robust nigral transduction (lower panel) and expression at the terminal level (upper panel). Panel b illustrates a mistargeted vector injection due to the misplacement of the glass capillary tip that failed to transduce the nigral DA neurons (lower panel) and therefore the striatal projections (upper panel). In this latter case the medial lemniscus has served as a barrier for diffusion of the vector ventrally toward the SN. Asterisks mark the approximate site of vector injection. ML, medial lemniscus; SNr, SN pars reticulata.

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capillary used for the delivery. In cases where the injection is misplaced dorsally, the surrounding white matter tracks (the medial lemniscus in par­ ticular) can act as a physical barrier for the vector solution to diffuse into the SN and lead to partial or no transduction of DA neurons, while other populations in the thalamus express the trangene (compare Fig. 8a with b). Thus, not only accurate coordinates need to be determined but also pre­ cise microscope-aided surgical techniques should be utilized and post-mortem histological analysis of gene transduction should be used as means to screen the variability in targeting AAV vectors to the area of interest, in order to allow exclusion of injection failures in the experimental groups.

Concluding remarks The viral vector model of a-syn overexpression has been slow in gaining wide acceptance. One reason for this is probably that models that involve more precise stereotaxic surgery are cumbersome to apply in routine screening work. In addition, the access to high-quality AAV vectors has been a limiting factor, and the work with viral vectors for in vivo delivery in the brain has so far been a technique that has been developed in a limited number of specialized laboratories. The situation is changing, however. Standardized and validated AAV vectors are now readily available commer­ cially for routine use, and the basic procedures for AAV-a-syn delivery to the SN in mice and rats are now well worked out. The model offers unique opportunities to induce and study the develop­ ment of PD-like functional and neurodegenerative changes in midbrain DA neurons, in a wide vari­ ety of species, including both rats and primates, and is so far the only model that replicates the profound, progressive a-syn-related neuropathol­ ogy in nigrostriatal neurons that develops over time in PD patients. The progressive feature of this model provides an interesting new tool for the study neuroprotec­ tive and disease-modifying therapeutic inventions.

As discussed above, models replicating the progres­ sive a-syn-related changes characteristic for human PD will be an essential complement to the toxinbased models currently used in such studies. AAVa-syn delivery offers the opportunity to overex­ press a-syn not only in the nigrostriatal projection neurons but also in the possibility to target other, non-DAergic systems in the brain. This property allows us to replicate selected aspects of the more widespread synucleinopathy seen in more advanced cases of PD, or so-called PD-plus syn­ dromes. For good and consistent results in the AAV-a-syn model, however, it is necessary to pay attention to a number of technical issues, dis­ cussed above. In particular, it is essential to check the quality (and possible toxicity) of the vector batches prior to use, and to make separate tests to select the optimal working titers of both the AAVa-syn and the control vectors. In mice, in particular, precise and reproducible targeting of the vector deposits in the SN has to be ensured and checked in post-mortem immunostained sections. A presently unsolved shortcoming of the AAVa-syn model is that the extent of behavioral defi­ cits is quite variable. Only a fraction of the AAVa-syn-treated animals show significant long-term motor deficits (usually in the order of 25% in AAV-a-syn-treated rats, and even less in mice). This limits the use of the model for studies where functional recovery or functional sparing is the main focus. In AAV-a-syn-treated rats the magni­ tude of DA neuron cell loss is on average about 50–60%, which we know is a borderline for induc­ tion of significant impairments in the standard drug-induced and spontaneous motor tests. Efforts are now being made to combine a-syn overexpression with “a second hit” that will increase a-syn-mediated neurodegeneration and induce more robust behavioral deficits. Such sec­ ond hits may include the use of more toxic variants of a-syn or the induction of a more prominent or long-lasting inflammatory response. Application of AAV-a-syn overexpression in transgenic mice may also offer interesting opportunities for more refined disease modeling.

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 6

Modeling neuroinflammatory pathogenesis of Parkinson’s disease Christopher J. Barnum and Malú G. Tansey
Department of Physiology, Emory University School of Medicine, Atlanta, GA, USA

Abstract: The molecular mechanisms underlying the pathogenesis of idiopathic Parkinson’s disease (PD) have not been completely elucidated; however, some progress has been made in identifying factors that compromise survival of the dopaminergic neurons in the substantia nigra (SN) the death of which give rise to the motor symptoms that enable clinicians to diagnose the disease in its mid- to late stages. The prevailing theory regarding processes that are likely to account for degeneration of the nigrostriatal system centers around mitochondrial dysfunction, oxidative stress, excitotoxicity, and neuroinflammation. Of these, neuroinflammation is one candidate that appears to accumulate more support with each passing year. A number of researchers have attempted to manipulate inflammation in various animal PD models with varying levels of success. Still others have used inflammatory stimuli to elicit nigral cell death (NCD), a disturbing finding that has prompted much interest. In this chapter, we attempt to integrate what is known about the role of neuroinflammation in PD with the factors we feel are critical for understanding how inflammation modulates disease progression. Keywords: Parkinson’s disease; inflammation; TNF; substantia nigra; neurotoxin; LPS; stress

statistic that will likely balloon as baby-boomers reach the mean onset age of 55. The pathological hallmark of PD is the death of noradrenergic neu­ rons within the locus coeruleus (LC), death of dopamine (DA) neurons within the nigrostriatal system, and the presence of proteinaceous inclusions called Lewy bodies (LB). It is estimated that by the time clinical symptoms present, nearly two-thirds of the DA cells within the substantia nigra pars compacta (SN) are dead, leading to deficits in movement typified by resting tremor, poverty of movement,

postural instability, and freezing (Weiner, 2006).

Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disease that afflicts more than 1 million individuals over the age of 60 within the United States alone (Jankovic and Stacy, 2007). Additionally, the number of new cases increases by about 50,000 annually (Dauer and Przedborski, 2003), a


Corresponding author.

Tel.: þ1-404-7276126; Fax: þ1-404-7272648; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84006-3

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Because symptoms do not arise until the majority of DA cells are already destroyed, there has been little success in determining the etiology of the disease. In some instances there is a direct genetic link (called familial PD); however, this accounts for less than 5% of observed PD cases, signifying that the etiology of greater than 95% of PD cases (called “sporadic or idiopathic PD”) remains unknown (Thomas and Beal, 2007). The molecular mechanisms underlying the pathogenesis of idiopathic PD have not been com­ pletely elucidated. The prevailing theory regarding processes that are likely to account for degenera­ tion of the nigrostriatal system centers around mitochondrial dysfunction, oxidative stress, excito­ toxicity, and neuroinflammation (Jenner and Ola­ now, 2006). Of these, neuroinflammation is one candidate that appears to accumulate more sup­ port with each passing year (see McGeer and McGeer, 2004; Nagatsu and Sawada, 2005; Whitton, 2007, for review). The origin of this stemmed from an initial observation that microglia are in an active, ramified state within nigral tissue of post­ mortem PD patients (McGeer et al., 1988). Micro­ glia play an integral role in the coordination of neuroinflammation (Block and Hong, 2005; Gao and Hong, 2008; Nagatsu and Sawada, 2005) sug­ gesting that inflammatory factors might also be increased in PD brains. Subsequent studies identi­ fied an increase in numerous inflammation-related enzymes and cytokines within the substantia nigra (SN), striatum, and cerebral spinal fluid of PD patients, including tumor necrosis factor (TNF) interleukin-1 beta (IL-1b), interferon-g, cyclooxy­ genase-1 and -2 (COX-1, COX-2), and inducible nitric oxide synthase (iNOS) (Hirsch et al., 1998; Knot et al., 2000; Mogi et al., 1994, 1995; see Tan­ sey et al., 2007, for review). These factors have been shown to cause cell death directly by binding “death receptors”, which activate extrinsic cell death pathways, or indirectly via the production of reactive oxygen/nitrogen (ROS/RNS) species, which converge on mitochondrial dysfunction and activation of intrinsic cell death pathways (Ferrari et al., 2006; Kim and Joh, 2006; Rothwell, 2003;

Rothwell and Luheshi, 2000; Viviani et al., 2004; Wilms et al., 2003a, b). These data suggest that PD might lead to, or result from, widespread inflam­ mation within the CNS and/or periphery (Knot et al., 2000; Mogi et al., 1994, 1995). A number of researchers have attempted to manipulate inflam­ mation in various animal PD models with varying levels of success. Still others have used inflamma­ tory stimuli to elicit nigral cell loss, a disturbing finding that has prompted much interest. In this chapter, we discuss and integrate what is known about the role of neuroinflammation in PD with the factors we feel are critical for understanding how inflammation modulates the death of nigral DA neurons underlying disease progression.

Rodent models of PD and their inflammatory contribution MPTP The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model is one of the oldest and most widely used neurotoxin to mimic PD in mice and primates. MPTP was first identified as a protoxin that gets converted to N-methyl-4 phenylpyridi­ nium (MPPþ) by monoamine oxidase-B enzyme within astrocytes (Ransom et al., 1987). It is sub­ sequently taken up by DA neurons and interacts with the mitochondrial respiratory chain and damages complex-1, leading to cell death (Williams and Ramsden, 2005). Concomitantly, inflammatory cytokines such as TNF and ROS/ RNS species are increased (i.e., Miller et al., 2009; Smeyne and Jackson-Lewis, 2005) suggesting that inflammation plays a critical role in MPTPinduced nigral cell death (NCD). However, con­ flicting findings on the effects of anti-inflammatory agents suggest the role of inflammation in MPTP models is complex. At the forefront of this is minocycline, a microglial inhibitor whose ability to block cell death in the MPTP model is only sometimes observed (Du et al., 2001; O’Callaghan et al., 2008; Wu et al., 2002; Yang et al., 2003)

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even though it retains its ability to attenuate inflammatory cytokines (O’Callaghan et al., 2008). There are a variety of reasons for these discrepan­ cies, most notably experimental procedure. In the two studies showing protection, minocycline was given multiple times throughout the same day that MPTP was given (Du et al., 2001; Wu et al., 2002); whereas in the studies where protection was not observed, minocycline administration was spread out over a couple of days (O’Callaghan et al., 2008; Yang et al., 2003). Thus, one potential expla­ nation is that minocycline interfered with the metabolism of MPTP to MPPþ. The inability of minocycline to block cell death might also result from its reported selectivity for microglia over astrocytes. This would therefore suggest that the cytokine response in the aforementioned MPTP studies were astrocyte-derived rather than micro­ glia-derived. Indeed, this last scenario is sup­ ported by a study showing that minocycline increases the toxicity of MPTP (Yang et al., 2003). Regardless, support for inflammatory involvement in MPTP-induced DA cell death has been demonstrated in the form of iNOS. For instance, mice injected with MPTP showed increased upregulation of iNOS and mice lacking the iNOS gene were considerably more protected from MPTP NCD than wild-type mice (Liberatore et al., 1999). Other studies have also shown that attenuation of iNOS results in reduced NCD fol­ lowing MPTP (Dehmer et al., 2000). While there is still much unknown regarding the role of inflam­ mation in MPTP-induced NCD, it is likely that inflammation plays some modulatory role.

6-OHDA The 6-hydroxydopamine (6-OHDA) model of PD has been the gold standard rat model since it was first used more than 40 years ago (Ungerstedt, 1968). A neurotoxic analog of DA, 6-OHDA selectively kills DA and norepinephrine (NE) neurons when it is retrieved from the extracellular matrix by their respective transporters DAT and

NET (Luthman et al., 1989). The primary mechanism by which 6-OHDA induces cell death is through oxidative stress, although inhibition of mitochondrial respiration has also been noted (see Schober 2004, for review). While 6-OHDA applied directly to human SH-SY5Y cells in vitro demonstrate the ability of 6-OHDA to kill DA cells without inflammatory involvement (Shih et al., 2009), there is considerable evidence that in vivo 6-OHDA is toxic to DA neurons in part through inflammatory mechanisms. PET imaging using a ligand that binds to the upregulated ben­ zodiazepine receptor in activated microglia (Wilms et al., 2003a, b) showed increased micro­ glial activity within the SN following 6-OHDA injections (Cicchetti et al., 2002). This observation is supported by other studies in which microglia and inflammatory mediators have been increased following 6-OHDA lesion (McCoy et al., 2006; Mogi et al., 1999; Nagatsu and Sawada, 2005; Wilms et al., 2003a, b). Importantly, both minocy­ cline and the COX-2 inhibitor celecoxib have been shown to attenuate NCD (He et al., 2001; Koprich et al., 2008; Quintero et al., 2006), likely through inhibition of free radicals such as H2O2 (Lin et al., 2003). The timing of administration appears to be important as DA cell loss was not as great when minocycline was given after 6-OHDA injection (Quintero et al., 2006). The inflammatory profile observed in 6-OHDA­ lesioned animals may also depend upon the site of injection. For example, when 6-OHDA is injected into the striatum, microglial activation was more robust within the striatum than the SN both 7 and 28 days after lesion (Armentero et al., 2006). In contrast, Na et al. (2010) reported that intrastriatal 6-OHDA resulted in an increase in inflammatory-related gene expression within both the striatum and SN 7 days post-lesion, an effect that persisted within the SN, albeit to a lesser magnitude, for 14 days. In the latter study, Na et al. (2010) used a convection-enhanced deliv­ ery of 6-OHDA, which they report provides a more uniform DA lesion (Oiwa et al., 2003). These data suggest that inflammatory processes

116

play a secondary but important role in NCD fol­ lowing a 6-OHDA lesion.

Pesticides A variety of environmental toxicants (Fig. 1) have been postulated to increase the risk of PD, includ­ ing the herbicide paraquat, the fungicide maneb, and the pesticide rotenone. These compounds have been postulated to cause PD due to their high selectivity for nigrostriatal dopamine neurons (Cannon et al., 2009; Drolet et al., 2009; ManningBog et al., 2002; McCormack et al., 2002; Peng et al., 2004; Purisai et al., 2007; Thiruchelvam et al., 2000) and the increased incidence of PD in farmers who routinely handle them (Baldereschi et al., 2008; Dhillon et al., 2008; Hancock et al., 2008; Kamel et al., 2006). In animal models, suc­ cessful use of pesticides such as rotenone recapi­ tulate a majority of the pathology observed in PD (Greenamyre et al., 2010) and are attractive to researchers due to their high external validity (i.e., wealth of studies linking exposures to these agents to PD). While the mechanisms that

Psychological stress

facilitate cell death with these toxins are beyond the scope of this chapter, interfering with mito­ chondrial respiration and generation of ROS likely play a critical role (Cicchetti et al., 2009). Early studies examining the potential role of inflammation in toxin/toxicant-induced DA degeneration suggested an important role for microglia (see Liu et al., 2003, for review). For instance, when administered to neurons in vitro, rotenone was markedly more toxic in neuron–glia cultures than in neuron-only cultures (Gao et al., 2002a). Other studies have shown that high levels of superoxide induce DA cell death (Radad et al., 2006), further suggesting a role for microglia as they are a primary generator of superoxide in the CNS. Similar studies using paraquat/maneb and dieldrin have also shown increased inflammatory activity of microglia or increased toxicity to tyro­ sine hydroxylase positive (THþ) neurons in the presence of microglia (Mao and Liu, 2008; Wu et al., 2005). In vivo, a variety of studies have shown that chronic administration of rotenone/ paraquat/maneb is associated with the presence of activated microglia (Liou et al., 1996; McCormack et al., 2002; Purisai et al., 2007; Saint-Pierre et al., Environmental factors

Chronic systemic disease

Aging

Inflammation SNPc DA neuron

Apoptotic Signaling

Cytotoxicity & DA cell death Fig. 1. Proposed model of potential triggers that contribute to chronic neuroinflammation and its role in nigral cell death (NCD). The delayed and progressive nature of NCD in PD may be mediated by multiple stimuli (including aging, physiological stress, environmental factors, and chronic systemic disease) that converge to create chronic neuroinflammatory load, thereby enhancing apoptotic signaling and accelerating cytotoxicity and death of DA neurons.

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2006; Thiruchelvam et al., 2000), an observation that has been shown to precede NCD (Sherer et al., 2003). Importantly, two compounds believed to selectively inhibit microglia, minocy­ cline and iptakalim, have been shown to rescue NCD following rotenone treatment (Casarejos et al., 2006; Zhou et al., 2007). The pro-inflamma­ tory effects of rotenone appear to be specific to the CNS as rotenone administered to a variety of cells within the periphery have been shown to suppress TNF (Basuroy et al., 2009; Ichikawa et al., 2004; Ko et al., 2001). Of course, many questions remain regarding the exact role of inflammation in rotenone models.

Lipopolysaccharide In animal models, inflammatory stimuli have been shown to be deleterious to DA neurons (Castano et al., 1998; Ferrari et al., 2006; Hunter et al., 2007). Some of the first evidence for this comes from a cell culture study by Bronstein and collea­ gues (1995) in which they observed increased cell death of THþ rat mesencephalic cultures follow­ ing lipopolysaccharide (LPS) treatment. A Gramnegative bacterial endotoxin, LPS activates the inflammatory response through the TLR4 recep­ tor located on glia. This in turn promotes inflam­ mation through mechanisms that include a shift in microglial morphology from a passive to an acti­ vated amoeboid state that includes proliferation of microglia and increased production of inflamma­ tory cytokines, chemokines, and ROS. Over the years, researchers have administered LPS in a variety of ways in vivo to elicit NCD. LPS has been injected within the CNS (intraventricular, intrastriatal, intranigral) and systemically (intra­ peritoneal), acutely and/or chronically, and even pre-natally (described in greater detail below). In each instance, researchers were able to get selec­ tive loss of DA neurons. For example, a single intranigral injection of LPS was sufficient to reduce the number of DA cells and increase the number of OX42 positive microglia presenting an

active ramified morphology 15 days later (Castano et al., 1998). In a subsequent study, Castano and colleagues (1998) demonstrated that NCD via intranigral LPS administration was attenuated when dexamethasone, a synthetic glucocorticoid receptor antagonist with potent anti-inflammatory properties, was given peripherally. Dexametha­ sone is a large peptide that has extremely poor brain penetrance (De Kloet et al., 1998), suggest­ ing that the blood–brain barrier (BBB) may be compromised as a result of central LPS adminis­ tration. Similarly, intrastriatal injection of LPS induced an inflammatory response that preceded and contributed to DA cell loss within the SN (Choi et al., 2009). NCD can also be attained from a single injection of LPS peripherally (Qin et al., 2007) or pre-natally (Carvey et al., 2003). These last two findings are particularly important as they demonstrate that a nonspecific immuno­ genic stimulus can selectively kill DA neurons. Because these paradigms reproducibly elicit delayed and selective death of 30–70% of nigral DA neurons, models similar to these may present a window of opportunity during which to study the molecular mechanisms that may contribute to pro­ gressive loss of DA neurons akin to that occurring in patients with PD. Furthermore, DA cell loss is relatively permanent (>1 year) and is specific to DA cells while sparing GABAergic and seroto­ nergic cells (Herrera et al., 2000). More recently, chronic low-dose intranigral administration of LPS via osmotic pumps in rats has been used to induce neuroinflammation in the CNS that trig­ gers delayed and progressive loss of nigral DA neurons in vitro and in vivo (Gao et al., 2002b). In addition, our group has used a chronic low-dose intraperitoneal LPS paradigm previously shown to accelerate Alzheimer’s-like pathology in 3×TgAD transgenic mice (Kitazawa et al., 2005) to induce nigral DA neuron loss in mice in which the gene Parkin has been knocked out. Importantly, these studies have uncovered a novel role for Parkin in protecting against inflammation-related NCD (Frank-Cannon et al., 2008). Together, multiple studies indicate that chronic neuroinflammation

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can directly trigger and hasten selective and pro­ gressive loss of nigral DA neurons in pre-clinical rodent models of PD.

Are DA neurons uniquely vulnerable to inflammation? One of the most important findings regarding DA neurons is their apparently selective vulnerability to inflammation. This is supported by data show­ ing that dopaminergic, but not GABAergic or serotonergic cells were killed by LPS (Herrera et al., 2000). This same group later showed that the rate-limiting enzyme for DA synthesis TH may facilitate these outcomes as inhibition of TH attenuated DA cell loss and microglial activation (De Pablos et al., 2005). Nigral cell death is likely carried out by cyto­ kines such as IL-1b and TNF (Ferrari et al., 2006; McCoy et al., 2006). For instance, pre-natal administration of LPS led to increased IL-1b more than 3 months later and was correlated with DA cell loss (Ling et al., 2006). In a more specific test, Ferrari et al. (2006) used a recombi­ nant adenovirus to express IL-1b for 60 days. This chronic expression of IL-1b elicited most of the pathological and behavioral characteristics of PD, providing compelling support for a role of IL-1b in PD-like degeneration. More recent data from this group suggest that although IL-1b may play a role in NCD, it is not necessary (De Lella Ezcurra et al., 2010). Specifically, they demonstrated that that chronic nigral TNF overexpression resulted in NCD without changing IL-1b levels. Other groups, including our own, have shown that TNF is sufficient for NCD. For instance, mice lacking the TNF receptor show reduced DA cell death following MPTP treatment compared to controls (Sriram et al., 2002). Similarly, McCoy et al. (2006) demonstrated that neutralizing soluble TNF increased the number of surviving DA cells by greater than 50% following a striatal 6-OHDA injection. Similar results have been observed following increased production of IL-1b.

Although neuroinflammation is common to most neurodegenerative diseases, it is likely to have the most effect on progressive neuronal loss in PD due to the high density of microglia in regions impli­ cated in the development of PD. Although micro­ glia are present throughout the CNS, they are not evenly distributed. Regional differences in micro­ glial localization have been observed in mice, rats, and humans (Kim et al., 2000; Lawson et al., 1990; Mittelbronn et al., 2001). In mice, microglia account for approximately 5% of the total cells in any given brain region. However, in the SN and striatum, microglia account for greater than 12% of total cells in these structures (Lawson et al., 1990) ren­ dering them more susceptible to microglialmediated tissue damage during microglial activa­ tion. Thus, chronically activated midbrain microglia are likely to play a critical role in the generation of ROS and may serve as the link between inflamma­ tion, oxidative stress, and DA cell death. For instance, LPS induces DA cell loss in neuron–glia, but not neuron-enriched, mesencephalon cultures (Gao et al., 2003a, b). A more focused examination of microglia and associative factors has demon­ strated that nitric oxide and ROS are also involved in NCD. For example, a single intranigral injection of LPS led to increased OX-42 expression (with typical microglia morphology) that was co-localized with iNOS (Arimoto and Bing, 2003). When L-MNA, an iNOS inhibitor, was injected into the SN prior to LPS injection, it reduced DA cell loss and OX-42 immunostaining from 60 and 20%, respectively (Arimoto and Bing, 2003). It has been hypothesized that ROS-mediated DA neuro­ toxicity observed following LPS injection is induced by NADPH oxidase (Qin et al., 2004). In support of this idea, varying concentration of neuron–glia mixtures from mice in which the key subunit of the NADPH oxidase (lacking a functional gp91 protein) has been deleted (PHOX-/-) or wild-type littermates (PHOXþ/þ) mice were examined for DA cell loss following LPS administration. Ventral mesencephalon neuron–glia cultures of PHOX/ mice showed an 18% reduction in the number of THþ cells compared to 44% in PHOXþ/þ controls.

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Importantly, DA uptake was not affected in neuron–neuron cultures or cultures enriched with neurons, implicating microglia as necessary for the observed DA cell loss. Microglial-derived superox­ ide production was also dose-dependently increased in neuron–glia and glia-enriched cultures from PHOXþ/þ, but not PHOX/, mice (Qin et al., 2004). Lastly, minocycline has been shown to attenuate DA cell loss in MPTP (see above; Du et al., 2001; Wu et al., 2002) and 6-OHDA (He et al., 2001) models of PD. Taken together, these data provide strong evidence that inflammation has deleterious consequences on nigrostriatal DA cells and support the hypothesis that infection and/or inflammation may facilitate the progressive loss of nigral DA neurons characteristic of this disease (Perry et al., 2007).

Considerations regarding an inflammatory model of PD Do they produce pathology? Ideally, the best inflammatory model of PD will also show the complement of pathology observed in humans: cell death (SN, LC, and olfactory bulb (OB)), a-synuclein aggregation, motor impair­ ments, inflammation, gastrointestinal dysfunction, as well as the psychological aspects of the disease, such as depression. Unfortunately, no single PD model recapitulates all these symptoms and each model has its strengths and weaknesses (Schober, 2004). For instance, 6-OHDA is excellent for destroying catecholaminergic neurons and can be used to destroy striatal DA terminals, SN/LC/OB if used in specific ways. 6-OHDA models also provide consistent motor deficits. MPTP, on the other hand, can also destroy striatal DA terminals and SN cell bodies, and induce a-synuclein aggregation (Scho­ ber, 2004), but motor deficits typically resolve over time. As described above, rotenone models can also recreate a majority of PD symptoms (Greenamyre et al., 2010) although their reproducibility in rodents is the biggest concern involving the use of this

pesticide. Interestingly, the most commonly used inflammatory agent, LPS, has been shown to elicit other PD-like pathology besides NCD, including motor deficits (Choi et al., 2009), LC and OB dys­ function (i.e., Kaneko et al., 2005; Molina-Holgado and Guaza, 1996; Mori et al., 2005; Ota et al., 2008), and aggregation of a-synuclein within the cyto­ plasm of THþ neurons (Choi et al., 2009). For instance, Choi et al. (2009) demonstrated that intrastriatal LPS could elicit motor deficits in the amphetamine-induced rotation and cylinder tests. Importantly, motor asymmetry was observed within the first week of LPS injection and coincided with a significant loss of nigral DA neurons. That these motor deficits appeared alongside (or prior to) DA cell loss is a critical feature that is not observed in all LPS studies (Frank-Cannon et al., 2008). Although these deficits were still observed 4 weeks following LPS injection, whether or not this deficit is permanent is still unknown. LPS-related changes have also been observed within LC and OB (Kaneko et al., 2005; Molina-Holgado and Guaza, 1996; Mori et al., 2005; Ota et al., 2008), suggesting that this model might indeed recreate a variety of PD pathology. While chronic LPS expo­ sure studies have yet to be investigated, acute injec­ tion of LPS has been shown to influence neuronal activity and inflammation within the OB and LC. For instance, administration of LPS intraperitone­ ally was shown to increase NE turnover within the brainstem (Cho et al., 1999) resulting in a reduction of striatal NE and DA within 24 h. Inflammatory factors (TNF and IL-1b) linked to NCD were also increased within the LC shortly after peripheral LPS injection (Kaneko et al., 2005), an observation that could have deleterious consequences for LC neuronal viability. Together, these data suggest that LPS may have greater utility as a model for PD-like pathology besides NCD.

Are they easy to implement? As described above, not all PD models produce the same degree and complement of pathology.

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While protocols exist to elicit PD for each of the aforementioned models, we propose that LPS is ideally suited for studying the mechanisms by which neuroinflammation contributes to progres­ sive neurodegeneration. In particular, three important factors regarding implementation should be considered when selecting a particular model to study inflammatory pathogenesis of PD: (i) reproducibility, (ii) route of administration, and (iii) ability to work in multiple species. Some PD models produce greater variability of pathology. For instance, when successful, the rote­ none model can reproduce the majority of PD symptoms (Greenamyre et al., 2010). However, reproducibility and robustness of nigral degenera­ tion has been variable (Schmidt and Alam, 2006). Importantly, however, the variability associated with this model may provide a unique opportunity to explore why, in a homogeneous group, some organisms are more likely than others to develop PD. The comparison between two animals (one with higher susceptibility) may shed light on a number of PD-related issues, including the extent of the inflammatory response. However, an important fact is that the rotenone model may not be the best suited for experimentally testing the direct role of inflammation in PD-like pathol­ ogy because although rotenone elicits microglia activation, it also has direct toxic effects on DA neurons as a result of its inhibition of electron transport components. A second consideration concerns route of administration. While direct CNS injections of LPS are both reliable and con­ ventional, we contend that these methods are not likely to shed light on how environmental factors that trigger neuroinflammation promote nigral degeneration. Indeed, central administration of anything is a serious concern as cranial surgery often induces the very same inflammatory factors one is studying and therefore confounds and in essence compounds the initial challenge. Thus, these types of studies require a higher level of control to tease apart the background noise. In this sense, peripheral administration of LPS may be an ideal way to study the role of inflammatory

processes in PD that may initiate outside the CNS but could spread into the CNS in a manner con­ sistent with the Braak hypothesis (Braak et al., 2003). Finally, we suggest that being able to exam­ ine inflammation in multiple species will provide the most valuable information. Only LPS and rotenone provide that opportunity as neither 6-OHDA nor MPTP work well in both rats and mice. In summary, LPS models are the only ones to meet all three of these criteria. Nevertheless, because LPS itself is unlikely to directly trigger idiopathic PD, identification of specific systemic inflammatory triggers in human populations that trigger neuroinflammation and compromise survi­ val of vulnerable neuronal populations is where the focus needs to be going forward so animal models can be developed to test the extent to which these triggers contribute to development of PD-like pathology.

Can the PD model be used with similar results both in vivo and in vitro? In vitro experiments provide a level of control not attainable in vivo. With that said, a major criticism of in vitro studies is that the environment does not accurately reflect what happens within the whole organism. Thus, there is rightful trepidation to extrapolation of results from in vitro experiments to the in vivo situation. The goal therefore, is to identify a model system that closely reflects what is observed in vivo. Although not without its lim­ itations and issues, primary mesencephalon rodent cultures provide an opportunity to examine many factors related to PD, including inflammatory con­ sequences. An excellent example of this comes from a study by Gao et al. (2002a, b) in which LPS was given chronically in vivo and in vitro. In both instances, chronic LPS led to an increase in microglia activation and a delayed, selective reduction in THþ neurons. The contiguity of results between the in vivo and in vitro studies by Gao and colleagues is an important observa­ tion that lends greater credibility to the use of

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LPS for studying inflammatory pathogenesis of PD. Similarly, the ability to demonstrate rescue of vul­ nerable neuronal populations with anti-inflamma­ tory agents in vitro and in vivo (McCoy et al., 2006) strengthens the claim that inflammatory processes do in fact contribute to the development of PD-like pathology.

What inflammatory features need to be examined? One of the most challenging aspects in studying the inflammatory pathogenesis of PD is teasing apart the protective versus detrimental aspects of immune activation. As the resident immune cell, microglia are at the forefront of this issue. Upre­ gulation of certain receptors (i.e., Cd11b, MHC-II, CD68, etc.) and adoption of an amoeboid mor­ phology are often used to describe a state upon which microglial-derived inflammatory mediators exert deleterious effects on neurons (Ransohoff and Perry, 2009), but in cases of infection the same changes may have a protective function. Thus, at what point do microglia become toxic to endogenous tissue and what activation states (morphology) and products (cytokines, chemo­ kines, ROS, etc.) confer protection versus destruc­ tion? While this is likely determined by a variety of factors, the precipitating stimulus, the timing of that stimulus, and the duration of the inflamma­ tory response appear to be critical. For example, IL-1b has been shown to be both detrimental (Ferrari et al., 2006) and protective (Saura et al., 2003) to DA cells. For instance, chronic intranigral IL-1b expression (60 days) resulted in a marked reduction in THþ cells. However, when adminis­ tered 5 days prior to 6-OHDA, DA cell death was attenuated (Saura et al., 2003). This is further complicated by the observation that time lag between the first and second stimulus is important. For example, Mangano and Hayley (2009) reported that LPS given 7 days prior to paraquat resulted in very little NCD whereas LPS given 2 days prior to paraquat sensitized to NCD.

While the reasons for this difference are currently unknown, the outcome is likely dictated by the activation state of microglia and their secreted factors at the time of the second challenge. Indeed, being able to manipulate microglial phe­ notypes in these types of studies in order to evaluate their effect on neuronal survival would yield valuable information. In summary, a better understanding of which microglia activities have neurotoxic versus protective effects is paramount and pre-requisite for development of new anti­ inflammatory therapies to treat PD.

Can the model mimic the progressive nature of PD? One benefit of the LPS model is that it allows us to explore the progressive nature of PD in response to low, sub-threshold doses of endotoxin, which may be encountered periodically throughout the lifespan of an organism, yet do not lead to any immediate noticeable trauma. Indeed, intriguing results have come from experiments in which exposure to LPS occurred pre-natally and led to delayed NCD (Carvey et al., 2003; Qin et al., 2007). Not surprisingly, microglial activation mar­ kers and inflammatory mediators such as TNF were associated with progressive NCD in these studies, suggesting inflammation might be the potential “silent driver” of progressive degenera­ tion. While these results need to be replicated, these initial findings provide an important founda­ tion from which to explore the role of pre-natal exposure to inflammation on development of idio­ pathic PD.

Considerations Chicken or the egg? One of the most important questions surrounding the role of inflammation in PD is whether inflam­ mation causes PD or perpetuates the disease as a

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response to some “other” stimulus. The observa­ tion that systemic LPS selectively kills nigral DA neurons demonstrates that inflammation is at least sufficient to cause NCD. On the other hand, LPS enhances NCD when given in conjunction with or following another toxin/toxicant (see below). This synergistic action of inflammation likely perpetu­ ates an ongoing inflammatory response that is best described by the multiple-hit hypothesis of PD. The multiple hit hypothesis stipulates that PD results from the cumulative toll of many known and unknown risk factors (see Carvey et al. 2006, for review). In this scenario, each time an organ­ ism is exposed to a risk factor, or “hit”, you get a reduction in DA cells. After a certain number of hits, a threshold for PD symptoms is reached lead­ ing to a clinical diagnosis. Over the years, a num­ ber of risk factors have been implicated including aging, exposure to environmental toxins, genetics, and more recently, inflammation. Indeed, support for this hypothesis comes from studies in which multiple exposures to inflammatory stimuli, envir­ onmental toxins, and/or genetic mutations lead to decreased DA neurons (Carvey et al., 2006; Sulzer, 2007). Importantly, the number of “hits” is inversely related to the number of viable DA cells. Although it has been demonstrated that each of these risk factors can lead to DA cell death, there is a need to identify which of these factors may be the most pervasive for the devel­ opment of PD. One hypothesis regarding the mechanisms by which inflammation leads to the progression of PD is through the priming of microglia, such that a sensitized pro-inflammatory response occurs fol­ lowing future (second “hit”) challenges (Perry, 2004; Perry et al., 2007). In this scenario, the initial insult (i.e., an environmental toxicant, genetic mutation, infection, etc.) is suggested to elicit microglia activation. Once primed, microglia are more susceptible to respond in an exaggerated manner. They may become alternatively activated (Colton, 2009), chronically activated releasing increased amounts of pro-inflammatory factors (i.e., IL-1b and TNF) that compromise cell

viability (Gifford and Lohmann-Matthes, 1987). A good example of this comes from an ME7 mur­ ine model of prion disease. Prion disease, or trans­ missible spongiform encephalopathy, contains an inflammatory component that is pathologically similar to many neurodegenerative diseases, includ­ ing PD (Cunnigham et al., 2005). In this study, mice were injected intracerebrally or intraperitoneally with LPS almost 5 months following ME7 inno­ culation. Both prion alone and prion-plus-LPS mice showed similar ramified microglial morphol­ ogy. However, far greater (sensitized) IL-1b, iNOS, and neutrophil infiltration was observed in the prion-plus-LPS group compared to animals treated with prion alone. Furthermore, LPS chal­ lenge resulted in double the number of dead cells compared to those found in mice treated with only prion (Cunnigham et al., 2005). Similar results have been demonstrated in animal models of PD. In these studies, animals were administered a combination of LPS and neurotoxin (i.e., rotenone (Gao et al., 2003a, b), MPTP (Gao et al., 2003a, b), or 6-OHDA (Ling et al., 2004)) separated by many months. The combination of neurotoxin/ LPS administration consistently led to two impor­ tant outcomes: (1) a sensitized pro-inflammatory response and (2) augmented DA cell loss. For example, pregnant Sprague–Dawley rats injected with LPS during gestation and later given 6-OHDA into the right lateral ventricle (on PD99) showed a 62% reduction in DA neurons (Ling et al., 2004). Administration of either 6-OHDA or LPS alone led to 46 and 33% reduc­ tion in DA cells, respectively, when examined on P120. Furthermore, striatal TNF was increased by 82% following combined LPS/6-OHDA adminis­ tration, which was more than 50% greater than the effect produced by each treatment individu­ ally (Ling et al., 2004). While the jury is still out on whether inflammation can cause PD, there is substantial data that an inflammatory challenge in a susceptible organism may exacerbate or potenti­ ate an inflammatory response and subsequent nigral DA neuron death. Indeed, this may have direct implications for exposure to inflammatory

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stimuli during critical periods of development such as bacterial vaginosis infections during labor on fetal predisposition for idiopathic PD later in life.

What is next – identifying stimuli that might facilitate PD through inflammation Aging On a nonpathological level, there are a variety of immunological changes that occur during aging, called immunosenescence (Franceschi et al., 1999). Aging leads to a shift in metabolic demands that is carried out by and greatly affects the immune system (Pawelec et al., 2002). Some of these changes include a decrease in the number of newly generated immune cells due to a reduc­ tion in thymus activity and size (Spoor et al., 2008) as well as impaired clearing of old immune cells (De Martinis et al., 2005). Part of what facilitates these changes is chronic antigenic load due to life­ long exposure to antigens that may or may not be associated with infection. Chronic antigenic load increases the number and variety of memory and effector cells, which reduce immunological space and increase inflammatory status (De Martinis et al., 2005). Similar changes in animals have also been observed. For instance, in young rats, anti­ inflammatory and neurotrophic factors such as IL-10 and transforming growth factor are released following systemic inflammatory challenge. This phenotypic response shifts in older rats in the form of IL-1b and results in increased BBB per­ meability, an effect that can have detrimental effects within the CNS (Wu et al., 2005, 2007, 2008). This general shift toward an increased inflammatory status has led to the molecular inflammatory hypothesis of aging put forth and reviewed by Chung et al. (2009). General changes in inflammation as a result of aging likely contri­ bute to an immunological status inclined to respond in an unregulated manner and increase susceptibility to inflammatory-provoking stimuli. Indeed, 15-month-old rats were more sensitive to

the toxicity of rotenone compared to their 4-month-old counterparts (Phinney et al., 2006). Thus, aging itself is likely a liability factor toward the development of PD (Fig. 1). Incorporating age into the current inflammatory models of PD might reveal novel mechanisms by which immuneprovoking stimuli interact with an inflammatory­ dysregulated system that ultimately leads to NCD. Indeed, more studies aimed at understanding the aging–inflammation connection are needed.

Systemic (chronic) disease If an activated immune system is believed to underlie the development of PD, then one needs to consider the impact of systemic diseases on the development of PD (Fig. 1). In an attempt to answer this question, researchers in Denmark examined medical records of 13,695 PD patients to determine whether they also had a diagnosis of a selected group of autoimmune diseases, at least 5 years prior to being diagnosed with PD (Rugbjerg et al., 2009). Although no significant risk for PD was observed in patients also diagnosed with autoimmune disease, a reduced risk for PD was observed in patients ailing from rheumatoid arthritis. While speculative, reduced risk in this population may result from chronic use of anti­ inflammatory drugs used to treat this disease and would be consistent with the protective effects of chronic NSAID use in human populations (Chen et al., 2003, 2005; Samii et al., 2009). Researchers have been interested in parkinson­ ism resulting from viral infection (see Jang et al., 2009, for review) since the dramatic increase in post-encephalitic parkinsonism was observed fol­ lowing the influenza pandemic between 1914 and 1918 (Dale et al., 2004), a phenomenon believed to account for half of all PD cases in the ensuing decades (Josephs et al., 2002). Japanese encepha­ litis virus can lead to post-encephalitis parkinson­ ism that manifests pathologically in a manner similar to PD (Shoji et al., 1993), and has been used to induce PD pathology in rodents (Hamaue,

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et al., 2006; Ogata et al., 1997). More recent empirical evidence linking influenza to PD-like pathology was recently demonstrated by Smeyne and colleagues (Jang et al., 2009) in which mice infected intranasally with the avian influenza A virus H5N1 showed neurological impairments, a-synuclein phosphorylation and aggregation, acti­ vated microglia, and protracted NCD even though the infection itself lasted about 10 days. Together, the studies provide compelling evidence that idio­ pathic PD may arise from, or be perpetuated by, viral infections.

Psychological stress Psychological stress is perhaps the most ubiqui­ tous of human experiences. Stress is often used as an umbrella term for any internal or external stimulus that poses some challenge to an organ­ ism. This includes exposure to antigens (see Aging section) and toxicants such as MPTP (see MPTP section) and will not be discussed here. Instead, this section will review the growing body of evi­ dence illustrating that stress may play an impor­ tant role in the development and/or progression of PD based on observations in both humans and rodents. Lastly, we will examine studies in which neuroinflammation is observed following expo­ sure to stressful stimuli. Although not often discussed in the literature, PD patients often report that their symptoms become worse during stressful life events (Macht et al., 2005). Stressful stimuli that provoke emo­ tional responses such as anger and anxiety have been reported to increase behavioral deficits such as bradykinesia and freezing (Macht and Ellgring, 1999; Marsden and Owen, 1967; Schwab and Zieper, 1965; Smith et al., 2002). Whether or not stress affects all persons suffering from PD is unknown and is likely influenced by a number of factors such as how much the disease has pro­ gressed. However, one study found that 70% of PD patients reported a worsening of symptoms as a result of stress (Macht et al., 2005), an indication

that an increase in symptoms following stress may be quite pervasive. Finally, it is important to con­ sider the psychological impact of having Parkin­ son’s disease. Indeed, the distress associated with motor dysfunction might also lead to enhanced stress that may, in turn, further exacerbate PD symptoms. The observation that humans experience a worsening of symptoms during stressful times is also supported by rodent studies. One of the first studies to examine the relationship between stress and PD was conducted by Snyder and colleagues (1985). This group investigated stress-induced impairments of 6-OHDA-lesioned rats. Rats were administered 6-OHDA into the cisterna magna, lateral ventricle, SN, nucleus accumbens, or striatum. Following recovery, rats were subjected to one of six stressors (i.e., 2-deoxyglucose, insulin-challenge, food depriva­ tion, hypertonicity, cold stress, or tail shock) and observed over the course of the next 24 h for the development of motor impairments. The authors reported that regardless of the stressor imposed, motor impairment was increased in all stressed rats. This is important as the nature, duration, and intensity of each stressor differed remark­ ably, suggesting that the mechanism underlying these changes must be a fairly universal response to all stressors. This same group reported similar findings in a subsequent study by demonstrating that restraint stress followed immediately by a 6-OHDA lesion led to greater behavioral deficits than either alone (Smith et al., 2002). Using a sub-threshold 6-OHDA lesion model, Seroogy and colleagues (2006) demonstrated, for the first time, that chronic variable stress leads to a marked reduction in THþ immunoreactive cells within the SN. Together, these data suggest that, in impaired rats, exposure to stressful stimuli can exacerbate both the behavioral and neuro­ chemical deficits associated with rodent models of PD. While these behavioral studies have yielded interesting findings, the mechanisms governing exacerbation of PD as a result of stressor exposure

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remain unknown. Although prior studies have looked toward classic stress hormones such as corticosteroids and catecholamines (epinephrine and NE) as the key culprits (Deak, 2007), more recent studies suggest that activation of inflamma­ tory signaling pathways as result of stressor expo­ sure might provide the missing mechanistic link between stressor exposure and liability toward the development of PD. Historically speaking, the idea that psychological stress can induce an inflammatory response is relatively new. Recent studies have demonstrated that stressor exposure can increase the production of inflammatory fac­ tors including TNF, IL-1b, and IL-6. Importantly, some of these factors have also been observed within the brain, including the hypothalamus and hippocampus (Blandino et al., 2006; Deak et al., 2005; Frank et al., 2006; Morrow et al., 1993; Nguyen et al., 1998; O’Connor et al., 2003; Suzuki et al., 1997). The most studied of these factors, IL-1b, has been shown to increase following immobi­ lization, tail shock, foot shock, and oscillation stress (restraint stress on a rotating platform) (Deak et al., 2005; Nguyen et al., 1998; Suzuki et al., 1997). Interestingly, our recent work demonstrated that IL-1b and IL-6 mRNA (but not TNF) was increased within the SN and stria­ tum following foot shock, suggesting that these structures might also be sensitive to stress-related neuroinflammation (Barnum et al., in revision). Importantly, foot shock exposure did not increase inflammatory factors in the amygdala, hippocampus, pituitary, or cortex (Deak et al., 2003; O’Connor et al., 2003), indicating that neuroinflammatory consequences of stressor exposure may (i) occur in a regionally selective manner rather than occurring widely throughout the brain, and (ii) be a downstream consequence of stress-dependent activation of microglia (Blandino et al., 2009). It should be noted however that acute stress has been shown to increase prosta­ glandins within the cortex (Garcia-Bueno et al., 2008), suggesting that neuroinflammation may manifest in region-specific ways across the CNS. Taken together, these data raise the interesting

possibility that chronic psychological stress may also be a silent driver of neuroinflammation in PD-relevant areas within the CNS (Fig. 1).

Conclusion In the past 10 years, the role of inflammation in PD pathogenesis has become less controversial and more firmly established as a result of a multi­ tude of pre-clinical, clinical, and epidemiological studies, which have gone beyond demonstrating the mere presence of inflammatory mediators in the SN to show the ability of anti-inflammatory interventions to attenuate or delay degeneration of nigral DA neurons. These interventions have provided proof of concept that identification of neuroinflammatory mediators that directly elicit death of nigral DA neurons may present opportu­ nities for therapeutic development to modify dis­ ease progression. But we are also at a critical juncture where we must carefully evaluate how to best protect selectively vulnerable brain regions without global suppression of microglia activation given that their role in immune surveillance is critical for neuroimmune homeostasis. Lastly, if chronic neuroinflammation in fact contributes to the delayed and progressive loss of nigral DA neurons (Fig. 1) and development of PD in animal models, we must seriously consider the possibility that chronic systemic inflammatory diseases in humans that begin in middle age and are highly prevalent in industrialized countries may be trig­ gering chronic neuroinflammation and increasing PD risk. In the next 10 years, development of animal models to test this exciting possibility should be the focus of investigators seeking to establish a link between inflammation and devel­ opment of idiopathic PD. Acknowledgment We thank members of the Tansey lab for useful discussions.

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Abbreviations 6-OHDA BBB COX DA IL-1b/10 INF LC LPS MPPþ MPTP NCD NE NOS OB PD ROS/RNS SN TNF TH

6-hydroxydopamine blood–brain barrier cyclooxygenase dopamine interleukin-1/10 interferon locus coeruleus lipopolysaccharide N-methyl-4 phenylpyridinium 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine nigral cell death norepinephrine nitric oxide synthase olfactory bulb Parkinson’s disease reactive oxygen/nitrogen species substantia nigra tumor necrosis factor tyrosine hydroxylase

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 7

The MPTP-lesioned non-human primate models of Parkinson’s disease. Past, present, and future Susan H. Fox
,†,‡ and Jonathan M. Brotchie‡ ‡

† Division of Neurology, University of Toronto, Ontario, Canada Toronto Western Research Institute, Toronto Western Hospital, Toronto, Ontario, Canada

Abstract: Non-human primate (NHP) models of Parkinson’s disease (PD) have been essential in understanding the pathophysiology and neural mechanisms underlying PD. The most common toxin employed, MPTP, produces a parkinsonian phenotype in NHPs that is very similar to human PD with excellent response to dopaminergic drugs and development of long-term motor complications. Over the past 25 years, MPTP-lesioned NHP models, using several species and a variety of MPTP administration regimens, have been used to understand disease pathophysiology, investigate several stages of the disease progression, from pre-symptomatic to advanced with motor complications, and apply knowledge gained to develop potential therapeutics. Many treatments in common use in PD patients were developed on the basis of studies in the MPTP model, in particular dopamine agonists, amantadine, and targeting the subthalamic nucleus for surgical treatment of PD. Continued development of novel therapies for PD will require improving methods of evaluating symptoms in NHPs to ease translation from NHP to patients with homogenized scales and endpoints. In addition, recent studies into non-motor symptoms of PD, especially in response to chronic treatment, is expanding the usefulness and impact of MPTP-lesioned NHP models. Despite these obvious successes, limitations still exist in the model, particularly when considering underlying mechanisms of disease progression; thus, it appears difficult to reliably use acute toxin administration to replicate a chronic progressive disorder and provide consistent evidence of Lewy-like bodies. Keywords: Non human primate; MPTP; Parkinson’s disease; non-motor

Discovery of MPTPa new dawn fades Prior to the discovery of 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine (MPTP), non-human primate (NHP) models for investigating PD were limited by lack of specificity for the dopaminergic system


Corresponding author. Tel.: þ1-416-6036422; Fax: þ1-416-603-5004; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84007-5

133

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and consequent phenotype. The earliest models were generated using acute administration of choli­ nergic agonists, carbachol and harmaline, that resulted in a tremor that lasted for the duration of the drug action (Everett et al., 1956; Poirier et al., 1974). Longer-lasting models were attempted by electrolytic lesions of the midbrain, resulting in hypokinesia and tremor (Pechadre et al., 1976; Poirier, 1960). However, these lesions also encroached onto the red nucleus and thus symptoms were not entirely due to lesioning of the substantia nigra pars compacta (SNC). The successful use of the synthetic neurotoxin 6-hydro­ xydopamine (6-OHDA) in rodents to generate a unilateral lesion of the SNC (Ungerstedt, 1976) was applied to NHPs. A unilateral model was attempted with stereotactic infusion into the medial forebrain bundle of baboons (Apicella et al., 1990) and marmosets (Annett et al., 1995) which resulted in unilateral hypokinesia. Multiple stereotaxic injections of 6-OHDA into the primate striatum are required to reduce spontaneous recovery that may occur after a few weeks (Eslamboli, 2005; Eslamboli et al., 2003). The advantage of the unilateral deficit is that the contralateral brain can be used as a control and animals are less severely compromised in the early stages and can thus feed themselves. A bilateral model was also tried that resulted in profound hypokinesia that required intensive care of the animals (Mitchell et al., 1995). Use of the 6-OHDA-lesioned NHP has not been widespread due to the practical difficulties of surgical infusions. The discovery that 1-methyl-4-phenyl-1,2,3,6-tet­ rahydropyridine (MPTP) was able to induce human parkinsonism (Langston and Ballard, 1983) was therefore a critical development in modeling parkin­ sonism in animals. MPTP is a protoxin that crosses the blood–brain barrier and is converted to 1-methyl-4-phenylpyridium ion (MPPþ), predomi­ nantly in serotonergic neurons and glia, via the action of monoamine oxidase B (MAO-B) (Chiba et al., 1984; Westlund et al., 1985). The mechanism whereby MPPþ is released from glia remains unclear (Inazu et al., 2003), but once in the extracellular

space, MPPþ is selectively transported by the dopa­ mine transporter (DAT) into dopaminergic neurons (Javitch et al., 1985). The relative selectivity of some dopaminergic neurons to MPPþ-induced cell death, i.e., the SNC rather than the ventral tegmental area (VTA), may relate to the higher concentration of DAT in the midbrain (Kitayama et al., 1993). Cell death occurs following MPPþ uptake into mitochondria and inhibition of complex 1 function (Ramsay et al., 1986). Other factors involved include superoxide radicals and nitric oxide that are pro­ duced secondary to MPPþ intoxication, and com­ bine to produce perooxynitrite that nitrates tyrosine residue in intracellular proteins, including tyrosine hydroxylase with resultant loss of dopamine cells (Przedborski et al., 2000). Microglial activation in the SNC occurs following MPTP administration, and glial cells also produce free radicals and nitric oxide synthase (Vazquez-Claverie et al., 2009). Removal of MPPþ from the cytoplasm into synaptic vesicles occurs via the vesicular monoamine trans­ porter (VMAT2), which prevents further toxic action (Miller et al., 1999). Loss of striatal VMAT2 may be a factor in MPTP toxicity; thus, a recent position emission tomography (PET) study using a chronic dosing schedule of MPTP reported VMAT loss in the striatum of asymptomatic primates, 2 months before changes in dopamine receptors and DAT (Chen et al., 2008). The ability of MPTP to produce nigral and striatal dopamine cell loss similar to that of human PD has led to multiple investigations into potential disease processes. (For an update on neurodegenerative pro­ cessessee Section I (Genetic and molecular mechanisms of neurodegeneration in PD) of Volume 183). To date, the understanding of MPTP toxicity has led to the assessment of MAO-B inhibitors, such as selegiline and rasagiline, as potential neuropro­ tective agents (PSG, 1989; Olanow et al., 2009). However, the real power of the MPTP NHP model probably lies in the link between the pathol­ ogy and the clinical phenomenology of the disease. In this respect, the model remains unique in neuro­ logical disease research and continues to be the gold standard in drug development for PD.

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Update on practicalities of the MPTP model A variety of NHP species have been used to induce a parkinsonian model, with macaques being the most common (including rhesus (sp mulatta) and cymologous (sp fascicularis) (Burns et al., 1983)), followed by common marmosets (Jenner et al., 1984), squirrel monkeys (Langston et al., 1984), African green monkeys (Taylor et al., 1997), and baboons (Todd et al., 1996). The most common implementations of the model produce bilateral parkinsonism that mimics the phenotype of human PD and is created using repeated systemic adminis­ tration of MPTP (e.g., 1–2 mg/kg) over several days to months (Burns et al., 1983; Fox et al., 2002; Visanji et al., 2009b). In such models, parkinsonian features develop over 2–3 months until stable. Recovery may occur after a few months, requiring further MPTP to maintain the model. Individua­ lized dosing is often required due to inter-animal variability in vulnerability to MPTP. One such fac­ tor in MPTP sensitivity is age of the animals, with older animals being more sensitive to MPTP (Ova­ dia et al., 1995). Older animals (> 5-year old) may be better in terms of modeling the human disease as PD becomes more common with aging, and like humans, older normal NHPs have age-related loss of striatal dopamine and reduced markers of tyro­ sine hydroxylase positivity (Collier et al., 2007). More chronic delivery of MPTP with lower daily doses (0.2 mg/kg) over 2–3 weeks has been pro­ posed as a means of modeling a progressive loss of dopamine that does not recover (Bezard et al., 1997; Meissner et al., 2003). Indeed, in such models symptoms develop over time and there is a period, up until 8–12 administrations have been made, when there are no motor symptoms, though there is demonstrable loss of dopaminergic functions. This “preclinical” stage may be a useful model to investigate potential pre-symptomatic compensa­ tory mechanism and thus neuroprotective strategies (Bezard et al., 2001a,b). Even longer treatment schedules over weeks to months have been used to extend the use of the model. Thus intermittent chronic dosing of low-dose MPTP, 1–2 times per

week every 1 or 2 weeks for several weeks or months has been proposed as a model of a more progressive onset of parkinsonism with recovery between injections to investigate compensatory mechanisms (Hantraye et al., 1993; Mounayar et al., 2007). However, some studies have failed to demonstrate a delayed neurodegenerative process in dopaminergic neurons after concluding MPTP injections, suggesting this dosing schedule does not initiate a truly progressive degenerative process (Garrido-Gil et al., 2009). Shorter treatments using MPTP (1 mg/kg for 3 days) have also been used to generate partial lesions, e.g., 60% tyrosine hydro­ xylase cell loss compared to the usual 90%, in an attempt to model a milder stage of the disease (Iravani et al., 2005). These animals have less moto­ ric problems and do not respond to levodopa, in contrast to models described above. Hemiparkinsonism can also be modeled by intracarotid infusion of a single low dose of MPTP to induce a unilateral parkinsonian syndrome (Bank­ iewicz et al., 1986). The advantage of hemiparkin­ sonian animals is that they are less severely affected, thus can be maintained more easily with­ out a need to initiate symptomatic therapy, as well as providing a contralateral side of the brain that can be used as a control. However, recent reports of necrotic basal ganglia lesions and the possibility of effects of the lesion being apparent on the injected side of the brain, may limit the use of these models (Emborg et al., 2006). Detailed information on the practical use of MPTP and safety issues have been reviewed else­ where (Emborg, 2007; Przedborski et al., 2001).

Pathology of MPTP-parkinsonism in the NHP Dopamine and other monoamine cell loss Dopamine cell loss in the SNC is the key patholo­ gical feature of MPTP-induced parkinsonism. The pattern of destruction of dopaminergic cells in the SNC is similar to human PD with a ventro-lateral

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predominance (Burns et al., 1983; Gibb et al., 1987). Depending on dosing and age of the ani­ mal, other dopaminergic systems may be affected. Thus cortical and limbic dopamine (Perez-Otano et al., 1991) and VTA cell loss may occur (Mitchell et al., 1985; Rose et al., 1989), although to a much lesser extent than in the SNC. In contrast to human PD, the pattern of dopamine loss in the striatum is usually more uniform, rather than the preferential loss in the putamen (Perez-Otano et al., 1994; Pertwee and Wickens, 1991). A sec­ ondary effect of loss of striatal dopamine is a reduction in spine density, with up to 50% reduc­ tion in spines in both the caudate nucleus and putamen, with the sensorimotor post-commis­ sural putamen being the most severely affected region for both dopamine depletion and spine loss (Villalba et al., 2009). Such loss of spines may be a compensatory effect of excessive cor­ tico- or thamalo-striatal glutamatergic activity (Garcia et al.). Other monoamines can be affected by MPTP, although to a lesser degree than dopamine. Thus 5-HT levels are reduced by 75–90% in the cingulate and frontal cortex, with less reduction in the stria­ tum (Perez-Otano et al., 1991; Russ et al., 1991). One study has reported no changes in brainstem serotonergic neurons (Gaspar et al., 1993). Cell loss within the locus coeruleus has been reported in the MPTP macaque (Forno et al., 1986; Mitchell et al., 1985) with reduction in noradrenaline in the frontal cortex (Alexander et al., 1992; Pifl et al., 1991). To date, the role of these monoamines has been inves­ tigated as potential therapeutic targets for motor symptoms of PD and in particular levodopa­ induced motor fluctuations (see below). However, recent pathophysiology studies in human PD are highlighting the potential role of such neurotrans­ mitters in many non-motor symptoms experienced by PD patients, e.g., mood disorders, psychosis, and autonomic problems (Lim et al., 2009). Thus future studies into these monoamine systems in the MPTP-primate should focus on investigating non-motor aspect of PD that may involve these non-dopaminergic systems.

Other non-dopaminergic neurotransmitters The MPTP-primate has been used to investigate the neuropharmacology of parkinsonism and levodopa­ induced dyskinesia, in particular the role of nondopaminergic systems. These have been reviewed in several recent publications (Brotchie, 2005; Fox et al., 2006a). Table 1 summarizes changes in nondopaminergic neurotransmitters and pharmacologi­ cal studies performed to date in the MPTP-primate. Alpha synuclein Alpha synuclein pathology occurs in MPTP-pri­ mates, but not to the extent seen in human PD. Thus, there is increased intraneuronal alpha synu­ clein immunoreactivity within the SNC; however, this is not in the usual structural form of a Lewy body (Kowall et al., 2000). Following a single injec­ tion of MPTP, there is an increase in phosphory­ lated alpha synuclein after 1 week that is associated with 10% dopamine nigral cell loss, while after 1 month dopamine cell loss progresses to 40% with alpha synuclein within cell bodies, suggesting a direct link between cell death and alpha synuclein deposition (McCormack et al., 2008; Purisai et al., 2005). The absence of Lewy bodies in MPTPprimates has been suggested to relate to the relatively short time post MPTP that pathological studies are performed; however, a recent study in two animals confirmed no Lewy bodies even 10 years post MPTP (Halliday et al., 2009). The lack of Lewy bodies in MPTP-primates is important in understanding the pathogenesis of PD in humans. Thus, the recent pathological studies reporting Lewy body pathology in fetal tissue transplanted in PD patients has been suggested to be due to factors such as inflammation and excitotoxicity (Kordower et al., 2008). However, both inflammation and excitotoxicity occur in the MPTP-primate suggesting other causes for the development of Lewy bodies occurring in human brain. One suggestion may be age. In the human post-mortem studies, only tissue transplanted after

Table 1. Non-dopaminergic neurotransmitters in MPTP-lesioned primates Motor symptoms Receptor class

Receptor subtype

Changes in receptors

Acetyl­ choline Muscarinic (mAChR) antagonists

M1, M4, possibly M3

[3H]-QNB (M1) binding increased in GPi in dyskinesia (Griffiths et al., 1990)

Acetyl­ choline Nicotinic (nAChR) agonists

Adenosine antagonists

Drug

Parkinsonian signs

Trihexy-phenydyl

þ Enhance effect of levodopa (Domino and Ni, 1998)

Biperidin

þ Enhance effect of levodopa (Domino and Ni, 2008)

Wearing-off

Non-motor symptoms

þ; May reduce levodopa-induced dystonia but worsens chorea (Pearce et al., 1999)

þ (Quik et al. 2007)

Non-selective agonists

Decreased in striatum in PD (Kulak et al., 2002)

Nicotine

beta2
–beta4
a4b2 nAChRs

Decreased in striatum and cortical regions, e.g., cingulate gyrus in PD (Bordia et al., 2007); (Kulak et al., 2007)

SIB-1508Y

þ Enhanced effect of levodopa (Schneider et al., 1998)

A2A

Increased in striatum dyskinesia (Morissette et al., 2006)

Istradefylline

þ (Grondin et al., 1999a, Kanda et al. 1998), (Bibbiani et al. 2003)

þ (Kanda et al. 2000)

ST1535

þ (Rose et al. 2006)

þ (Rose et al. 2006)

ASP5854

þ (Mihara et al. 2008)

A2A and A1A

Dyskinesia

(Continued)

Table 1 (Continued ) Motor symptoms Receptor class

Receptor subtype

Glutamate NMDA antagonists

Non-selective

Changes in receptors

Drug

Parkinsonian signs

Amantadine MK801, LY235959

 Can worsen at high doses (Gomez-Mancilla and Bedard, 1993); (Rupniak et al., 1992)

NR2A-NMDA antagonist

No changes (Ouattara et al., 2009) Increased NR2A subunit in dyskinesia (Hallett et al., 2005)

MDL 100,453

NR2B-NMDA antagonist

Decreased in PD; increased in striatum and cortical regions in dyskinesia (Hurley et al., 2005); (Ouattara et al., 2009)

Ifenprodil; CP-101,606

Synaptosomal cycling of NR2B (Hallett et al., 2005)

Co 101244

Ro 25-6981

CI 1041

Wearing-off

Dyskinesia þ Can reduce chorea but also worsen dystonia (Blanchet et al., 1998); (Papa and Chase, 1996); (Visanji et al., 2006)  Worsened dyskinesia (Blanchet et al., 1999)

þ (Nash and Brotchie 2000) þ Potentiated action of levodopa (Nash et al., 2004); (Steece-Collier et al., 2000).

Exacerbates dyskinesia (Nash et al., 2004; SteeceCollier et al., 2000)

þ Potentiated action of levodopa (Loschmann et al., 2004) þ (Blanchet et al. 1999) Prevent dyskinesia (Hadj Tahar et al., 2004)

Non-motor symptoms Increases psychosislike behavior (Visanji et al., 2006)

AMPA antagonists

Metabotropic glutamate receptor (mGLuR)

AMPA

mGluR2/3

No change (Silverdale et al., 2002) Increased in striatum in dyskinesia (Calon et al., 2002)

LY300164

Alpha adreno­ receptors

þ (Konitsiotis et al. 2000)

GYKI-47261 þ (Combined with amantadine) (Bibbiani et al., 2005)

Decreased in striatum and GP dyskinesia (Samadi et al., 2008) þ

mGluR4 agonist mGluR5 antagonist

þ Potentiated effects of levodopa (Konitsiotis et al., 2000)

Increased in putamen and GP in dyskinesia (Samadi et al., 2008); (SanchezPernaute et al., 2008) Possibly increased in striatum (Ouattara et al., 2010)

þ (Morin et al. 2010) þ But possible reduced parkinsonism (Johnston et al., 2010)

MPEP/MTEP

þ

Alpha2 agonist

Alpha2A/2c antagonist

Idazoxan

Fipamezole Alpha1 adreno­ receptor antagonist

þ (Gomez-Mancilla and Bedard 1993)

Yohimbine

Prazosin

þ (Bezard et al. 1999)

þ (Henry et al. 1999), (Domino et al. 2003, Fox et al. 2001)

þ (Bezard et al. 1999), (Fox et al. 2001)

þ (Savola et al. 2003)

þ (Savola et al. 2003) Reduced L­ dopa-induced hyperactivity (Visanji et al., 2009b) (Continued)

Table 1 (Continued ) Motor symptoms Receptor class

Receptor subtype

Changes in receptors

Serotonin

5-HT1A agonists

Increased in striatum and motor cortex (Huot et al., in submission-b)

5-HT1B agonists

5-HT2A antagonists

Increased in striatum and motor cortex (Huot et al., 2010)

Drug

Parkinsonian signs

þ But worsened PD (Iravani et al., 2006)

Sarizotan

þ (Bibbiani et al. 2001, Gregoire et al. 2009)

SKF-99101

þ But worsened PD (Jackson et al., 2004)

MDMA

þ (Iravani et al., 2006; Johnston et al. 2009)

Methy-sergide

þ But worsens PD (Gomez-Mancilla and Bedard, 1993) þ (Vanover et al. 2008) þ (Visanji et al., 2006), can worsen PD at higher doses (Grondin et al., 1999b) þ (Oh et al. 2002, Visanji et al. 2006)

ACP 103

Quetiapine

Exogenous cannabinoids

CB1 agonist CB1 agonist

þ (Fox et al. 2002)

Nabilone Increased CB1 binding in striatum in untreated parkinsonism that reverses with

Dyskinesia

R)-(þ)-8­ OHDPAT

Clozapine

5-HT2C receptor antagonists (mixed)

Wearing-off

Rimonabant

þ (van der Stelt et al. 2005)/ (Meschler et al. 2001)

Non-motor symptoms

No change in psychosis-like behaviours

Reduces psychosis-like behaviors (Visanji et al., 2006)

chronic levodopa (Lastres-Becker et al., 2001) Enhanced endo­ cannabinoids in the GPe in untreated parkinsonism (Di Marzo et al., 2000) Carboxylic acid amide benzenesulfonate (CE) Opioid

d-opioid agonist

�-Opioid agonist

þ Enhanced action of levodopa (Cao et al., 2007) þ (Hille et al. 2001)

PPEA mRNA; enkephalin protein increased in striatum in PD; further increased in dyskinesia; PPE-B mRNA and dynorphin decreased in PD and increased in dyskinesia (Bezard et al., 2001b); (Herrero et al., 1995); (Morissette et al., 1997; Quik et al., 2002) Enadoline

U50,488

þ

þ/ (Maneuf et al. 1995), (Hill and Brotchie 1995) þ Worsens PD (Cox et al., 2007)

(Continued)

Table 1 (Continued ) Motor symptoms Receptor class

Histamine

Key: þ = improves  = worsens

Receptor subtype

Changes in receptors

Drug

Parkinsonian signs

Wearing-off

Dyskinesia

Opioid-like receptor (ORL­ 1) antagonist

J113397

þ/ Mild effect (Viaro et al., 2008) þ enhanced effect of L-dopa but worsened dyskinesia (Visanji et al., 2008)

Non-selective antagonist

Naloxone/ naltrexone

þ/ (GomezMancilla and Bedard 1993), (Henry et al. 2001, Klintenberg et al. 2002, Samadi et al. 2003)

m-Opioid antagonist

Increased mopioid receptors in dyskinesia (Chen et al., 2005; Hallett and Brotchie, 2007)

Cyprodime ADL5510

þ (Henry et al. 2001); (Fox et al. 2010b)

d-Opioid antagonist

Increased dopioid receptors (Hallett and Brotchie, 2007)

Nor-BNI

þ (Henry et al. 2001)

Histamine H3 agonist

þ Reduces chorea not dystonia (Gomez-Ramirez et al., 2006)

H2 antagonist

þ Reduces chorea, increases dystonia (Johnston et al, 2010)

Non-motor symptoms

143

at least 11 years contained Lewy bodies. The NHP study only investigated animal to 10 years (Halliday et al., 2009). There is an effect of age on the level of alpha synuclein in NHPs; thus, older animals are more likely to have higher levels, similar to human PD (Chu and Kordower, 2007). The effect of age is thought to be due to increased stabilization of the alpha synuclein protein allowing accumulation, rather than increased mRNA expression (Li et al., 2004). However, this process also occurs in MPTP-primates thus suggesting other processes are required to initiate Lewy body formation in PD patients. One suggestion has been termed “permissive templating” and may occur with prions, amyloid, and tau whereby a concentration-dependent for­ mation of a pathogenic protein oligomer occurs, followed by a non-concentration-dependent pro­ cess of further aggregation onto the oligomeric template (Hardy, 2005). The MPTP-primate thus continues to be useful in understanding the pathology of human PD.

to Braak (Braak et al., 2003) starts in the dorsal motor nucleus of the vagus and olfactory tract and extends through brainstem structures into cortical regions. Premotor symptoms in PD are known to include anosmia, sleep disorders, constipation, and mood problems all suggesting extranigral pathology that may involve such brainstem structures. In addi­ tion, the non-motor symptoms of advanced PD, including psychiatric, sleep, and autonomic also implicate many non-dopaminergic systems. To investigate these features of PD, appropriate mod­ els are needed. The MPTP-primate may fulfill the need for some of these systems (see later); however, to date there have been few pathological or imaging studies. Table 2 summarizes pathological studies performed to date. Investigation of non-motor symptoms is discussed below.

Updates on phenomenology of the model; motor and non-motor features MPTP-parkinsonismmotor phenotype

Pathological changes beyond the basal ganglia The pathology of idiopathic PD extends beyond the dopaminergic cells of the SNC (Lim et al., 2009). Indeed, the proposed progression of PD according

MPTP-lesioned NHPs exhibit the typical motor signs of PD seen in patients, including bradykine­ sia, rigidity, tremor, and postural instability (Hughes et al., 1992). The cardinal feature is bra­ dykinesia or akinesia when animals become

Table 2. Extranigral pathology and behavioral consequences in the MPTP-primate Region

Pathological changes induced by MPTP

Behavioural observations

Olfactory system

TH positive cells in glomerular layer of olfactory bulb increased by 100% compared to controls (Belzunegui et al., 2007) Loss of dopamine transporter-positive fibers in the PPN compared with control animals (Rolland et al., 2009)

Olfactory impairment reported in MPTP-lesioned marmoset (Miwa et al., 2004) Microinjections of GABA antagonist bicuculline into the PPN reverses akinesia (Nandi et al., 2008) None reported

Pedunculopontine nucleus (PPN) region Gastrointestinal tract

Cardiovascular

Increase in nitric oxide synthase immunoreactive (IR) neurons in myenteric plexus vs. controls; decrease in tyrosine hydroxylase-IR neurons by 70% compared to controls, no change in cholinergic or vasoactive peptide (Chaumette et al., 2009) MPTP does not mimic changes seen in PD (Goldstein et al., 2003)

None reported

144

slower in all movements, particularly walking. In addition, some animals will have episodes of “freezing” with an inability to move for a few seconds, as if stuck in one place. Bradykinesia is also evident in an overall reduced range of move­ ment with less spontaneous movement, less exploratory behavior, and less head movement. A reduced blinking rate may occur, in a similar manner to PD patients with the classical masked facies. Postural abnormalities are seen with a for­ ward head tilt that can often reach to the floor. However, unlike PD patients, animals rarely fall. The classical 4–6 Hz resting tremor of PD is not usually observed in the MPTP-primate but may occur in the African green monkeys (Bergman et al., 1998). More commonly, a postural tremor may be seen when an animal is walking and reach­ ing for objects. Several rating scales have been published for measuring parkinsonian disability in the MPTP­ lesioned NHP (Gomez-Ramirez et al., 2006; Imbert et al., 2000; Visanji et al., 2009b). The strength of the MPTP-primate models is that these scales are similar to rating scales used to assess PD patients such as the Unified Parkinson’s Disease Rating Scale (UPDRS) (Goetz et al., 2008). The NHP scales consist of subjective clinical assessment of severity, and possibly disability, of range of move­ ment, bradykinesia, posture, alertness, and tremor. Rigidity is harder to assess, particularly in smaller primates. A recent objective method using EMG, force, and elbow angle measures has been pro­ posed (Mera et al., 2009). Due to the time-consuming nature of this ana­ lysis and possibility of subjectivity, other more objective measures of total or global motor activ­ ity have been proposed. Video analysis systems where images of freely moving animals are captured at half-second intervals and movement is quantified as the number of pixel changes between consecutive images have been shown to correlate with portable accelerometers and infra­ red activity counting (Togasaki et al., 2005). Hemiparkinsonian primates have also been evaluated using such video systems (Liu et al.,

2009). Although potentially useful for objective measures for overall level of motor activity, such systems generally fail to distinguish movement due to reversal of parkinsonism and increased movement due to dyskinesia. Other non-validated quantitative methods pro­ posed include video recordings of animals in a “behavioral observation hallway” and measure­ ment of a range of activities including displace­ ment time across the hallway, reaching time towards rewards, number of rewards obtained, and level of the highest shelf reached for rewards before and after levodopa, called the Hallway task (Campos-Romo et al., 2009). Further beha­ vioral tests in marmosets have been reported including a measure of akinesia using the marmo­ set’s natural jumping behavior, called the “Tower”, and a measure of axial rigidity using the marmoset’s natural righting reflex, the “Hourglass”; both are impaired with MPTP (Verhave et al., 2009). However, the effects fol­ lowing treatment with dopaminergic drugs is not clear and further validation of these tests are required. To date, clinical observation is still the gold standard to fully evaluate motor features of parkinsonism, in particular the presence of bradykinesia. Levodopa-induced motor complications Long-term treatment of MPTP-lesioned NHPs with levodopa results in the development of both choreiform and dystonic dyskinesias which are essentially identical to dyskinesia in humans (Clarke et al., 1987; Jenner, 2003b). There are species differences in the expression of dyskinesia. Thus, Old World species have less overall motor activity and exhibit dyskinesia easily distinguish­ able as either chorea or dystonia (Boyce et al., 1990a, b). However, practically, such large pri­ mates provide logistical challenges and thus the marmoset model of levodopa-induced dyskinesia has been developed to facilitate the conduct of studies with robust statistical outcomes (Henry

145

et al., 1999; Pearce et al., 1995). The marmoset tends to be overall more active and often chorea and dystonia may be difficult to distinguish unequivocally. In all species used, in a similar manner to patients with PD, the severity of dyskinesia relates to the severity of parkinsonism (Schneider et al., 2003), although not consistently (Guigoni et al., 2005), and the dose and duration of levodopa ther­ apy (Smith et al., 2003). The dyskinesia is stable and consistent on separate days of dosing (Pearce et al., 1995; Visanji et al., 2006, 2009a). Likewise, chronic levodopa alters the dose–response curve to levodopa, or so-called short-duration response (Nutt et al., 2002) in a similar way to PD patients. Thus in de-novo animals, there is a dose response in reversal of PD motor disability and production of dyskinesia, whereas following chronic treatment with levodopa, this changes to a shorter latency to reversal of PD (“switch-on”) and an all-or-none response with no increase in dyskinesia severity with increased doses (Mestre et al., 2010). Dyski­ nesia experienced by MPTP-lesioned primate ani­ mals is commonly present when the levels of levodopa are maximal, i.e., “peak-dose” dyskinesia (Fox et al., 2001). PD patients with dyskinesia can also experience dyskinesia at the onset and end of a dose of levodopa termed “diphasic dyskinesia” and often experience dystonia in the off-state (Obeso et al., 2000); these are rarely described in NHPs, though it is clear that they do occur (Boyce et al., 1990b). Other motor fluctuations appear in the longterm levodopa-treated MPTP-lesioned primate. Thus, reduction in duration of action of levodopa on successive treatment days, “wearing off” occurs (Fox et al., 2010a; Jenner, 2003a). Animals can also exhibit what is termed “beginning and endof-dose worsening”, in a similar way to PD sub­ jects (Quinn, 1998). Thus, following an acute dose of levodopa, there is a transient worsening of motor function before improvement, and then as the beneficial response to levodopa is declining there is a rebound worsening of parkinsonism to below-baseline values (Kuoppamaki et al., 2002).

The advantage of recognizing such additional levodopa-induced motor fluctuations in the MPTP-primate improves the ability to evaluate efficacy of novel drugs for treating fluctuations in PD and enhances the ability to design phase II and phase III clinical studies to better improve positive outcomes. Non-motor phenotypes Appreciation of non-motor problems in PD has now been reflected in developing NHP models to investigate pathophysiology and novel treatments for these issues. Psychosis-like behaviors as a model of neuropsychiatric symptoms PD patients experience a range of neuropsychiatric symptoms both due to disease-related pathology and as side-effects of medications. These symptoms include psychosis, ranging from illusions, wellformed visual hallucinations to delusions and hypo­ mania. Side-effects of dopaminergic agents include impulsive and compulsive disorders, psychomotor agitation, and complex motor stereotypies (Voon and Fox, 2007). MPTP-lesioned primates treated with levodopa and dopamine agonists also exhibit abnormal repetitive, exaggerated, and driven gross motor behaviors which are distinct from dyskinesia and parkinsonism and may represent behavioral correlates of neural processes of these neuropsy­ chiatric symptoms in PD. Prior studies in both MPTP-lesioned marmosets and macaques have commented on some of these behaviors, including agitation (Pearce et al., 1995), climbing behavior (Boyce et al., 1990b), “hallucinatory-like beha­ vior” (Blanchet et al., 1998), and hyperactivity (Akai et al., 1995) but with limited quantification. Recent study of abnormal psychotomimetic beha­ viors seen in the levodopa-treated MPTP-lesioned marmoset has demonstrated that four behavioral

146

categories exist: hyperkinesia (fast movements), response to non-apparent stimuli (possible hallucinatory-like behaviors), repetitive grooming (representing compulsive activity), and stereotypies (including pacing, repetitive side-to-side jumping, and running in circles). These can be rated using a neuropsychiatric-like behavior rating scale (Fox et al., 2006b, 2010; Visanji et al., 2006). The parti­ cular strength of this model is that it has predictive validity in terms of response to treatments that both exacerbate or attenuate psychosis-like behaviors in PD patients. Thus in the model, the atypical antipsychotics, clozapine and quetiapine, reduce psychosis without worsening PD, in con­ trast to the effects of haloperidol that worsen PD, while amantadine increased psychosis (Visanji et al., 2006). The subjective nature of psychotic behaviors can clearly not be assessed in the MPTP-lesioned marmoset; rather, these psychosis-like behaviors might be a physical manifestation of similar pro­ cesses in the NHP brain. The advantage of using the MPTP model in asses­ sing the risk of developing psychiatric problems is that impulse control disorders were only appreciated after many years of use of dopamine agonist (Voon et al., 2006). Recent clinical studies investigating potential agents for PD now routinely include assess­ ment of impulse control disorders as part of the evaluation of side-effects and the updated UPDSR rating scales for PD patients include questions on behavioral issues (Goetz et al., 2008). Sleep disorders Sleep disorders are a common feature of PD. Patients can experience nocturnal issues due to disease pathology including disturbance of the sleep–wake cycle with insomnia and excessive daytime sleepiness, as well as specific sleeprelated issues such as REM sleep behavior disor­ ders (RBD). Such problems can arise before the motor features of PD appear; in particular, exces­ sive daytime sleepiness and RBD and are thought to be due to early brainstem dysfunction (Postuma

et al., 2009). Sleep problems can also be sideeffects of antiparkinsonian medications in PD. To date there have been limited investigation of these issues in MPTP monkeys. One study measured hormone levels and reported no circa­ dian changes in cortisol, but possible changes in melatonin and prolactin in MPTP-lesioned animals compared to controls, although no corre­ lation with sleep states was performed (Barcia et al., 2003). More recent studies of sleep architecture in MPTP-lesioned primates using long-term continu­ ous electroencephalographic monitoring via implanted miniaturized telemetry device has shown that decreased dopamine turnover following a single MPTP intoxication completely suppressed REM sleep, while chronic MPTP with develop­ ment of parkinsonism resulted in progressive sleep deterioration, fragmentation, and reduced sleep efficacy with a corresponding increased slee­ piness during the day by about 50%. However, there was no evidence of RBD, i.e., REM sleep without atonia (Barraud et al., 2009). Thus, the MPTP-primate model does experience some of the sleep disorders encountered in PD and can be used to further study these problems as well as identify side-effects of new medications. Cognitive impairment A range of cognitive problems are encountered in PD subjects from mild cognitive impairment to dementia (Hely et al., 2008; Mamikonyan et al., 2009). Modeling such symptoms in the MPTP-pri­ mate has been attempted using behavioral para­ digms and has shown evidence of fronto-striatal cognitive deficits that are consistent with PD patients (Kulisevsky and Pagonabarraga, 2009). Thus many studies have shown chronic deficits in executive and attentional tasks including delayed response, delayed matching-to-sample, visual dis­ crimination, and object retrieval/detour tasks that are impaired even in MPTP-treated primates that have minimal motor deficits (Pessiglione et al.,

147

2004; Schneider and Kovelowski, 1990; Taylor et al., 1990). In addition, measuring self-initiated and visually-triggered saccades in MPTP-lesioned primates have shown that errors such as number of GO mode (no-response, location, and early release) increased after MPTP treatment and per­ severative errors, e.g., switching from the GO to the NO-GO mode, are also consistent with frontal deficits (Slovin et al., 1999). In a similar fashion to PD patients, treatment with levodopa does not reverse these findings and can often worsen cog­ nitive problems (Decamp and Schneider, 2009). The MPTP-primate has thus shown promise as a model of cognitive deficits in PD; however, none of the currently used agents for cognitive pro­ blems in PD, such as acetylcholinesterase inhibi­ tors, have been evaluated in this model.

Emerging concepts on the use of MPTP-lesioned NHP in translational medicine The key role of the MPTP-primate model for more than 25 years has been to increase under­ standing of the basic neural mechanisms under­ lying PD and levodopa-induced dyskinesia. Thus, the seminal studies, especially using MPTP­ lesioned macaques, performed by the groups of Delong (DeLong et al., 1985) and Crossman (Crossman et al., 1985) were instrumental in deli­ neating the role of the direct and indirect striato­ pallidal pathways and subthalamic nucleus (STN) in control of the output regions of the basal gang­ lia in motor symptoms of PD and dyskinesia. From an understanding of these basal ganglia pathways, many novel targets/concepts for treat­ ing PD and dyskinesia have been evaluated in the MPTP-primate, including non-dopaminergic neu­ rotransmitters (Brotchie, 2005; Gomez-Mancilla and Bedard, 1993) and STN lesioning (Aziz et al., 1991; Bergman et al., 1990) (Table 1). Many have progressed into routine clinical use, e.g., the glutamate antagonist amantadine for dyskinesia and STN DBS for advanced PD (Pahwa et al., 2006).

Improving measurements in the MPTP-NHP to mimic clinical endpoints in trials The MPTP-primate remains an excellent model to assess agents with potential to improve parkinsonian disability, either as monotherapy or as an add-on to levodopa. In addition, agents that can reduce levo­ dopa-induced dyskinesia or extend the duration of action of levodopa, i.e., treat wearing-off, are com­ monly assessed (Jenner, 2003a). The strength of the model is the phenomenology of motor features (see above) that enables rating scales for parkinson­ ism and dyskinesia to be broadly equivalent to human rating scales in PD (Brotchie and Fox, 1999). Many agents can thus be tested using similar rating scales in primates and then at the phase II level (Fox et al., 2006a). However, several drugs have failed in the trans­ lation process from phase II to phase III clinical studies (e.g. Goetz et al., 2007; Manson et al., 2000). One reason may be lack of equivalent end­ points employed in primate studies that are then used in Phase III studies. Recent attempts to improve this include the concept of using a clinical measure of quality of a treatment’s benefit in NHP studies rather than just a measure of severity. One suggestion has been to incorporate measures of “good” on time, when there is reversal of PD with either no or non-disabling dyskinesia in con­ trast to “bad-on time” when the animal has a reversal of parkinsonism but with disabling dyski­ nesia (Johnston et al., 2009). Such measures are then equivalent to typical endpoints used in phase III studies which provide some measure of pro­ portion of time for which dyskinesia is present (UPDRS part IV, item 32, or MDS-UPDRS item 4.1) (Goetz et al., 2008) and diary measures of “on-time” which incorporate the impact of trou­ blesome dyskinesia such as proportion of “on­ time” without troublesome dyskinesia (Hauser et al., 2000). New endpoint measurements of neu­ ropsychiatric and cognitive problems, as discussed above, will potentially allow the MPTP-primate to more fully evaluate potential drugs for PD and include measures of potential adverse effects.

148

Use of the MPTP-NHP model in developing drugs for neuroprotection The use of the MPTP-primate to evaluate poten­ tial neuroprotective agents has been less success­ ful to date (Bezard, 2006), for example, the failure to replicate the positive effects of infusion of GDNF into the MPTP-lesioned NHP in PD patients (Kordower et al., 2000; Lang et al., 2006). The use of the low-dose chronic MPTP protocols has been one means of attempting to replicate the progression of disease (as discussed above). However, logistical issues of large num­ bers of animals required to perform such studies have resulted in use of lower-order animals in these settings, e.g., MPTP-lesioned mice. With respect to modeling this aspect of the disease other approaches need to be considered and are currently being evaluated. The most promising of these is the use of alpha synuclein-expressing vec­ tors. Kirik and colleagues have introduced recom­ binant adeno-associated viral vector (AAV) coding wild-type alpha synuclein or A53T mutated alpha synuclein into the SNC (unilateral) of marmosets (Eslamboli et al., 2007; Kirik and Bjorklund, 2003). The resultant phenotype was spontaneous rotations in animals overexpressing wild-type alpha synuclein while animals expres­ sing A53T mutation had gradual impairment of hand motor tasks and coordination tasks for up to 52 weeks. Pathological studies revealed degen­ eration of dopaminergic fibers in the striatum and dopamine loss in the ventral midbrain, more pro­ minent in the A53T group than in the wild-type group; alpha synuclein aggregates were also posi­ tive for ubiquitin. Further studies are needed to evaluate the potential uses of such models. On the other hand, it is clear the MPTP model still has much to offer in the search for diseasemodifying therapies, for instance, in the under­ standing of how imaging might provide biomar­ kers of disease progression that could be used in clinical development. Thus, imaging can deter­ mine serial changes in markers of nigrostriatal dopamine function in MPTP-primates. Several

centers are developing these techniques to mea­ sure markers of striatal dopamine, dopamine transporters (DAT), vesicular monoamine trans­ porter-type 2 (VMAT2), and D2-dopamine recep­ tors (Collantes et al., 2008; Doudet et al., 2006; Nagai et al., 2007; Tabbal et al., 2006). Such tech­ niques will enable use of the MPTP-lesioned NHP in assessing potential neuroprotective drugs by combining a biomarker with clinical assessment of the parkinsonian phenotype.

Conclusion The MPTP-lesioned NHP remains the gold-standard in modeling motor symptoms and complications of long-term levodopa therapy in PD. Improving out­ come measures for translating preclinical findings into potentially useful drugs for PD will continue to maximize the potential of this model. Future uses include understanding non-motor symptoms of PD, such as neuropsychiatric and sleep issues that occur in this model to increase understanding and develop novel treatments for PD.

Abbreviations NHP PD MPTP STN DBS MPPþ MAO-B DAT SNC VTA VMAT2 5-HT RBD

Non-human primate Parkinson’s disease 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine Subthalamic nucleus deep brain stimulations 1-methyl-4-phenylpyridium ion Monoamine oxidase B Dopamine transporter Substantia nigra pars compacta Ventral tegmental area Vesicular monoamine transporter 5-Hydroxytryptamine (serotonin) Rapid eye movement sleep behavior disorder

149

REM STN UPDRS MDS-UPDRS

6-OHDA AAV GABA A mAChR nAChR NMDA AMPA mGLuR PPEA PPEB PPN IR

Rapid eye movement Subthalamic nucleus Unified Parkinson’s disease rating scale Movement disorder society Unified Parkinson’s disease rating scale 6-Hydroxydopamine Adeno-associated viral vector Gamma aminobutyric acid A Muscarinic acetylcholine receptor Nicotinic acetylcholine receptor N-methyl D-aspartate a-amino-3-hydroxyl-5-methyl­ 4-isoxazole-propionate Metabotropic glutamate receptor Preproenkephalin-A Preproenkephalin B Pedunculopontine nucleus Immunoreactivity

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SECTION II

Exploring PD with brain imaging

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 8

Abnormal metabolic brain networks in Parkinson’s disease: from blackboard to bedside Chris C. Tang† and David Eidelberg
,†,‡ †

Center for Neurosciences, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, NY, USA ‡ Departments of Neurology and Medicine, North Shore University Hospital, Manhasset, NY, USA

Abstract: Metabolic imaging in the rest state has provided valuable information concerning the abnormalities of regional brain function that underlie idiopathic Parkinson’s disease (PD). Moreover, network modeling procedures, such as spatial covariance analysis, have further allowed for the quantification of these changes at the systems level. In recent years, we have utilized this strategy to identify and validate three discrete metabolic networks in PD associated with the motor and cognitive manifestations of the disease. In this chapter, we will review and compare the specific functional topographies underlying parkinsonian akinesia/rigidity, tremor, and cognitive disturbance. While network activity progressed over time, the rate of change for each pattern was distinctive and paralleled the development of the corresponding clinical symptoms in early-stage patients. This approach is already showing great promise in identifying individuals with prodromal manifestations of PD and in assessing the rate of progression before clinical onset. Network modulation was found to correlate with the clinical effects of dopaminergic treatment and surgical interventions, such as subthalamic nucleus (STN) deep brain stimulation (DBS) and gene therapy. Abnormal metabolic networks have also been identified for atypical parkinsonian syndromes, such as multiple system atrophy (MSA) and progressive supranuclear palsy (PSP). Using multiple disease-related networks for PD, MSA, and PSP, we have developed a novel, fully automated algorithm for accurate classification at the single-patient level, even at early disease stages. Keywords: brain networks; glucose metabolism; Parkinson’s disease; differential diagnosis




Corresponding author. Tel: þ1-516-5622498; Fax: þ1-516-5621008; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84008-7

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162

Abnormal metabolic networks in Parkinson’s disease Parkinson’s disease (PD) is characterized by the insidious onset and inexorable progression of both motor and cognitive symptoms. Prodromal symp­ toms such as hyposmia or rapid eye movement (REM) behavior disorder might predate the appearance of the classic motor symptoms by years. Characteristic motor manifestations of PD typically appear only after more than 50% of nigrostriatal dopaminergic neurons have been lost (Bernheimer et al., 1973). Thus far there is a paucity of reliable biomarkers to identify preclini­ cal PD or monitor disease progression through its natural history or response to treatment. Functional positron emission tomography (PET) imaging methods have proven useful in fill­ ing this void, particularly at the system-wide level. Spatial covariance analysis of metabolic imaging data acquired with [18F]-fluorodeoxyglucose (FDG) PET has become an important means of detecting network-level functional abnormalities in neurodegenerative disorders such as PD, Huntington’s disease, and Alzheimer’s disease. The details of this approach have been summar­ ized elsewhere (see Eidelberg, 2009 for review). In brief, spatial covariance mapping utilizes principal component analysis (PCA), a multivariate method designed to isolate linearly independent sources of variability in large datasets. In typical multisubject, multi-voxel metabolic imaging data, this approach is applied to a combined sample of scans from patients and healthy subjects to identify one or more spatial covariance patterns that differenti­ ate between the two groups (e.g., Feigin et al., 2007b; Habeck et al., 2008; Ma et al., 2007). The expression of a given disease-related metabolic pattern can be quantified prospectively on an indi­ vidual scan basis through the operation of dot product computation. The resulting subject scores (i.e., pattern expression values) can be used in further investigations of group discrimination, dis­ ease progression, treatment effects, or correlations with independent clinical or physiological indices.

In this chapter we will briefly describe recent advances in imaging analysis and the potential ramifications of this approach on the investigation of PD and related neurodegenerative disorders. The PD-related motor pattern (PDRP) We have recently identified and validated several PD-related spatial covariance patterns involving metabolic changes at key nodes of the cortico­ striato-pallido-thalamocortical (CSPTC) loops and related anatomical/functional pathways (see Eidelberg, 2009; Hirano et al., 2009; Poston and Eidelberg, 2009 for review). By applying network analysis to FDG PET data in the rest state, we have found that the activity of an abnormal meta­ bolic network is elevated in PD patients relative to healthy control subjects (Ma et al., 2007). This pattern is associated with the motor manifesta­ tions of the disease and is characterized by covarying increases in pallido-thalamic and pon­ tine metabolic activity and relative reductions in the premotor cortex, supplementary motor area (SMA), and parietal association areas (Fig. 1a). To date, we have verified the presence of this abnormal PD-related motor pattern (PDRP) in seven independent patient populations scanned under widely different rest-state imaging proto­ cols (Eidelberg, 2009). We have additionally demonstrated that quantitative measures of PDRP expression are highly reproducible in indi­ vidual patients undergoing repeat imaging proce­ dures (Ma et al., 2007). It is important to note that not only does PDRP expression accurately discriminate between PD patients and healthy controls, but individual sub­ ject scores consistently correlate with composite Unified Parkinson’s Disease Rating Scale (UPDRS) motor ratings in different PD popula­ tions (Asanuma et al., 2006; Eidelberg et al., 1994, 1995; Feigin et al., 2002, Lozza et al., 2004) (Fig. 1b). In particular, abnormal elevations in PDRP network activity have been linked to the akinetic-rigid manifestations of the disease but

163

not to tremor (Antonini et al., 1998; Isaias et al., 2010, cf. Eidelberg et al., 1990, 1994, 1995). Parkinsonian tremor has recently been found to be associated with a discrete spatial covariance pattern, independent of the PDRP topography. Specifically, in a recent study, we identified a candidate PD tremor-related metabolic pattern (PDTP) using a novel within-subjects network mapping approach (Habeck and Stern, 2007). Using supervised principal components analysis (PCA), we detected a highly significant metabolic pattern with consistently lower expression during ventro-intermediate (Vim) thalamic nucleus sti­ mulation (“on”) relative to baseline (“off”). The expression of this pattern, which was characterized by metabolic increases in the cerebellum/dorsal pons, caudate/putamen, and primary motor cortex (Fig. 1c), correlated with tremor severity (Fig. 1d) but not with akinesia/rigidity measures. PDTP expression measured in PD patients scanned with 99m Tc-ECD single photon emission computed tomography (SPECT) perfusion imaging (Isaias et al., 2010) was selectively elevated in tremordominant PD patients as compared to their aki­ netic-rigid counterparts and to healthy control subjects. As will be described below, elevations in PDRP expression are specific for idiopathic PD and can be used to differentiate this condition from atypi­ cal parkinsonian syndromes (Tang et al., 2010b). Moreover, the activity of this network may pre­ cede the onset of motor symptoms by several years (Tang et al., 2010a). That said, other PDrelated metabolic networks can be expressed as part of the natural history of this disorder. The PD-related cognitive pattern (PDCP) Cognitive deficits and behavioral abnormalities are also well documented in PD and can have a major impact on quality of life (Aarsland et al., 2005; Schrag et al., 2000), but the pathological basis of cognitive impairment in PD remains con­ troversial (Emre, 2003). Ample evidence exists for

Alzheimer’s disease (AD)-type changes in cogni­ tively impaired PD patients (Jellinger et al., 2002). More recent studies utilizing a-synuclein (aSN) immunostaining have demonstrated that cortical Lewy body pathology is likely to be the most critical feature of this clinical syndrome (Braak et al., 2003; Hurtig et al., 2000). Indeed, the minimental status examination, a coarse description of cognitive status, correlates with the magnitude and distribution of Lewy body pathology at post­ mortem examination (Braak et al., 2005). Metabolic imaging in the resting state has proved useful in investigating the basis for impaired cognitive functioning in PD. Using reststate FDG PET and network analysis, we identi­ fied a distinct cognition-related spatial covariance pattern in non-demented PD patients (Huang et al., 2007a). This PD-related cognitive pattern (PDCP) is characterized by covarying reductions in metabolic activity involving the rostral supple­ mentary motor area (pre-SMA), prefrontal cor­ tex, precuneus, and parietal association regions, with relative increases in the cerebellar vermis and dentate nuclei (Fig. 1e). Quantitative mea­ sures of PDCP expression have been found to correlate with subject performance on neuropsy­ chological tests of executive functioning such as the California and Hopkins Verbal Learning Tests (CVLT, HVLT), Trails B, and Stroop (color) tests (Fig. 1f) (Huang et al., 2007a). Like PDRP scores, PDCP expression exhibits excellent test–retest reproducibility in patients undergoing repeat FDG PET over an 8-week period. Moreover, by computing PDCP expression in a prospective group of non-demented PD patients with and without minimal cognitive impairment (MCI) on neuropsychological testing, we found that PDCP scores were higher in the PD patients with MCI than in those who were cognitively intact (Huang et al., 2008, see Eidelberg, 2009 for review). The relationship between abnormal PDCP expression in the resting state and changes in brain deactiva­ tion during the performance of cognitive tasks (Argyelan et al., 2008) is a topic of ongoing investigation.

164

Network activity evolves with disease progression The prolonged asymptomatic state in PD patients with extensive brainstem Lewy body pathology suggests that the brain can summon effective com­ pensatory mechanisms for quite some time (Bezard et al., 2003; Smith and Zigmond, 2003). That said, the functional changes in CSPTC loops

PD-related motor pattern

Gp/Put

B

50

Motor UPDRS (total)

A

and related pathways that develop as a conse­ quence of pre-symptomatic loss of SNc neurons are not well understood (e.g., Buhmann et al., 2003; Hirano et al., 2008, cf. Moeller and Eidel­ berg, 1997), and little is known about metabolic changes associated with the onset of clinical symp­ toms of this disease. To approach these questions, we made use of the fact that PD typically presents

PMC

Thalamus D

Putamen

Pre-SMA PMC

Cerebellum/DN

Fig. 1 (continued)

Parietal

2 3 4 PDRP expression

5

12

8

4

0

−4

0

F California verbal learning test (sum)

Precuneus

PD-related cognitive pattern

10

16

SMC

E

20

1

Motor UPDRS (tremor)

PD-related tremor pattern

30

0

Parietal C

40

1

2 3 4 PDTP expression

5

80

60

40

20

0

−1

0

1 2 3 4 PDCP expression

5

6



165

unilaterally (i.e., hemiparkinsonism) to assess metabolic changes in the brain hemisphere con­ tralateral to the unaffected side. In a recent study (Tang et al., 2010a), we assessed hemispheric progression in a cohort of 15 hemiparkinsonian patients undergoing serial imaging with FDG PET at baseline and again after 2- and 4-years’ follow-up. We separately quantified the activity of the PDRP and PDCP metabolic networks in the cerebral hemispheres ipsilateral and contralateral to the initial clinical signs at each of the three longitudinal time points. These measurements allowed for the determina­ tion of the time course of network progression in each hemisphere, and on the ipsilateral side, the specific metabolic changes associated with symp­ tom onset. Contrary to expectation, we found sig­ nificant baseline elevations in PDRP expression on the ipsilateral (“preclinical”) side (Fig. 2a), preceding the appearance of motor signs on the opposite body side by approximately 2 years. By contrast, expression of the PDCP network did not

reach abnormal levels until the last time point (Fig. 2b), which was approximately 4 years after PDRP elevation. Notably, significant PDCP eleva­ tions were evident in both hemispheres several years before the typical onset of mild cognitive impairment (MCI) in PD (Eidelberg, 2009; Huang et al., 2008). Furthermore, examination of whole-brain net­ work activity in this longitudinal cohort demon­ strated that PDRP expression increases linearly over time (Fig. 2c) and is accompanied by com­ mensurate increases in UPDRS motor ratings. PDCP expression also increases over time, but at a significantly slower rate than for PDRP scores. Interestingly, the longitudinal changes in PDTP expression are yet more gradual, corresponding to the relatively slower rate of progression docu­ mented for tremor in PD (Louis et al., 1999). The three PD-related metabolic networks (PDRP, PDCP, and PDTP) thus appear to capture unique clinical and mechanistic features of the disease process.

Fig. 1. Abnormal metabolic networks in Parkinson’s disease. (a) PD-related motor pattern (PDRP) identified by spatial covariance analysis of [18F]-fluorodeoxyglucose (FDG) PET scans from 33 PD patients and 33 age-matched normal volunteer subjects. This pattern is characterized by relative hypermetabolism (red) in the globus pallidus/putamen (GP/Put), thalamus, pons, cerebellum, and sensorimotor cortex, associated with metabolic decreases (blue) in the lateral premotor cortex (PMC) and parieto-occipital association regions (Ma et al., 2007). Representative slices of the covariance map were overlaid on a standard MRI brain template. (b) PDRP expression correlated with composite Unified Parkinson’s Disease Rating Scale (UPDRS) motor ratings in each of the three independent, prospectively imaged patient groups (circles: n = 27; r = 0.66; p < 0.001; squares: n = 15; r = 0.65, p < 0.01; triangles: n = 23; r = 0.76, p < 0.001), as well as in the combined cohort (n = 65; r = 0.68, p < 0.001). In each group, PDRP scores correlated with subscale ratings for akinesia/rigidity but not with tremor ratings (Asanuma et al., 2006, Lozza et al., 2004, Eidelberg et al., 1995). (c) PD-related tremor pattern (PDTP) identified by supervised principal components analysis (PCA) (Habeck and Stern, 2007) of FDG PET scans from nine tremor-predominant PD patients scanned at baseline and during high-frequency deep brain stimulation (DBS) of the ventralintermediate (Vim) thalamic nucleus. This pattern is characterized by relative hypermetabolism of sensorimotor cortex (SMC), cerebellum, pons, and putamen. Representative slices of the covariance map were overlaid on a standard MRI brain template. (d) PDTP expression correlated with UPDRS tremor ratings in a prospective group of PD patients (n = 35; r = 0.53, p = 0.001). (e) PD-related cognitive pattern (PDCP) identified by spatial covariance analysis of FDG PET scans from 15 non-demented PD patients with mild-to­ moderate motor symptoms. This pattern is characterized by relative hypometabolism (blue) in the rostral supplementary motor area (preSMA), precuneus, premotor cortex (PMC), posterior parietal and prefrontal regions, associated with metabolic increases (red) in the cerebellar/dentate nucleus (DN) (Huang et al., 2007a). Representative slices of the covariance map were overlaid on a standard MRI brain template. (f) PDCP expression correlated with performance on California Verbal Learning Test (sum) in the original group for pattern derivation (circles; n = 15: r = 0.71, p < 0.005) and in each of the two prospective validation groups (squares; n = 25: r = 0.53, p < 0.01; triangles; n = 16: r = 0.80, p < 0.001) (Huang et al., 2007a). The correlation was also significant in the combined cohort (n = 56; r = 0.67, p < 0.001). Subject scores for each of the three networks were z-transformed so that the normal mean is zero and standard deviation is 1. [a, b, e, and f: Reprinted from Trends Neurosci, Metabolic brain networks in neurodegenerative disorders: a functional imaging approach, 548–557, Copyright 2009, with permission from Elsevier; c and d: courtesy of Dr. H. Mure]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this book.)

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Fig. 2. Changes in network activity with disease progression. (a) Mean PDRP expression in the hemispheres contralateral (circles) and ipsilateral (triangles) to the initially affected limbs in 15 hemiparkinsonian patients who underwent FDG PET at baseline, 2, and 4 years (Tang et al., 2010a). Relative to healthy controls, PDRP expression in the PD patients was abnormally elevated (p < 0.05) in both hemispheres relative to controls at baseline when motor symptoms only appeared on one side of the body; network activity continued to increase in parallel over the course of the study (p < 0.001). (b) By contrast, mean PDCP expression reached abnormally elevated levels (p < 0.01) in both the contralateral (circles) and ipsilateral (triangles) hemispheres only at the final time point. In both hemispheres, PDCP network activity increased in parallel over time (p < 0.005). For both PDRP and PDCP, subject scores in each hemisphere were z-transformed so that the normal mean is zero and standard deviation is 1. (c) Mean activity of the PD-related motor (PDRP), cognitive (PDCP), and tremor (PDTP) spatial covariance patterns at baseline, 2, and 4 years. Network activity increased significantly over time for all three patterns (PDRP: p < 0.0001; PDCP: p < 0.0001; PDTP: p = 0.01), but at different rates (p < 0.01). Of the three patterns, PDRP expression progressed most rapidly while PDTP progression was the slowest, corresponding to the concurrent clinical changes observed in this cohort. Subject scores for each of the three networks were z-transformed so that the normal mean is zero and standard deviation is 1. [a and b: Adapted from J Neurosci, Abnormalities in metabolic network activity precede the onset of motor symptoms in Parkinson’s disease, 1049–1056, Copyright 2010, with permission from the Society for Neuroscience. PDTP, courtesy of Dr. H. Mure].

Assessing treatment effects with network activity Dopaminergic treatment Quantitative imaging measures such as metabolic network activity can also be valuable for the objective assessment of treatment efficacy. To qualify as treatment biomarkers in clinical trials, metabolic networks should exhibit consistent change with therapeutic interventions, ideally at the individual subject level. This attribute has been demonstrated for PDRP expression, in that treatment-mediated changes in network activity have been shown to correlate with clinical improvement in UPDRS motor ratings in patients undergoing dopaminergic treatment (Feigin et al., 2001), deep brain stimulation (Asanuma et al., 2006; Fukuda et al., 2001), and gene therapy (Feigin et al., 2007a) (Fig. 3a).

In general, network expression values derived from metabolic scans such as FDG PET correlate closely with those derived from measures of cere­ bral blood flow (H15 2 O PET, arterial spin-labeled MRI) in the same subjects. It is thus particularly interesting that patients on levodopa/carbidopa–– but not other therapies, including DBS––show a significant dissociation between changes in PDRP expression measured in cerebral metabolism scans and that measured in cerebral blood flow scans (Hirano et al., 2008). Specifically, patients receiv­ ing levodopa/carbidopa show reductions in PDRP expression in the former (FDG PET), but increases in the latter (H15 2 O PET) following acute treatment. Notably, levodopa-mediated dissociation of cerebral blood flow and metabolism was found to be greatest in PD patients with levo­ dopa-induced dyskinesias (LID). We propose that the observed flow–metabolism dissociation results

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Fig. 3. Assessment of treatment effects with network activity. (a) Treatment-mediated changes in mean PDRP expression. Left panel shows network modulations after the levodopa infusion (LD, gray), the bilateral deep brain stimulation of the subthalamic nucleus (STNBi DBS, black), and for the test–retest PD controls (CN, white) (Asanuma et al., 2006). Right panel shows network modulations after the unilateral DBS (filled black) or lesioning (stripe black) of the internal globus pallidus (GPi) or STN (Fukuda et al., 2001; Trost et al., 2006). Error bar represents the SEM.
p < 0.05,

p < 0.01. (Adapted from Brain, Network modulation in the treatment of Parkinson’s disease, 2667–2678, Copyright 2006, with permission from Oxford University Press.) (b) Changes in mean PDRP network activity over time for the operated (filled circles) and unoperated (open circles) hemispheres after gene therapy. There was a significant difference (p < 0.005) in the time course of PDRP activity across the two hemispheres. In the unoperated hemisphere, network activity increased continuously over the 12 months following surgery. By contrast, in the operated hemisphere, network activity declined during the first 6 months and then increased in parallel with values on the unoperated side over the subsequent 6 months. The dashed line represents one standard error above the normal mean value of 0. (c) By contrast, there was no change (p = 0.72) in PDCP network activity in either of the two hemispheres over time. The dashed line represents one standard error above the normal mean value of zero. [b and c: Reprinted from PNAS, Modulation of metabolic brain networks after subthalamic gene therapy for Parkinson’s disease, 19559–19564, Copyright 2007, with permission from the National Academy of Science]. Subject scores for each network were z-transformed so that the normal mean is 0 and standard deviation is 1.

from neurovascular alterations (i.e., dopaminergic vasodilation) that likely underlie LID in PD patients. Further study is needed to understand the cause of LID, whether flow–metabolism dissociation occurs with other forms of dopaminergic

therapy, and whether this side effect can be miti­ gated by antidyskinetic agents. Levodopa has been found not to modulate PDCP expression at the group mean level in PD patient populations (Huang et al., 2007a). However, by

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analogy to our observations in PD patients scanned while learning motor sequences (Argyelan et al., 2008), PDCP modulation is likely to be baseline dependent. Indeed, preliminary evidence suggests that cognition-related resting-state cerebral func­ tion at both the regional and network levels can be pharmacologically modulated based upon the extent of the metabolic abnormality present in the unmedicated condition. Currently, prospective stu­ dies are underway to investigate the effect of base­ line PDCP expression on the cognitive response to medication on an individual patient basis. Deep brain stimulation and microlesion effect Deep brain stimulation (DBS) of the subthalamic nucleus (STN) has proven to be highly effective for advanced PD motor symptoms (Benabid et al., 2009). Indeed, DBS interventions at STN and internal globus pallidus (GPi) have been shown to modulate the activity of the PDRP metabolic network (Fig. 3a), with significant correlations between reductions in pattern expression and clin­ ical improvement in motor function (Asanuma et al., 2006; Fukuda et al., 2001; Trost et al., 2006). Notably, there is an association between PDRP expression and spontaneous firing rates recorded during stereotaxic surgery (Lin et al., 2008, cf. Eidelberg et al., 1997). This likely reflects the pathophysiological basis of this disease-related motor network abnormality (Eberling et al., 2008). It is worth noting that the magnitude of PDRP modulation is comparable for STN DBS and levodopa treatments but that combining the two therapies confers no additional benefit. This is consistent with the notion that the two interven­ tions exert their therapeutic benefits through the same mechanistic pathway. It is also worth noting that electrode implanta­ tion itself, without stimulation, can induce a microlesion effect on pallido-thalamic brain function (Pourfar et al., 2009) analogous to that seen following therapeutic STN lesioning (subthala­ motomy) (Trost et al., 2003, 2006). However, the

magnitude of this highly localized metabolic change is not strong enough to produce consistent changes in PDRP expression or significant clinical benefit (Pourfar et al., 2009). These data suggest that a minimum threshold for PDRP modulation exists and is necessary for a positive outcome to occur following treatment. Moreover, treatment effects on network activity appear to be highly selective. For instance, we have recently noted that high-frequency stimula­ tion of the Vim thalamic nucleus, while highly effective for parkinsonian tremor, had little effect on akinesia or rigidity. Accordingly, Vim DBS was found to have a significant effect on PDTP but not PDRP activity (H. Mure, personal communica­ tion). STN DBS, by contrast, improved both PD tremor and akinesia/rigidity and was associated with substantial reductions in the activity of both metabolic networks. Gene therapy Animal studies suggested that transfer of the glu­ tamic acid decarboxylase (GAD) gene into the STN can suppress spontaneous neural activity in this region and increase GABA release in down­ stream areas (Lee et al., 2005; Luo et al., 2002), leading to improvement in parkinsonian motor manifestations (Emborg et al., 2007). In a subse­ quent Phase I clinical trial, adeno-associated virus (AAV) was used to deliver the GAD gene uni­ laterally into the STN of advanced PD patients (Kaplitt et al., 2007). Each subject underwent clin­ ical evaluation and FDG PET at three time points: before surgery, then at 6 and 12 months after surgery (Feigin et al., 2007a). At baseline, hemi­ spheric PDRP expression was elevated bilaterally. Following gene transfer, this network abnormality was suppressed on the treated side (Fig. 3b), with concomitant improvement in contralateral limb motor ratings. In the unoperated hemisphere, however, network activity increased over time following surgery, consistent with disease progres­ sion (cf. Huang et al., 2007b). Gene therapy did

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not alter PDCP expression in either hemisphere (Fig. 3c), in accordance with the absence of cogni­ tive change in these patients. Given these findings, a blinded sham-surgery controlled Phase II study of bilateral STN AAV-GAD gene therapy is cur­ rently underway for advanced PD motor symp­ toms. FDG PET studies are being conducted under the blind, with results to become available in the latter half of 2010. Together, the findings of these studies suggest that abnormal metabolic networks can be used as imaging biomarkers for assessing clinically mean­ ingful treatment effects as well as for understand­ ing the pathophysiological mechanisms underlying these therapies. Network analysis may also be useful in evaluating novel treatments in clinical trials for PD.

Differential diagnosis of parkinsonian conditions The challenge of early parkinsonian symptoms The classic parkinsonian clinical triad of rigidity, resting tremor, and bradykinesia is not limited to PD; other atypical parkinsonian syndromes (APSs), such as multiple system atrophy (MSA) and progressive supranuclear palsy (PSP), can produce very similar clinical signs especially in the early stages when signs are mild. Pathological studies indicate that up to 25% of patients clini­ cally diagnosed to have PD actually have a differ­ ent disease; approximately 80% of misdiagnoses turn out to be MSA and PSP (Hughes et al., 2001a, 2002). Conversely, clinically misdiagnosed MSA and PSP cases are often found to display Lewy body changes at postmortem (Osaki et al., 2002, 2004). Clinicopathologic studies of patients with par­ kinsonism have found that the positive predictive value (PPV) for an initial clinical diagnosis of PD can be as low as 75%, although the PPV improves drastically to 98.6% after patients are followed over 2 years by movement disorders specialists (Hughes et al., 1992a, 2001b, 2002). While strict

diagnostic guidelines have improved the PPV for a diagnosis of MSA or PSP, the sensitivity for these diagnoses at initial clinical visit with a movement disorder specialist remains low (<70%) for both disorders (Osaki et al., 2002, 2004). More problematically, up to 15% of patients enrolled in large clinical trials for early PD can end up with a different diagnosis after long-term clinical follow-up (see e.g., Fahn et al., 2004; Parkinson Study Group, 2002; Whone et al., 2003). It is clear that diagnostic biomarkers to differentiate PD from APS, if validated, will help assure the accuracy of clinical trials to assess disease modification in early-stage PD patients (Tang et al., 2010b). Most neuroimaging techniques for the differen­ tial diagnosis of parkinsonism have not been shown to reliably discriminate between idiopathic PD and APSs in early-stage patients, particularly before the diagnosis is achieved by clinical evalua­ tion. Some imaging techniques, such as SPECT and transcranial sonography (TCS), are able to discriminate patients with parkinsonism from nor­ mal volunteer subjects, or to establish group mean level separation between diagnostic classes––but have not achieved accurate individual diagnosis in de novo patients (see e.g., Doepp et al., 2008; Vlaar et al., 2007). In part, this is because such techniques measure nigrostriatal dopaminergic projections and/or localized structural/functional changes in the basal ganglia, which are also evi­ dent in atypical neurodegenerative syndromes such as MSA and PSP. Differentiating PD, MSA, and PSP Pattern analysis of metabolic images can provide an unbiased whole-brain evaluation of functional changes in the basal ganglia and in interconnected brain regions. This is potentially a more accurate means for differentiating between parkinsonian syndromes by capturing abnormal metabolic changes in network regions known to be specifi­ cally related to each disease process. As such, we

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hypothesized that pattern-based imaging classifi­ cation can provide accurate differential diagnosis for patients with parkinsonism several years before a final clinical diagnosis is made. First, we identified specific disease-related metabolic patterns for MSA and PSP (termed MSARP and PSPRP, respectively) (Eckert et al., 2008). The MSARP is characterized by bilateral metabolic reductions in the putamen and cerebel­ lum (Fig. 4a, left); the PSPRP is characterized by metabolic decreases predominately in the upper brainstem, medial prefrontal cortex, and medial thalamus (Fig. 4c, left). These patterns accurately discriminate between patients and healthy con­ trols on a prospective single-case basis (Fig. 4b and d) (Eckert et al., 2008; Spetsieris et al., 2009). To determine whether these patterns would prove useful in discriminating between PD, PSP, and MSA in patients with early-stage disease, we recruited and scanned 167 patients with parkin­ sonism who were referred to our center for FDG PET by movement disorders specialists because of uncertain clinical diagnosis (Tang et al., 2010b). After PET, all patients were followed clinically by movement disorders specialists for an average of 2.6 + 2.4 (mean + SD) years before a final clinical diagnosis was reached. For every scan, we com­ puted the expression (i.e., subject scores) of the PDRP, MSARP, and PSPRP separately on an individual scan basis. Utilizing these pattern scores in conjunction with logistic regression mod­ els, we then developed a two-level, automated algorithm to classify the individual subjects between PD, MSA, and PSP. For first-level analysis, we combined the clini­ cally diagnosed MSA and PSP patients into a single APS group. We then employed a logistic regression model to discriminate between the PD and APS groups and to compute the probabilities for having PD and APS for all individuals. These probabilities were used to determine the diagnos­ tic criteria for the image-based classification of PD vs. APS for each subject. For second-level analy­ sis, subjects classified as APS were further ana­ lyzed to differentiate between MSA and PSP.

Logistic regression models were also used for dif­ ferentiation of MSA vs. PSP and for computation of probabilities for these two disorders in each APS subject. Subsequently, these probabilities were used to determine the diagnostic criterion for the image-based classification of MSA vs. PSP for each subject. By comparing the imagebased classification for each patient to the final clinical diagnosis obtained after several years of clinical follow-up, we calculated discriminative measures––sensitivity, specificity, PPV, and nega­ tive predictive value (NPV)––for each of the three disorders. We found that in our patient cohort, the imagebased classification results were highly specific and accurate in discriminating among PD, MSA, and PSP. Imaging classification exhibited 84% sensitivity, 97% specificity, 98% PPV, and 82% NPV for the clinical PD subjects. It exhibited 85% sensitivity, 96% specificity, 97% PPV, and 83% NPV for the MSA subjects, and 88% sensi­ tivity, 94% specificity, 91% PPV, and 92% NPV for the PSP subjects. We further divided the subjects into subgroups according to the duration of their disease at the time of scanning and by the duration of follow-up. The classification results remained excellent (>90% specificity) even in the subgroups of early patients with very short symptom durations (i.e., <2 years), whose clinical diagnoses were sub­ sequently confirmed after more than 2-year followup by movement disorders specialists. Moreover, there was excellent agreement between the imaging classifications obtained in a group of patients who underwent repeat PET imaging separated by an average of 3.1 + 2.2 (SD) years. Imaging classifica­ tion was also confirmed in a small subgroup of subjects at autopsy (Fig. 4a and c, right). The results of this study indicate that our auto­ mated algorithm with FDG PET can accurately differentiate PD, MSA, and PSP in patients with parkinsonism prior to the development of clini­ cally diagnostic symptoms and signs. Our findings support the use of FDG PET and pattern analysis in the identification of patients with PD or APS

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Fig. 4. Abnormal metabolic networks in atypical parkinsonian syndromes and postmortem findings. (a). Multiple system atrophy-related pattern (MSARP; left) identified by spatial covariance analysis of FDG PET scans from 10 MSA patients and 10 healthy controls. This pattern is characterized by covarying metabolic decreases (blue) in the putamen and the cerebellum (Eckert et al., 2008). Representative slices of the covariance map were overlaid on a standard MRI brain template. Neuropathological findings (right) from a patient classified as MSA with a likelihood of 98% by the automated differential diagnosis algorithm based on an FDG PET scan performed 3 years before death (Tang et al., 2010b). Autopsy revealed characteristic changes of neuronal loss and gliosis in the putamen (top) and cerebellum (bottom). Both regions displayed glial cytoplasmic inclusions (Gallyas stain, 200×). Insets show areas of higher magnification (putamen, 400×; cerebellum, 630×). (b) MSARP expression was significantly elevated (p < 0.001) in the training group of 10 MSA patients (open diamonds) relative to the 10 healthy controls (open circles) that were used for pattern derivation. Similarly, pattern expression was elevated (p < 0.001) in the two testing groups of MSA patients (closed diamonds) relative to a testing group of healthy controls (closed circles). Mean and standard deviation are also displayed for each group. (c) Progressive supranuclear palsy-related pattern (PSPRP; left) identified by spatial covariance analysis of FDG PET scans from 10 PSP patients and 10 healthy controls. This pattern was characterized by covarying metabolic decreases (blue) in the medial prefrontal cortex, the frontal eye fields, the ventrolateral prefrontal cortex, the caudate nuclei, the medial thalamus, and the upper brainstem (Eckert et al., 2008). Neuropathological findings (right) from a patient classified as PSP with a likelihood of 99% by the automated differential diagnosis algorithm based on an FDG PET scan performed 3.9 years before death (Tang et al., 2010b). Postmortem examination confirmed this diagnosis, with characteristic histopathological changes in the pons (top) and frontal cortex (bottom). Argyrophilic globosum neuronal tangles were noted in the basis pontis (Bielschowsky stain 400×). A neuronal tangle with cytoplasmic inclusions and neuropil threads is displayed from the fifth cortical layer of the prefrontal region (AT8 stain, 630×). Tufted astrocytes (not shown) were present in this cortical region, the amygdala, globus pallidus, and claustrum. (d) PSPRP expression was significantly elevated (p < 0.001) in the training group of 10 PSP patients (open diamonds) relative to the 10 healthy controls (open circles) that were used for pattern derivation. Similarly, pattern expression was elevated (p < 0.001) in the two testing groups of PSP patients (closed diamonds) relative to a testing group of healthy controls (closed circles). Mean and standard deviation are also displayed for each group. a and c: Reprinted from Lancet Neurol, Differential diagnosis of parkinsonism: a metabolic imaging study using pattern analysis, 149–158, Copyright 2010, with permission from Elsevier; b and d: Reprinted from Mov Disord, Abnormal metabolic networks in atypical parkinsonism, 727–733, Copyright 2008, with permission from John Wiley & Sons, Inc. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this book.)

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(MSA or PSP) who would be ideal candidates for clinical trials, particularly those aimed at evaluat­ ing disease-modifying agents in the earliest phase of the disease. This technique could also be bene­ ficial for diagnostic confirmation of PD prior to invasive treatments, such as DBS and gene trans­ fer therapy, which are likely to be less effective or possibly deleterious in atypical patients (Shih and Tarsy, 2007). In addition, the incorporation of a fully automated algorithm enables our approach to be unbiased by clinical impression and does not require special technical expertise in carrying out the test. Future directions There are three main conclusions we want to draw from the results discussed here. First, network analysis on human FDG PET data has revealed three specific disease-related spatial covariance patterns that are associated with distinct clinical manifestations in PD and faithfully trace the pro­ gression and treatment response of these different aspects of the disease (akinetic-rigid motor mani­ festations, tremor, and cognitive dysfunction). Second, comparison of networks for IPD, MSA, and PSP within a fully automated algorithm enables surprisingly accurate differential diagnosis of individual patients with parkinsonism even in early stages of disease. Third, these studies shed light on the mechanisms of compensatory changes in preclinical period and potentially those that mediate the side effects of chronic treatment such as LID. In this vein, it will be interesting to use this approach to study individuals at risk of developing PD, such as those with REM sleep behavior disorder (Albin et al., 2000; Eisensehr et al., 2000). In particular, it is relevant to know whether they display abnormalities in the expression of established metabolic networks, or whether they exhibit novel patterns that are specific to this preclinical PD population. It would also be interesting to investigate the pathophysiological basis of PDCP expression in

non-demented PD patients. For example, do the metabolic reductions observed regionally in fron­ tal and parietal association cortices reflect local histopathological change within these brain areas, or the functional effects of the loss of ascending dopaminergic and/or cholinergic pro­ jections? The involvement in the PDCP of the neo-cortical regions with the earliest histopatholo­ gical changes (Braak et al., 2003, 2005) is consis­ tent with the first possibility, although the latter cannot be excluded in individual patients with sig­ nificant pharmacological modulation of network activity. Moreover, other histopathological changes can also contribute to the regional abnormalities observed within this network. For instance, some of these regions may be directly or indirectly involved by coincident Alzheimer-type pathology (Jellinger et al., 2002). To address these issues, a multi-tracer PET approach is being con­ ducted to compare the cortical metabolic reduc­ tions seen in non-demented PD patients with the estimates of local protein aggregation in the same subjects. The goal of the study will be to deter­ mine whether abnormal cortical metabolic activity is associated with the accumulation of aggregated protein deposits, and whether those changes impact upon the response of PD patients to phar­ macological interventions targeting the cognitive manifestations of this disorder. Our automated differential diagnosis algorithm can potentially be expanded to include other atypical parkinsonian conditions, e.g., cortico­ basal ganglionic degeneration (CBGD). Although MSA and PSP account for the majority of APSs (Hughes et al., 1992b, 2002), CBGD accounts for approximately 5% of APSs. Because of the rarity of this and other APS conditions, it will take longer to identify and validate disease-related metabolic patterns for these unusual conditions. That said, the availability of antemortem FDG PET scans from patients with autopsy-proven diagnoses has facilitated the characterization of new and highly specific network biomarkers for CBGD and other less frequently encountered par­ kinsonian variant disorders. It will certainly be

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valuable to “update” the present differential diag­ nosis algorithm with new disease-related patterns as they become available. More relevant perhaps will be to test the performance of this pattern recognition method in a broader multi-center con­ text. The ultimate validity of this and related diag­ nostic methods rests on the study of strictly defined populations under blinded conditions. Most critically, rigorous case ascertainment proce­ dures will be required over long-term clinical fol­ low-up, ideally with pathological confirmation.

Vim

Acknowledgments This work was supported by the National Insti­ tutes of Health [NINDS R01 NS 35069 and P50 NS 38370 to D.E.] and the General Clinical Research Center of The Feinstein Institute for Medical Research, North Shore-LIJ Health Sys­ tem [National Center for Research Resources (NCRR), a component of the National Institutes of Health, M01 RR018535]. The authors wish to thank Dr. Vicky Brandt, Mr. Noam Gerber, and Ms. Toni Fitzpatrick for valuable editorial assistance.

List of Abbreviations AAV APS CBGD CSPTC DBS FDG GAD LID MCI MSA MSARP NPV

PET PD PDCP PDRP PDTP PPV PSP PSPRP REM STN UPDRS

adeno-associated virus atypical parkinsonian syndromes cortico-basal ganglionic degeneration cortico-striato-pallido­ thalamocortical deep brain stimulation [18F]-fluorodeoxyglucose glutamic acid decarboxylase levodopa-induced dyskinesias mild cognitive impairment multiple system atrophy MSA-related pattern negative predictive value

positron emission tomography Parkinson’s disease PD-related cognitive pattern PD-related motor pattern PD-related tremor pattern positive predictive value progressive supranuclear palsy PSP-related pattern rapid eye movement the subthalamic nucleus Unified Parkinson’s Disease Rating Scale ventral-intermediate

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 9

Imaging the nigrostriatal system to monitor disease progression and treatment-induced complications Renju Kuriakose and A. Jon Stoessl
Pacific Parkinson’s Research Centre, University of British Columbia and Vancouver Coastal Health,

Vancouver, BC, Canada

Abstract: Radiotracer imaging (RTI) techniques such as positron emission tomography (PET) allow the in vivo assessment of nigrostriatal DA function in Parkinson’s disease and have provided valuable insights into the mechanisms of nigrostriatal degeneration and the consequent compensatory changes. Moreover, functional imaging serves as an excellent tool in the assessment of the progression of PD and the evolution of treatment-related complications. However, various studies have shown discordance between clinical progression of PD and nigrostriatal degeneration estimated by PET or SPECT, and no RTI technique can be reliably used as a biomarker for progression of PD. Presynaptic dopaminergic imaging has consistently demonstrated an anterior–posterior gradient of dopaminergic dysfunction predominantly affecting the putamen, with side-to-side asymmetry in tracer binding. Dopaminergic hypofunction in the striatum follows a negative exponential pattern with the fastest rate of decline in early disease. Evaluation of central pharmacokinetics of levodopa action by PET has demonstrated the role of increased synaptic dopamine turnover and downregulation of the dopamine transporter in the pathophysiology of levodopa­ induced dyskinesias. In PD with behavioral complications such as impulse control disorders, increased levels of dopamine release have been observed in the ventral striatum during performance of a positive reward task, as well as loss of deactivation in orbitofrontal cortex in response to negative reward prediction errors. This suggests that there is a pathologically heightened “reward” response in the ventral striatum together with loss of the capacity to respond to negative outcomes. Overall, functional imaging with PET is an excellent tool for understanding the disease and its complications; however, caution must be applied in interpretation of the results. Keywords: Positron emission tomography; dopamine turnover; dopamine transporter (DAT); fluorodopa; vesicular monoamine transporter type 2 (VMAT2); biomarker; compensation; dopamine receptors; fluctua­ tions; dyskinesias; impulse control disorders
Corresponding author.

Tel.: þ1-604-822-7967;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84009-9

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Introduction Parkinson’s disease (PD) is one of the most common neurodegenerative disorders, with a prevalence rate of more than 1 in 100 among affected persons above the age of 65 years (de Rijk et al., 2000). Clinically PD is characterized by symptoms of bradykinesia, resting tremor, rigidity, and postural instability (Calne et al., 1992). The pathophysiological hallmark of PD is degeneration of nigrostriatal pathway leading to dopamine (DA) deficiency (Hornykiewicz, 1998). PD symptoms appear when 80% of the striatal DA or 50% of nigral cells are lost (Bernheimer et al., 1973; Fearnley & Lees, 1991). Dopamine replace­ ment therapies, which include the DA precursor levodopa and DA agonists, are very effective in treating motor symptoms, but can cause substantial motor and behavioral adverse events. These side-effects include motor fluctuations and levodopa-induced dyskinesia (LID) and non-motor symptoms such as mood and anxiety fluctuations, psychosis, and impulse control disorders (ICDs) (Voon et al., 2009). LIDs are defined as involuntary, purposeless, irregular but sometimes repetitive movements, which are mainly choreic, and generally coincide with the peak anti-parkinsonian effect of levodopa (Obeso et al., 2007). LIDs affect at least 90% of patients with PD after 10 years of levodopa treat­ ment (Fabbrini et al., 2007) and are a major cause of disability. ICDs (i.e., pathological gambling, compulsive shopping, hypersexuality, and binge eating), punding (i.e., abnormal repetitive non-goal-oriented behaviors), or hobbyism, and compulsive medication use are associated with dopaminergic therapy and are increasingly recog­ nized in PD (Avanzi et al., 2006; Grosset et al., 2006; Miyasaki et al., 2007; Pezzella et al., 2005; Voon et al., 2006; Weintraub et al., 2006). Radio­ tracer imaging (RTI) techniques such as positron emission tomography (PET) and single photon emission computerized tomography (SPECT) allow the in vivo assessment of nigrostriatal DA function and have provided valuable insights into the mechanisms of nigrostriatal degeneration and the consequent compensatory changes

(Nandhagopal et al., 2008). These techniques also help to assess the progression of disease and eva­ luation of treatment interventions (Au et al., 2005).

Neuroimaging of the nigrostriatal system Biochemistry of dopamine function A basic knowledge of biochemistry of DA meta­ bolism is essential to understand the imaging of nigrostriatal DA function. The first step in DA synthesis is the conversion of tyrosine to L-3-4­ dihydroxyphenylalanine (L-dopa). Exogenously administered L-dopa crosses the blood–brain bar­ rier via the large neutral amino acid transporter. Striatal uptake of L-dopa requires active transport and its further conversion to DA is catalyzed by L-aromatic amino acid decarboxylase (AADC). Vesicular monoamine transporter type 2 (VMAT2) pumps both newly synthesized and recycled DA into presynaptic vesicles. Vesicular storage helps to maintain the molecular integrity of neurotransmit­ ters by preventing their catabolism to potentially toxic compounds. Axonal depolarization leads to exocytotic release of DA into the extracellular space, where it interacts with pre- and post-synaptic DA receptors. The molecular effects of DA are terminated by conversion via methylation and oxi­ dative deamination to homovanillic acid and also (indeed primarily) by reuptake into presynaptic terminals from the synaptic cleft. The membrane DA transporter (DAT) mediates this reuptake, following which DA is recycled into storage vesi­ cles or converted to inactive metabolites.

Presynaptic imaging There are three different strategies to assess presy­ naptic dopaminergic integrity using radioligands that measure various aspects of striatal DA processing. 6-[18F]-fluoro-L-dopa (F-DOPA) is used as a marker to monitor the uptake and decarboxyla­ tion of F-DOPA to fluorodopamine (FDA), and

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the subsequent storage of FDA in synaptic vesi­ cles. It has been extensively characterized and is widely regarded as the “gold standard” for asses­ sing the integrity of the nigrostriatal DA system. F-DOPA uptake correlates well with nigral cell counts in humans (Snow et al., 1993) and in non­ human primates with MPTP-induced parkinson­ ism (Pate et al., 1993). It also reflects the clinical severity of PD, and correlates well with bradyki­ nesia but not with tremor (Vingerhoets et al., 1997). The standard approach is to scan for 90–120 min following tracer injection, during which time tracer uptake is unidirectional in the normal brain. Prolonged scan times of up to 4 h (during which there is tracer egress) can be used to assess effective DA turnover, which is increased in early PD (Sossi et al., 2002). Biochemically, DA turn­ over is defined as the ratio between DA metabo­ lites and DA. The concept of the effective dopamine turnover (EDT) that is measurable by F-DOPA PET has been developed to estimate DA turnover in vivo (Doudet et al., 1998). The blood to striatum dopa uptake rate constant Ki estimates the rate of DA synthesis and storage (Patlak et al., 1983). Ki reflects a combination of tracer uptake, decarboxylation to FDA, and sub­ sequent trapping in synaptic vesicles, and has been shown to correlate well with the number of DA neurons and the levels of striatal DA (Snow et al., 1993). During the first 90 min after tracer injec­ tion, F-DOPA behaves as an irreversibly bound tracer in healthy normal individuals. Ki is obtained from data acquired during this time. However, with prolonged scanning time, some degree of reversibility is observed in the data, which indi­ cates neuronal release of DA and subsequent metabolism. Such reversibility is quantified with the rate constant kloss. The rate constant kloss is a measure of the frequency of depletion of the trapped tracer component, and its inverse repre­ sents the mean dwell time of that component in brain tissue. The ratio kloss/Ki is a powerfully dis­ criminating indicator of the turnover of the trapped F-DOPA compartment (EDT). Its inverse Ki/kloss can be interpreted as an effective distribution

volume (EDV) of the specific compartment alone with respect to the plasma tracer concentration and is a similarly discriminating measure of the ability of the trapping mechanism to store tracer. In Parkinson’s disease, the rate of F-DOPA uptake decreases and the rate of loss increases. Thus both EDT and its inverse EDV are sensitive markers of disease severity and progression. [18F]- and [11C]-labeled antagonists can be used to determine the DAT density. DAT is a 620-amino acid protein, with 12 a-helical hydrophobic trans­ membrane domains, 2–4 extracellular glycosylation sites, and up to 5 intracellular phosphorylation sites, which is found exclusively in DA axons and dendrites (Hersch et al., 1997; Nirenberg et al., 1996). DAT levels correlate with striatal DA con­ centrations (Bezard et al., 2001). It is therefore a potential specific marker of DA nerve terminal density. The binding of DAT ligands correlates with the clinical severity of PD (Pirker, 2003; Seibyl et al., 1995). The reproducibility of scan results within subjects is also acceptable (Nurmi et al., 2000a; Seibyl et al., 1997; Volkow et al., 1995). [11C]dihydrotetrabenazine (DTBZ) can be used to determine the VMAT2 density. There are two forms of VMAT expressed in human: VMAT1 is found in the adrenal glands, while VMAT2 is expressed exclusively in brain. VMAT2 is a 515-amino acid protein responsible for the uptake of intracytoplasmic monoamines into the synaptic vesicles. Although VMAT2 is not specific for DA, it is responsible for the packaging of all monoamine neurotransmitters and more than 95% of striatal monoaminergic innervation is dopaminergic. Tetrabenazine binds to VMAT2 and blocks the uptake of monoamines into the vesicles. In rats, the binding of striatal VMAT2 with [3H] methoxytetrabenazine correlated with SNc den­ sity (Vander Borght et al., 1995). Since the mid­ 1990s, DTBZ has been used in humans to monitor the integrity of striatal monoaminergic nerve terminal density (Frey et al., 1996). The interpretation of dopaminergic scans is not straightforward. Indeed, early PD is characterized by relative increases in F-DOPA uptake compared

180

to the degree of denervation as assessed by DTBZ PET, probably reflecting compensatory upregula­ tion of AADC activity (Lee et al., 2000). Hence, F-DOPA uptake may underestimate the degree of dopaminergic denervation, particularly in early disease. On the other hand, VMAT2 expression per existing DA terminal is thought to be relatively resistant to regulatory changes resulting from denervation and pharmacotherapy. DTBZ binding correlates well with presynaptic vesicle density and hence, in turn, reflects the nerve terminal density, although it is subject to competition from cytosolic DA and extensive depletion of vesicular DA may therefore lead to apparent ele­ vation of VMAT2 binding (Boileau et al., 2008; Tong et al., 2008). Although DAT binding might be expected to reflect DA terminal density, the DAT is downregulated in early PD as a compen­ satory change (Lee et al., 2000) and may be further influenced by pharmacotherapy and age (Parkinson Study Group, 2002; Volkow et al., 1994). Therefore, DAT binding may tend to over­ estimate nigral cell loss.

pontine activity, and concurrent relative metabolic reductions in the cortical motor and association regions. The PD-related spatial covariance pattern expression is highly reproducible (Ma et al., 2007) and in addition to the accurate discrimination between patients with PD and healthy volunteers, this network measure was useful in the differential diagnosis of classic PD and atypical forms of parkinsonism (Eckert et al., 2007). Substantial evidence links the PD-related spatial covariance pattern to the motor manifestations of the disease. The activity of this network is associated with standardized motor ratings (Asanuma et al., 2006) and spontaneous firing rates of neurons in the motor pallidum (Eckert and Eidelberg, 2005). Moreover, PD-related spatial covariance pattern activity can be modulated by therapeutic lesioning or deep brain stimulation of the motor pallidum and the subthalamic nucleus (Asanuma et al., 2006; Trost et al., 2006). The reduction in network activity induced by these interventions is associated with the degree of post-operative motor benefit seen.

Post-synaptic imaging Parkinson’s disease-related spatial covariance pattern (PDRP) Functional brain imaging can provide other insights into possible mechanisms of therapy for PD and related disorders. In particular, metabolic imaging of the brain with 18F-fluorodeoxyglucose (FDG) PET has revealed useful information about disor­ dered functional connectivity in neurodegenerative disease (see chapter by Eidelberg, this volume). By mapping glucose metabolism at a voxel level, this imaging approach provides a measure of regional synaptic activity and the biochemical maintenance processes that dominate the rest state. The effects of localized pathology on these cellular functions can alter functional connectivity across the entire brain in a disease-specific manner. Parkinson’s disease is associated with the expres­ sion of an abnormal metabolic pattern that is characterized by increased pallidothalamic and

D1 and D2 receptors can be evaluated using dif­ ferent radiotracers. [18F]fallypride, [11C]FLB-457 (PET ligand), and [11C]epidepride (SPECT ligand) belong to the family of ultra high-affinity DA receptor antagonist radioligands, which allows quantification and visualization of low-density DA extrastriatal D2/D3 receptors as well as striatal receptors (de Paulis, 2003). The radioligands [11C]raclopride (RAC) (for PET) and [123I] IBZM (for SPECT) are widely employed to assess striatal DA receptor availability. Since these ligands have a lower affinity for D2/D3 receptors, quantification of extrastriatal receptors is not possible (Pinborg et al., 2007). RAC competes with endogenous DA for in vivo binding to D2 receptors and changes in binding can therefore be used to infer alterations in synaptic DA con­ centration. Tracer binding is also influenced by age and, to a lesser extent, the stage of PD and

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DA replacement therapy (DRT). While increased tracer binding is observed in the more affected putamen in early PD (Antonini et al., 1997; Kaasinen et al., 2000), advanced PD and chronic DRT result in normalization of binding in the putamen and decreased binding in the caudate (Antonini et al., 1997; Thobois et al., 2004). In early PD, increased D2 binding has also been demonstrated using [11C]-N-methylspiperone (Kaasinen et al., 2000). Unlike RAC, this ligand is not thought to be subject to displacement by endogenous DA and the findings therefore suggest that increased binding of D2 ligands to putaminal D2 receptors in early PD additionally reflects receptor upregulation, as opposed to increased receptor occupancy due to endogenous DA deficiency. In contrast, D1 binding as assessed by [11C]SCH23390 and PET is normal in PD (Rinne et al., 1990), but may be decreased in con­ ditions characterized by loss of striatal neurons, such as multiple system atrophy (MSA).

Assessment of progression F-DOPA uptake in early PD is most severely decreased in the dorsal part of the caudal putamen but significant decreases can be seen throughout the striatum. Even in patients with unilateral disease, the less severely affected putamen is abnormal, in keeping with subclinical loss of dopa­ minergic function (Bohnen et al., 2006; Lee et al., 2000; Marek et al., 1996). An anterior–posterior gradient of dopaminergic dysfunction has been demonstrated in the putamen, with side-to-side asymmetry in tracer binding between the more and less severely affected striatum. As the disease progresses, the anterior–posterior gradient for striatal dopa influx and presynaptic reuptake of DA (DAT function) are maintained, suggesting a similar relative rate of decline throughout the putamen, while the degree of asymmetry between less and more affected putamen becomes less pro­ minent (Bruck et al., 2009; Nandhagopal et al., 2009). Taken together, the findings support the

notion that while factors responsible for disease initiation may affect striatal subregions differently, disease progression could be due to non-specific mechanisms such as oxidative stress/free radical elaboration, excitotoxicity, mitochondrial damage, inflammation, etc (Muchowski, 2002; Schapira et al., 1998; Tatton et al., 2003) that might be expected to affect striatal subregions to a similar degree. This notion is supported by the observations of progres­ sion of parkinsonism many years after encephalitis lethargica (Calne and Lees, 1988) or after exposure to MPTP (Langston et al., 1999; McGeer et al., 2003), associated with active inflammation. Age-related alterations in striatal DA processing, neuronal attrition, mitochondrial perturbation, and oxidative stress may also play a role in disease pro­ gression (Braskie et al., 2008; Kraytsberg et al., 2006; Langston et al., 1999; McGeer et al., 2003;). Autopsy studies in PD have demonstrated a 45% decrease in nigral cell counts during the first decade of PD, 10 times greater than the loss associated with normal aging, with a tendency to approach the normal age-related linear decline in the later stages (Fearnley and Lees, 1991). This nonlinear pattern of nigral cell loss is supported by various PET studies (Bruck et al., 2009; Nandha­ gopal et al., 2009). Dopaminergic hypofunction in the putamen, as demonstrated by decline in F-DOPA uptake, is faster in the beginning of the disease than in the later stages, supporting the hypothesis of negative exponential decline (Nandhagopal et al., 2009; Nurmi et al., 2003; Schulzer et al., 1994). The caudate is affected much later and to a lesser degree than the puta­ men. Some studies, in which a linear decline is assumed, have estimated a slower rate of decline in the caudate compared to the putamen, but esti­ mates based on exponential models of decline suggest that the rate is similar, although the inter­ cept and asymptote of decline remain different. In tremor-dominant subjects, a significantly slower annual F-DOPA uptake decline has been noted in caudate than in other PD subtypes (0.6–1.3% compared with 4.3–6.5%). Estimation of preclini­ cal duration in PD from F-DOPA PET studies

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varies according to the model that is used but is approximately 6 years (Hilker et al., 2005; Morrish et al., 1998) with estimated losses ranging from approximately 30% (Hilker et al., 2005; Morrish et al., 1998) to 55% (Lee et al., 2000) of normal putaminal F-DOPA uptake at the time of symp­ tom onset, in broad agreement with post-mortem studies (Fearnley and Lees, 1991; Hilker et al., 2005; Morrish et al., 1998). The annual rate of decline in putamen DTBZ binding potential was 5.5% of baseline (Au et al., 2005). Given the limitations of the various PET measures, the stu­ dies so far suggest that nerve terminal loss in the nigrostriatal DA system progresses at an annual rate of 5–13% in the putamen (Morrish et al., 1998; Nurmi et al., 2000b, 2001, 2003). Changes in metabolic network activity with pro­ gression of PD have been studied (Huang et al., 2007). PDRP activity has been found to increase linearly with disease progression, and is signifi­ cantly elevated compared with control. The disease progression was associated with increasing metabolism in the subthalamic nucleus (STN) and internal globus pallidus (GPi), as well as in the dorsal pons and primary motor cortex. Advan­ cing disease was also associated with declining metabolism in the prefrontal and inferior parietal regions. PDRP expression was elevated at base­ line relative to healthy control subjects, and increased progressively over time. Changes in PDRP activity correlated with concurrent declines in striatal DAT binding and increases in motor ratings. Network analysis of metabolic imaging data showed a short preclinical period in PD, in which the dissociation of the normal relations between metabolic activity and age occurred about 5 years before the onset of symptoms.

Disparity between clinical and in vivo measures of disease progression RTI has been used as an in vivo biomarker to assess the effect of treatment on disease progres­ sion in various clinical trials. These studies include

the CALM-PD study (Parkinson Study Group, 2000) which compared the early use of L-dopa with pramipexole using b-CIT SPECT (a measure of DAT binding), the REAL-PET study (Whone et al., 2003), which compared the use of ropinirole and L-dopa in de novo PD patients using F-DOPA PET, the ELLDOPA study (Fahn, 1999), in which the effects of L-dopa on clinical progression of PD were studied, and bCIT SPECT was included, and studies on fetal nigral transplantation with F-DOPA PET as an imaging modality (Freed et al., 2001; Nakamura et al., 2001; Olanow et al., 2003; Stoessl, 2003). The effects of glial cell line-derived neuro­ trophic factor (GDNF) on clinical and imaging end­ points have also been reported (Gill et al., 2003). All these studies showed discordant results between clinical progression and the estimated disease progression as determined by PET or SPECT. In the CALM-PD (Parkinson study group, 2000, 2002) and REAL-PET studies (Whone et al., 2003), imaging findings suggested a slower rate of disease progression with pramipexole and ropinirole, respectively. However, the clinical improvement, based on the Unified Parkinson’s Disease Rating Scale (UPDRS), favored the L-dopa treatment group. In the ELLDOPA study (Fahn et al., 2004), the L-dopa treatment group had a slower rate of clinical progression compared to the placebo group when clinical assessments were performed after 2 weeks of wash-out. Although this most likely reflects inadequate washout of symptomatic effects even after 2 weeks, a more rapid rate of decline in the L-dopa treatment group was noted with b-CIT imaging. In the fetal nigral transplant studies (Freed et al., 2001; Nakamura et al., 2001; Olanow et al., 2003), there was a substantial increase in striatal uptake of F-DOPA following transplantation, but clinical improvement was disappointing. A recent rando­ mized controlled trial of intraputaminal GDNF infusion in PD did not confer the predetermined level of clinical benefit despite increased F-DOPA uptake (Lang et al., 2006). The phase 1 openlabel trial of intraputaminal stereotactic delivery of CERE-120 (adeno-associated virus serotype

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2-neurturin) to patients with idiopathic Parkinson’s disease demonstrated potential efficacy for treatment; however, no change in striatal uptake from baseline was seen with F-DOPA PET (Marks et al., 2008). The discordance between clinical progression and RTI markers could in part be due to the effects of the therapies on the surrogate markers rather than on the disease process. Moreover, clinical progression was measured using UPRDS, which reflects a composite of dopaminergic and non-dopaminergic dysfunctions in PD (Lang and Obeso, 2004) and the clinical sign that best reflects the severity of the nigrostriatal lesion is bradyki­ nesia (Vingerhoets et al., 1997). In the case of cellbased therapies such as transplantation, grafts may survive but fail to form synaptic connections with the host striatum. Thus, assessment of the nigrostriatal DA system alone may be inadequate to assess the overall disease progression in PD. Proper study design and analysis are needed, and the PET data must be interpreted with caution and in the context of the clinical outcome.

techniques do not reliably distinguish idiopathic PD from MSA or other forms of atypical PD, although FDG PET can potentially distinguish these groups using a discriminant function analysis (Antonini et al., 1998; Eidelberg et al., 1993) The interpretation of imaging data from these clinical trials is challenging because of the potential for direct pharmacologic regulation of the targets of these ligands (Albin & Frey, 2003; Ahlskog, 2003; Clarke & Guttam, 2002). The duration of these pharmacodynamic effects is often unknown, mak­ ing washout designs problematic (Albin and Frey, 2003; Ahlskog, 2003; Clarke and Guttman, 2002). Various clinical trials highlight the variable rela­ tionship between RTI measure and clinical effects. Therefore, no RTI technique can be considered as a surrogate endpoint in PD for clinical trials.

Neuroimaging of treatment-related motor complications Presynaptic mechanisms

Imaging as a biomarker Currently there is an increasingly important need for a biomarker to monitor the course of PD, as new therapies for this disorder are developed. RTI of the nigrostriatal dopaminergic system is a widely used but controversial biomarker in PD. Radiotracer-based imaging assessments of nigros­ triatal dopaminergic function are useful to diag­ nose early Parkinson’s disease and monitor the progression of the disease. However, the associa­ tion between these measures and clinical change has not always been straightforward (Ravina et al., 2005). These techniques do not assess the number or density of nigral dopaminergic neurons, and do not directly measure the biologic processes under study. Non-dopaminergic symptoms such as depression, cognitive impairment, and postural instability, which are major contributors to disabil­ ity in PD, are not captured by DA-related tracers (Karlsen et al., 1999; Schrag et al., 2000). These

Neuroimaging studies have provided in vivo sup­ port for the importance of pulsatile stimulation of DA receptors in the emergence of LID. Altera­ tion in central pharmacokinetics of DA can be assessed using PET with ligands that bind to the VMAT2, the plasmalemmal DAT (Au et al., 2005; Brooks et al., 2003) and indirectly by ligands that bind to post-synaptic DA D2 receptors. Addition­ ally, the fluorinated analog of levodopa, F-DOPA can be used to assess uptake and decarboxylation of levodopa to DA, as well as storage of DA in synaptic vesicles and, when prolonged scans (4 h, rather than the usual 90–120 min) are performed, DA turnover (Sossi et al., 2001). Dyskinesias tend to occur in more advanced PD. One might therefore anticipate a loose rela­ tionship between markers of presynaptic dopami­ nergic integrity and LID. With the possible exception of dyskinesias that emerge following fetal mesencephalic transplantation (see below), there is little evidence for this in the literature,

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apart from a report by Linazasoro and colleagues, who found an inverse relationship between F-DOPA uptake and dyskinesias (Linazasoro et al., 2004). Fluctuations in motor function, which commonly occur together with dyskinesias, are associated with reduced F-DOPA uptake (Fuente-Fernandez et al., 2000), but there is substantial overlap between patients with and without motor fluctuations, sug­ gesting that other factors play an important role. Traditional measures of presynaptic dopaminer­ gic integrity give only a rough estimate of striatal DA nerve terminal density. The critical factor in the emergence of motor complications is the pat­ tern of DA receptor stimulation. Thus, assessment of the central pharmacokinetics of levodopa action may provide greater insight. RAC labels D2/D3 receptors with relatively low affinity and its binding is subject to competition from endogen­ ous DA (Breier et al., 1997; Seeman et al., 1989). Thus, interventions such as levodopa therapy that result in increased synaptic DA will result in reduced RAC binding as assessed by PET (Tedroff et al., 1996). De la Fuente-Fernandez et al. found a greater magnitude but less sustained decline in RAC binding in PD patients who had a stable response to levodopa at the time of the PET study but who went on to develop motor fluctua­ tions within 3 years compared to those subjects who had stable response to medication 3 years later (Fuente-Fernandez et al., 2001). In a follow-up study, these authors found that the relative change in RAC binding 1 h after oral levodopa increases with disease duration and even after correction for this factor, is higher in subjects with LID compared to those with a stable response, while there is no difference between dyskinetic and non-dyskinetic subjects 4 h after levodopa (Fuente-Fernandez et al., 2004). This is compatible with a more pulsa­ tile pattern of levodopa-induced DA release in subjects with motor complications. Similar findings have been reported by Pavese et al. (2006). Another way of assessing the kinetics of DA release and metabolism is to estimate DA turn­ over using prolonged scans with F-DOPA. While uptake measured over the standard 90–120 min

scan reflects uptake, decarboxylation to FDA, and trapping of FDA in synaptic vesicles, prolonged scans also reflect the egress and subsequent meta­ bolism of this trapped radioactivity. The model used to analyze the acquired radioactivity data thus shifts from one that assumes unidirectional transport of tracer (i.e., the radioactivity is trapped) to a reversible model. The EDV that is derived from this reversible tracer model corre­ lates well with the inverse of the ratio of tracer loss to tracer uptake constants (Sossi et al., 2001), which in turn correlates with classical neurochemical mea­ sures of DA turnover (Doudet et al., 1998). DA turnover measured using this approach is increased early in PD (Sossi et al., 2002) and further increases occur with disease progression (Sossi et al., 2004). Even when one accounts for disease severity, the magnitude of the abnormality in DA turnover is greater in PD patients with younger disease onset than the abnormality of F-DOPA uptake (Sossi et al., 2006). This suggests that comparable degrees of denervation result in greater increases in DA turnover in younger individuals and is in keeping with the widely held view that such individuals are more prone to dyskinesias (Golbe, 1991; Grandas et al., 1999; Kumar et al., 2005; Quinn et al., 1987). The determinants of DA turnover are not fully understood. However, it appears that in patients with PD, downregulation of the DAT results in increased turnover, again even after correcting for disease severity (Sossi et al., 2007). One would therefore predict that downregulation of DAT beyond the degree expected based on disease severity (i.e., loss of DA nerve terminals) would be an independent predictor of the development of LID and this indeed appears to be the case (Troiano et al., 2009). Thus, while downregulation of the DAT may serve a useful function in early disease in order to conserve levels of DA in the synapse (Calne and Zigmond, 1991; Lee et al., 2000), in the long run such a compensatory mechanism may prove deleterious. Dyskinesias that occur following fetal mesence­ phalic transplantation may represent a special example, as they may occur either as an

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exaggerated form of LID or in some patients, may occur off medication (Freed et al., 2001; Olanow et al., 2003). Ma and colleagues reported post­ operative increases in F-DOPA uptake in the left posterodorsal putamen and left ventral striatum of patients who developed post-transplant dyskinesias (Ma et al., 2002). In contrast, using a combination of F-DOPA and RAC, Piccini et al. found no evidence for increased graft-derived DA release in subjects with dyskinesias (Piccini et al., 2005). Politis et al. (2010) have recently used PET to demonstrate increased serotonergic innervation in the grafted striatum of patients with post-transplant off-medication dyskinesias.

Post-synaptic mechanisms There is to date no convincing evidence for a clear relationship between the densities of either DA D1 or D2 receptors and motor complications, including dyskinesias, although prolonged treatment is asso­ ciated with normalization of D2 receptors in the putamen (increased in untreated patients), reduc­ tion of D2 receptors in the caudate nucleus, and possibly with reduction of D1 receptors in the puta­ men (Antonini et al., 1997; Turjanski et al., 1997). There is extensive evidence from animal models of alterations downstream to striatal DA receptors following chronic dopaminergic stimulation, thought to contribute to LID. These include upre­ gulation of immediate early genes and of several neuropeptides, including enkephalin and dynorphin (Cenci and Lindgren, 2007). There is very limited evidence available in the imaging literature, largely reflecting the paucity of informative tracers. Piccini and colleagues demonstrated reduced striatal bind­ ing of the opioid ligand [11C]diprenorphine in PD patients with LID, presumably reflecting occupancy of striatal opioid receptors due to increased opioid levels (Piccini et al., 1997). Whone and colleagues demonstrated in a preliminary study a reduction in thalamic NK1 neurokinin receptor binding in PD patients with LID (Whone et al., 2002). Whether this represents a loss of NK1 receptors or increased

receptor occupancy reflecting increased availability of endogenous substance P is unclear.

Cerebral blood flow studies Studies of cerebral blood flow can be used to infer changes in patterns of neuronal activity within the basal ganglia and its connections. In the rest state, tight correlations exist between regional cerebral metabolic rate and blood flow. However, because of their hemodynamic effects, dopaminergic treat­ ments may cause a dissociation of these parameters. A large increase in cerebral blood flow following administration of LDOPA has been noted in thala­ mus and basal ganglia in PD patients with dyskine­ sia (Hershey et al., 1998; Hirano et al., 2008). Because regional cerebral blood flow is thought to predominantly reflect synaptic activity (bearing in mind the above-noted caveat), this finding may be compatible with a sensitized response to levodopa in the internal segment of the globus pallidus and while it is not easily explained by standard “box and arrow” models of the basal ganglia (Albin et al., 1989), it is very much in keeping with the reduction in LID that is consistently reported following palli­ dotomy (Fine et al., 2000). Sanchez-Pernaute and colleagues have studied the hemodynamic response to a selective DA D3 receptor agonist using fMRI and found that the response was increased in rodent and non-human primate animals with LID (San­ chez-Pernaute et al., 2007), in keeping with in vitro and behavioral evidence (Bezard et al., 2003; Bordet et al., 1997; van Kampen and Stoessl, 2003).

Potential future applications With the few exceptions noted above, most studies performed to date have focused either on dopami­ nergic mechanisms or on patterns of cerebral acti­ vation in response to medication. Within the DA system, study of the D3 receptor may be of parti­ cular interest, but investigation has been hampered by the lack of selective positron-emitting tracers.

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Other neurotransmitters of interest with respect to their role in LID include 5-hydroxytryptamine, adenosine, excitatory amino acids, and GABA, but there are very few relevant imaging studies, in part reflecting the paucity of informative radioli­ gands. Studies of cell signaling pathways and of immediate early gene expression similarly await the development of better tools for in vivo imaging.

Neuroimaging of treatment-related behavioral complications While a number of other behavioral complications such as depression and cognitive impairment repre­ sent a major source of disability in PD, they pre­ dominantly reflect manifestations of the underlying disease rather than its therapy and are accordingly not discussed here (but see article by Brooks in this volume). The same is true for hallucinations and other psychotic features, which while potentially induced by medication, are often seen in association with diffuse Lewy body disease. The most impor­ tant treatment-induced behavioral side-effect is a group of related problems generally referred to as the impulse control disorders (ICDs). ICD affects approximately 10% of patients treated with dopa­ minergic agents, particularly those treated with DA agonists (Evans et al., 2009; Voon et al., 2009) and can include pathological gambling and shopping, hypersexuality, binge eating, punding (repetitive non-goal-oriented behaviors), and compulsive med­ ication use. While these behaviors may arise from an interaction between the underlying disease and its treatment, it is of interest that they have also been reported in the setting of DA agonist therapy of Restless Legs Syndrome, a condition where there is little direct evidence of meso-striatal/meso-limbic DA denervation (Tippmann-Peikert et al., 2007). Evans et al. (2006) used RAC PET to estimate levodopa-derived DA release in PD patients with compulsive medication use. In these subjects, DA release was much higher in the ventral striatum compared to PD patients without this complication. In contrast, both groups had comparable DA

release in the putamen. In keeping with other lit­ erature on drugs of abuse (Leyton et al., 2002), the degree of DA release correlated with the degree of “drug wanting” rather than the degree of “drug liking”. Steeves et al. (2009) recently used a similar approach to study DA release in PD patients with pathological gambling, but in response to a gam­ bling task with monetary reward, compared to a control task. The patients with pathological gam­ bling had higher relative DA release in the ventral striatum during performance of the card task. Inter­ estingly, however, the levels of RAC binding in the ventral striatum during performance of the control task were much lower in the gambling patients. This may suggest either a higher level of basal DA release in patients with this complication or reduced levels of DA D2/D3 receptors. Support for the latter possibility is derived from animal models of drug abuse, in which impulsive traits are associated with reduced D2/D3 receptor availability in rats (Dalley et al., 2007). In addition to this evidence for sensitized medica­ tion- and task-induced DA release, a key factor in ICD is the failure to stop, despite the conscious recognition of the deleterious effects these behaviors may have on the patient’s life. In this respect, it is relevant that DA release is thought to signal the error between predicted and actual delivery of reward (Schultz, 2001). Thus, dopaminergic therapy, while sufficient to improve the motor deficits seen in PD, cannot mimic the close temporal relationship between reward delivery (or lack thereof) and pha­ sic DA release thought to underlie the temporal difference model of learning. This is particularly true for DA agonists, which produce relatively con­ stant levels of dopaminergic stimulation. Using func­ tional magnetic resonance imaging, van Eimeren and colleagues (Van Eimeren et al., 2009) demon­ strated that use of the DA agonist pramipexole resulted in loss of deactivation in orbitofrontal cor­ tex in response to negative reward prediction errors. This suggests that in addition to pathologically heightened “reward” responses in the ventral stria­ tum of patients with ICD, there is likely to be loss of the capacity to respond to negative outcomes.

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Concluding comments Functional imaging with PET can detect DA defi­ ciency in PD and correlates loosely with disease severity, particularly with bradykinesia. However, despite its overall utility in assessing disease pro­ gression, caution must be used in the interpreta­ tion of the results, as disparity between imaging and clinical outcomes has been the rule in most studies of putative disease-modifying therapies. Functional imaging studies may be of particular benefit in studying the pathophysiology of disease and treatment-related complications in PD, parti­ cularly studies that take advantage of the dynamic capacity of PET to assess not only the functional integrity of the DA system, but also more detailed aspects of the response to pharmacological and behavioral stimuli known to modify DA release. In the future, the ability to perform analogous studies examining neurotransmitters other than DA should prove similarly fruitful.

FDA MPTP EDT EDV DAT DTBZ VMAT RAC DRT PDRP STN GPi UPDRS FDG

Fluoro dopamine 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine Effective dopamine turnover Effective dopamine volume dopamine transporter [11C]dihydrotetrabenazine Vesicular monoamine transporter [11C]raclopride Dopamine replacement therapy Parkinson’s disease-related spatial covariance pattern Subthalamic nuclei Globus pallidus interna Unified Parkinson’s disease Rating Scale 18 F-fluorodeoxyglucose

Acknowledgments References The authors’ work is supported by the Canadian Institutes of Health Research, the Canada Research Chairs program, the Michael Smith Foundation for Health Research, the Pacific Alzheimer Research Foundation, and the Pacific Parkinsons Research Institute.

List of Abbreviations PD DA LID ICD RTI PET SPECT F-DOPA

Parkinson’s disease Dopamine Levodopa-induced dyskinesia Impulse control disorders Radio tracer imaging Positron Emission Tomography Single Photon Emission Computerized Tomography 6-[18F]-fluoro-L-dopa

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Vander Borght, T. M., Sima, A. A., Kilbourn, M. R., Desmond, T. J., Kuhl, D. E., & Frey, K. A. (1995). [3H] methoxytetrabenazine: A high specific activity ligand for estimating monoaminergic neuronal integrity. Neuroscience, 68, 955–962. Vingerhoets, F. J., Schulzer, M., Calne, D. B., & Snow, B. J. (1997). Which clinical sign of Parkinson’s disease best reflects the nigrostriatal lesion? Annals of Neurology, 41, 58–64. Volkow, N. D., Ding, Y. S., Fowler, J. S., Wang, G. J., Logan, J., Gatley, S. J., et al. (1995). A new PET ligand for the dopamine transporter: Studies in the human brain. Journal of Nuclear Medicine, 36, 2162–2168. Volkow, N. D., Fowler, J. S., Wang, G. J., Logan, J., Schlyer, D., MacGregor, R., et al. (1994). Decreased dopamine trans­ porters with age in healthy human subjects. Annals of Neu­ rology, 36, 237–239. Voon, V., Fernagut, P. O., Wickens, J., Baunez, C., Rodriguez, M., Pavon, N., et al. (2009). Chronic dopaminergic stimula­ tion in Parkinson’s disease: From dyskinesias to impulse control disorders. Lancet Neurology, 8, 1140–1149. Voon, V., Hassan, K., Zurowski, M., de Souza, M., Thomsen, T., Fox, S., et al. (2006). Prevalence of repetitive and rewardseeking behaviors in Parkinson disease. Neurology, 67, 1254–1257. Weintraub, D., Siderowf, A. D., Potenza, M. N., Goveas, J., Morales, K. H., Duda, J. E., et al. (2006). Association of dopamine agonist use with impulse control disorders in Par­ kinson disease. Archives of Neurology, 63, 969–973. Whone, A. L., Rabiner, E. A., Arahata, Y., Luthra, S. K., Hargreaves, R., Brooks, D. J. (2002). Reduced substance P binding in Parkinson’s disease complicated by dyskinesias: An 18 F-L829165 PET study [abstract]. Neurology, 58, A488–A489. Whone, A. L., Watts, R. L., Stoessl, A. J., Davis, M., Reske, S., Nahmias, C., et al. (2003). Slower progression of Parkinson’s disease with ropinirole versus levodopa: The REAL-PET study. Annals of Neurology, 54, 93–101.

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 10

Brain imaging after neural transplantation Marios Politis
and Paola Piccini
Centre for Neuroscience and MRC Clinical Sciences Centre, Faculty of Medicine, Hammersmith Hospital,

Imperial College London, UK

Abstract: Functional imaging has provided objective evidence that human fetal ventral mesencephalic tissue implanted in the striatum of Parkinson’s disease patients can survive, grow, release dopamine, normalize brain metabolism, and restore striatal–cortical connections. Open-label clinical trials have shown robust clinical improvement in several PD patients but these results were not replicated in two double-blind sham-surgery controlled clinical trials. Graft-induced dyskinesias are serious adverse effects and a major roadblock for the further development of cell therapies, and functional imaging can help investigate the mechanisms underlying their cause. Functional imaging can also aid future trials by improving patient selection, assessing restoration of brain connectivity, and monitor inflammatory processes. Although functional imaging cannot currently be used as a primary endpoint in clinical transplantation trials, it can provide additional valuable information alongside clinical observations. Keywords: PET; SPECT; Parkinson; Neural grafts

Monitoring the presynaptic dopaminergic system and graft survival

(VM) tissue, stem cells, growth factors, or gene therapy. Trials assessing the safety and efficacy of intrastriatal transplantation of human fetal VM tissue were started in 1987. The clinical outcome and the pattern of recovery varied widely. Some PD patients experienced marked symptomatic relief with reduced “off” periods, reduced motor fluctua­ tions, and less need for dopaminergic medication alongside improvements in their quality of life. Some other patients experienced moderate or no improvement of their motor symptoms with a subset of cases experiencing postoperational “off”­ medication dyskinesias [known as graft-induced

The main goal of cell replacement therapies in Parkinson’s disease (PD) is to correct its main chemical defect by restoring the production of dopamine in the brain. These procedures include transplantation of human fetal ventral mesencephalic
Corresponding author. Tel.: þ44-20-83833773; Fax: þ44-20-83831783; E-mail: [email protected] Tel.: þ44-20-83833754; Fax: þ44-20-83831783; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84010-5

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dyskinesias (GIDs)] (Brundin et al., 2000; Cochen et al., 2003; Freed et al., 2001; Freeman et al., 1995; Hagell et al., 1999; Hauser et al., 1999; Lindvall et al., 1990, 1994; Mendez et al., 2000, 2002; Olanow et al., 2003; Peschanski et al., 1994; Piccini et al., 1999; Remy et al., 1995; Sawle et al., 1992; Wenning et al., 1997; ). Positron emission tomography (PET) allows regional changes in brain metabolism, blood flow, and receptor binding to be detected in vivo and quantified in PD. Current tomographs have a reso­ lution of 3–4 mm and therefore are potentially cap­ able of assessing the function of transplanted sites in the striatum. 18F-dopa PET by measuring striatal aromatic amino acid decarboxylase (AADC) activity allows an indirect measure of dopamine storage within the nigro-striatal dopaminergic terminals. Also, with PET radioligands such as 11C-CFT, 18 F-CFT, and 11C-RTI-32, or with single photon emission computed tomography (SPECT) radioli­ gands such as 123I-b-CIT, 123I-FP-CIT, 123I-altropane, and 99mTc-TRODAT-1 it is possible to assess the availability of presynaptic dopamine transporter (DAT) (the plasma membrane transporter respon­ sible for the high-affinity uptake of dopamine). 11 C-dihydrotetrabenazine PET can measure the density of the vesicular monoamine transporter (VMAT2), which is responsible for the transport of dopamine from the cytoplasm into the secretory vesicles. Moreover, 11C-RTI-121 PET studies (another dopamine terminal marker) in rats have demonstrated a correlation between the survival of striatal grafts and striatal binding of the tracer (Sullivan et al., 1998). To date, there are no 11 C-dihydrotetrabenazine PET data on the expres­ sion and survival of VMAT2 in VM tissue grafts. Also, imaging techniques have been unable to detect significant changes in DAT binding after transplan­ tation of VM tissue in humans, although putaminal 18 F-dopa uptake improved (Cochen et al., 2003). Also, two postmortem studies have reported oppo­ site results concerning DAT expression in grafted neurons (Kordower et al., 1996, 2008). 18 F-dopa PET was the first in vivo marker of dopamine terminal function to be developed

(Firnau et al., 1987). The uptake of 18F-dopa in the striatum reflects four different processes: (i) the transport of the radioligand through the blood–brain barrier, (ii) the uptake of the radioli­ gand into the presynaptic dopamineric neurons, (iii) the metabolism by AADC, and (iv) the vesi­ cular storage of 18F-dopamine. AADC activity and density of the dopaminergic presynaptic term­ inals are the principal determinants of 18F-dopa uptake as measured by the influx constant Ki. Therefore, the quantification of 18F-dopa uptake within the striatum of PD patients will reflect the number of surviving dopaminergic neurons. Reports assessing striatal pathology in PD have shown that striatal 18F-dopa uptake correlated with nigro-striatal degeneration (Pate et al., 1993; Snow et al., 1993). Furthermore, in PD patients putaminal 18F-dopa uptake inversely correlates with the degree of motor impairment (Morrish et al., 1996; Remy et al., 1995; Vingerhoets et al., 1997) whereas the correlation between motor symptoms and decreases in caudate 18F-dopa uptake is weaker (Morrish et al., 1996). Striatal 18 F-dopa uptake in vivo also correlates with the numbers of substantia nigra dopamine neurons and striatal dopamine levels observed at postmor­ tem (Snow et al., 1993). By using this technique, an average of 7–12% annual decline in baseline puta­ men dopaminergic function has been reported in early PD patients (Morrish et al., 1998). There­ fore, 18F-dopa PET can provide an in vivo means of objectively monitoring the progression of neu­ rodegenerative disorders and the functional effects of restorative treatments. Functional imaging with 18F-dopa PET has indeed been used early on in the European open trials to objectively monitor survival and growth of human fetal dopamine neurons grafted in the striatum of PD patients (Brooks, 2004; Lindvall and Björklund, 2004; Lindvall and Hagell, 2000). Several of these open trials have shown significant increases in 18F-dopa uptake after striatal grafts supporting the concept that human fetal mesence­ phalic dopamine neurons can survive and grow in the brain of PD patients (Brundin et al., 2000;

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Cochen et al., 2003; Freed et al., 1992; Freeman et al., 1995; Hagell et al., 1999; Hauser et al., 1999; Lindvall, 1990; Lindvall et al., 1994, 1999; Mendez et al., 2000, 2002; Peschanski et al., 1994; Piccini et al., 1999, 2000, 2005; Remy et al., 1995; Sawle et al., 1992; Widner et al., 1992; Wenning et al., 1997). Histopathological analyses have confirmed survival of the dopaminergic grafts and demon­ strated their ability to reinnervate the striatum (Kordower et al., 1995, 1996, 1998). Several transplantation studies with human fetal VM grafts express 18F-dopa data as percentage changes from baseline (Brundin et al., 2000; Freed et al., 2001; Hagell et al., 1999). However, in vivo imaging data are more adequately expressed as a percentage of mean 18F-dopa uptake in normal healthy controls. This allows more robust comparisons between different studies using different PET scanners. Available clinical data show that the outcome following transplantation is dependent on restora­ tion of the number of viable dopaminergic neurons innervating the striatum as assessed by 18 F-dopa PET. According to data from open trials it appears that an increase in 18F-dopa uptake of 50–60% of the normal mean is necessary to achieve clinically valuable antiparkinsonian effects. The Lund group has reported up to 6-year follow-up data on four PD patients who had received unilateral putaminal implants and on two PD patients who had received unilateral putaminal and caudate implants of human fetal VM tissue (Wenning et al., 1997). All these patients received immunosuppression therapy. After 1 year following transplantation, there was a mean 68% increase in putaminal 18F-dopa uptake on the implanted side but only 6% mean increase in the implanted caudate, whereas the nonimplanted putamen showed a corresponding 25% mean decrease in 18F-dopa uptake. The changes in 18F-dopa PET following implantation were mirrored by improvements in the “off”-state Unified Parkinson’s Disease Rating Scale (UPDRS) motor scores which was reduced by 18% in the first postoperative year while the mean time in

“off” fell by 34%. One of these PD cases was with­ drawn from any dopaminergic medication and his putaminal 18F-dopa uptake reached normal levels. Five of these six PD patients subsequently received transplantation of human fetal VM tissue on the side that had not been grafted originally (Hagell et al., 1999). At 12–18 months following the second graft, there was an 85% mean increase in 18F-dopa uptake in the more recently trans­ planted putamen but no further 18F-dopa change in the previously transplanted putamen. Clinical scores in the majority of the PD patients who received the second graft were further improved with reduced bradykinesia scores, reduced time spent in “off,” and lower dopaminergic medica­ tion requirements. A few years later the Lund group performed bilateral putamen and caudate transplantation of human fetal VM tissue in four more PD patients (Brundin et al., 2000). By 6 months postopera­ tively the mean 18F-dopa uptake had increased in these patients by 53% in putamen and by 24% in caudate compared to the baseline. At the 20­ month follow-up 18F-dopa uptake was increased by only 3% in the putamen and 4% in the caudate compared to the previous assessment. However, clinical parameters, such as the UPDRS motor scores, continuously improved over this period with sores falling by an average of 50%. These data suggest that graft function and integration continues despite the stabilization of dopamine storage capacity. Furthermore, dopaminergic medication requirements were reduced by a mean of 50% and one subject was withdrawn from any dopaminergic medication. Remy et al. (1995) reported up to 2-year 18 F-dopa PET follow-up data on three PD patients who had received unilateral putaminal transplan­ tation of human fetal VM tissue and on two who additionally received unilateral caudate implants. In these patients a 61% mean increase in 18F-dopa uptake in the grafted putamen was shown after 1 year postoperatively, whereas in the grafted caudate no significant changes were reported. Putaminal 18F-dopa uptake values correlated with

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percentage times in “on” and timed finger dexterity when “off.” The results of this study indicated that transplantation of human fetal VM tissue, by restor­ ing putaminal 18F-dopa uptake within normal levels, is able to restore normal limb function in patients with PD. An open-label study performed in Tampa assessed four PD patients who had received bilat­ eral putaminal transplantation of human fetal VM tissue (Freeman et al., 1995). They found that 6 months after transplantation there was 53 and 33% mean increases in right and left putaminal 18 F-dopa uptake, respectively. The clinical scores were also improved with a 37% mean decrease in UPDRS total scores, a 41% mean improvement in Schwab and England disability score, a 65% mean decrease in time spent “off,” and 92% mean decrease in “on” dyskinesias. Two of these trans­ planted PD cases died from unrelated causes and postmortem examination showed viable tyrosine hydroxylase staining graft tissue forming connec­ tions with host neurons (Kordower et al., 1995). Given the encouraging results of these open trials, two major double-blind sham-surgery con­ trolled trials assessing the efficacy of transplanta­ tion of human fetal VM tissue in PD were sponsored by the National Institute of Health (NIH) in the USA. The first double-blind trial involved 40 advanced PD patients with mean dis­ ease duration of 14 years and age ranging from 34 to 75 years (Freed et al., 2001). The subjects were randomized to receive either bilateral intraputam­ inal transplantation of human fetal VM tissue or sham-surgery and no immunotherapy was used. The primary endpoint of the study was not fulfilled as the patients who had received trans­ plantation did not show a significant improvement in the global clinical impression scale as reported by the patients themselves 1 year after surgery. However, there was a significant 18% mean improvement in the UPDRS motor scores in the “off” state when compared with the sham-surgery group. PET imaging showed a 40% mean increase in putaminal 18F-dopa uptake. When the trans­ planted patients were subdivided into two groups

according to age older or younger than 60 it was observed that patients younger than 60 had signifi­ cant clinical improvement (UPDRS motor score fell by 38%) in comparison to baseline. Unfortu­ nately, 15% of patients in this trial developed “off”-dystonia and “off”-medication dyskinesia. In a recent report the long-term clinical and PET outcome of 33 of the participants in the original trial of Freed and colleagues were retro­ spectively assessed. These subjects were followed for 2 years after transplantation while15 were fol­ lowed for two additional years (Ma et al., 2010). This study showed that UPDRS motor scores declined over time postoperatively and the differ­ ence in clinical improvement between younger and older patients seen at 1 year after transplanta­ tion was not evident at longer-term follow-up. Furthermore, putaminal 18F-dopa uptake corre­ lated with changes in clinical parameters suggesting that the clinical benefit and graft viability were sustained up to 4 years after transplantation and that clinical difference related to the age of the subjects may not persist over the long term. In the second double-blind trial, 34 PD patients were randomized to receive either bilateral intra­ putaminal transplantation of human fetal VM tis­ sue procured from four fetuses per side or tissue from one fetus per side or to undergo sham-sur­ gery (Olanow et al., 2003). All patients received immunosuppressant treatment for a period of 6 months after operation. The primary endpoint of the trial was set as the change in UPDRS motor score in the “off” state. Two patients died during the trial while another three died after the trial, all from unrelated causes. In total, 31 patients com­ pleted this trial. Postmortem findings showed sig­ nificantly greater tyrosine hydroxylase staining in the putamen of the patients who received trans­ plantation than the sham-surgery patients with evident graft innervation of intrinsic cells. 18F-dopa scans showed significant increases of 20 and 30% in mean putaminal uptake in patients who had received tissue from one and four fetuses, respec­ tively, while there were no differences in putam­ inal uptake in the control sham-surgery group.

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Similarly to the previous double-blind trial, this study did not show significant difference in the primary endpoint. The mean “off” UPDRS motor score in the patients transplanted with four fetus group improved by 0.7 points over 2 years while it deteriorated by 3.5 and 9.4 points in the patients transplanted with one fetus and in the shamsurgery control group, respectively. These clinical changes were not significantly different overall at the end of the study, but interestingly at 6 months and before discontinuation of immunosuppression the transplanted groups were both significantly improved. Also, transplanted PD patients with less severe disease who had received tissue from four fetuses per side improved by an adjusted mean score of 1.5. However, there were no signif­ icant differences in “on” time without dyskinesias, the total “off” time, Activities of Daily Living scores, and dopaminergic requirements needed between the groups. Furthermore, this study yet again highlighted the problem of “off”-medication dyskinesias, which were evident in more than half of the transplanted PD patients. Indirect assessment of dopamine release and graft function Both PET and SPECT ligands have been used to assess postsynaptic dopaminergic receptors. 11 C-raclopride, a low-affinity D2 receptor ligand, has been used in most PET studies. Postsynaptic dopamine receptors broadly fall into two classes, the D1-type (D1 and D5), which are adenyl cyclase-dependent and D2-type (D2, D3, and D4), which are not. The striatum contains mainly D1 and D2 receptors and both play a key role in the regulation of locomotor function. In early, dopaminergic therapy naïve PD patients, 123 I-IBZM SPECT studies reported normal levels of striatal binding, whereas 11C-raclopride PET studies demonstrated 10–20% increases in putam­ inal D2 binding contralateral to the more affected limbs (Brooks et al., 1992; Playford and Brooks, 1992; Rinne et al., 1993). Following chronic

exposure to levodopa medications 11C-raclopride putaminal D2 binding normalizes in PD patients (Antonini et al., 1994; Brooks et al., 1992). 11 C-raclopride and 123I-IBZM are both benza­ mide ligands with affinity for the D2 receptor in the low nanomolecular range and therefore are subject to competitive displacement by endogen­ ous dopamine (Chugani et al., 1988; Volkow et al., 1994). Acute administration of substances such as amphetamine, methylphenidate, or levodopa which are known to increase the levels of extracellular dopamine result in a reduction of striatal 11C-raclo­ pride and 123I-IBZM binding (de la Fuente-Ferna­ dez et al., 2001; Goerendt et al., 2003; Laruelle et al., 1995; ). In normal subjects, 0.3 mg/kg infusion of methamphetamine results in a 25% decrease of striatal 11C-raclopride binding, which has been esti­ mated to represent a 10-fold increase in extracellu­ lar dopamine levels (Morris et al., 1995). In patients with PD, a similar amount of methamphetamine induces only 40% of the dopamine release seen in normal subjects (Piccini et al., 2003). Studies with 11C-raclopride PET and pharma­ cological challenges have shown that human fetal VM tissue can restore dopamine release in the striatum of patients with PD (Piccini et al., 1999). In one patient who had received unilateral intrapu­ taminal transplantation of human fetal VM tissue 10 years previously and had subsequently showed major clinical improvement, 18F-dopa uptake was observed to be restored to normal levels in the grafted putamen, whereas 18F-dopa uptake was only about 10% of normal level in the nongrafted puta­ men. Dopamine release from the grafted cells was also shown to be within normal levels in the grafted putamen, suggesting that grafted neurons can con­ tinue for a decade to store, release dopamine, and give rise to substantial symptomatic relief. Assessment of restoration of striato-cortical circuitries PET studies can also provide measurements of changes in regional cerebral flow (rCBF) and

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regional cerebral metabolism associated with striatal transplantation of human fetal VM tissue. 18 F-FDG PET studies have reported H15 2 O and increased levels of resting oxygen and glucose metabolism in the contralateral lentiform nucleus of early-hemiparkinsonian PD patients, respec­ tively (Miletich et al., 1988; Wolfson et al., 1985). More advanced PD patients with established dis­ ease and bilateral symptoms have normal levels of lentiform metabolism (Eidelberg et al., 1994; Wolfson et al., 1985). However, covariance analy­ sis has shown that in PD patients with established disease, although the absolute 18F-FDG values are normal, there is an abnormal glucose metabolism profile consisting of increased resting lentiform metabolism and decreased frontal metabolism (Eidelberg et al., 1994). Additionally, the degree of these changes correlated with the severity of the clinical disease (Eidelberg et al., 1995). H15 2 O PET studies in normal subjects performing paced movements with a joystick in freely selected directions have showed rCBF increases in contral­ ateral sensorimotor cortex and lentiform nucleus and bilaterally in anterior cingulate, supplemen­ tary motor area (SMA), lateral premotor cortex, and dorsolateral prefrontal cortex (DLPFC) (Jahanshahi et al., 1995; Playford et al., 1992), whereas similar studies in PD patients “off” med­ ication performing the same task have observed significant rCBF decreases in the contralateral lentiform nucleus, anterior cingulate, rostral SMA, and dorsal prefrontal cortex. Interestingly, normalization of activation in SMA and DLPFC during joystick movements in PD patients can be restored after treatment with levodopa (Rascol et al., 1994), apomorphine (Jenkins et al., 1992), and deep brain stimulation of the subthalamic nucleus (Ceballos-Baumann et al., 1999) and inter­ nal globus pallidum (Fukuda et al., 2001). Four PD patients from the Lund series were studied in order to determine whether movementrelated activation in SMA and DLPFC could improve as a result of bilateral intrastriatal trans­ plantation with human fetal VM tissue. These patients were serially assessed with H15 2 O PET at

baseline and twice more over 2 years after surgery while they performed paced joystick movements in freely selected directions (Piccini et al., 2000). A gradual restoration of movement-related activation in frontal motor cortical areas was indeed observed in these patients alongside with significant clinical improvement. This study suggested that striatal transplantation of human fetal VM tissue is able to restore striatal–cortical networks in the host brain, driving major clinical improvements. Graft-induced dyskinesias Graft-induced dyskinesias (GIDs) are involuntary movements occurring in transplanted PD patients “off” medication, and they have been reported in subsets of PD patients in all clinical trials (Freed et al., 2001; Hagell et al., 2002; Olanow et al., 2003). Occurrence of GID was one of the major concerns raised in the two NIH-sponsored doubleblind sham-surgery controlled trials (Freed et al., 2001; Olanow et al., 2003). Indeed in both trials a significant number of patients (15 and 56.4%, respectively) developed GID. The Lund group also reported occurrence of GID in 14 trans­ planted patients (8 mild, 5 moderate), although only in one patient GID constituted a major clin­ ical problem (Hagell et al., 2002). Notwithstanding the several mechanisms having been proposed, the pathogenesis of GID remains unclear. In their initial report, Freed et al. (2001) suggested that GID could be related to fiber out­ growth from the graft causing increased dopamine release. This hypothesis was supported by two further observations. Ma and colleagues (2002) observed that PD patients with GID showed an imbalance between dopaminergic innervation in the ventral and dorsal putamen with asymmetric increases in 18F-dopa uptake in the nongrafted ventral striatum. Another study (Huang et al., 2003) reported that GID in one patient was asso­ ciated with an increased caudate 18F-dopa uptake and in two other patients with greater-than­ expected striatal 11C-raclopride displacement after

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a levodopa challenge. In these cases, 11C-raclopride binding in the “off” state was reduced below normal, suggesting that striatal D2 receptors were occupied by excessive release of dopamine both under base­ line conditions and after levodopa challenge. There are, however, several lines of evidence against this view. Olanow et al. (2003) reported no differences in either regional or global levels of striatal 18F-dopa uptake between patients with and without GID. In another study, the severity of GID was inversely correlated with baseline 18 F-dopa uptake before surgery rather than posttransplantation increases. The authors argued against GID arising as a consequence of excessive dopaminergic function (Hagell et al., 2002). In support to this view, in one of the double-blind sham-surgery trials (Freed et al., 2001), there was no obvious relationship between the severity of GID and increases in 18F-dopa uptake. Further­ more, a subsequent study using an 11C-raclopride paradigm with administration of placebo or methamphetamine reported no evidence that GID were caused by abnormal dopamine release from the grafts. Also these authors did not observe any correlation between baseline or after methamphetamine putaminal 11C-raclopride binding and dyskinesia severity scores in the con­ tralateral side of the body (Piccini et al., 2005). Observations from animal models of dyskine­ sias have indicated a possibility that failure of the grafts to restore a precise distribution of dopami­ nergic synaptic contacts on host neurons could result in abnormal gating of corticostriatal inputs, causing abnormal striatal signaling and synaptic plasticity which may result in increased dyskinesia (Cenci and Hagell, 2005). The occurrence of GID may be also induced by inflammatory and immune responses around the graft. In this context, it is interesting to note that in the study of Olanow et al. (2003), dyskinesias developed after discontinuation of immunosuppressive therapy, with signs of an inflammatory reaction around the grafts in autop­ sied cases. In agreement with this observation, withdrawal of immunosuppression at 29 months

after transplantation in the Lund subjects asso­ ciated with increased dyskinesias, which might have been caused by either growth of the graft or worsening of a low-grade inflammation around the graft (Piccini et al., 2005). Another contributing factor for the develop­ ment of GID could include the composition of the cell suspension. The composition of the graft derived from human fetal VM tissue contains a varied proportion of nondopaminergic neurons or nonneuronal cells with different properties such as firing pattern, transmitter release, and axonal growth capacity (Isacson et al., 2003). Among these nondopaminergic neurons, seroto­ nergic neurons have been found at postmortem in the grafted tissue of PD patients (Mendez et al., 2008). It was recently shown using PET with 11C­ DASB, a marker of presynaptic serotonergic terminals, an excessive serotonergic innervation in the grafted striatum of two patients with PD, who had exhibited major motor recovery after striatal transplantation of human fetal VM tissue but later developed GID (Politis et al., 2010). It was also shown that GID of these patients were significantly attenuated by systemic administra­ tion of a serotonin 1A receptor agonist, which dampens transmitter release from serotonergic neurons. These findings indicated that GID in these patients was caused by serotonergic hyper­ innervation and it was suggested that the ratio between serotonin and dopamine neurons could be the driving factor for the development of GID (Politis et al., 2010). Future directions Functional imaging can improve the objective assessment of future cell therapy trials in PD. 18 F-dopa PET can help facilitate patient selection and screening, as patients with baseline reductions in 18F-dopa uptake extending to the ventral part of the striatum and patients with reductions in 18F-dopa uptake consistent with atypical or secondary parkinsonism should be excluded from these trials.

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Combined diffusion-based tractography and functional magnetic resonance imaging (fMRI) studies as measures of structural and functional brain connectivity have contributed to reveal the complex brain organization into large-scale net­ works. Such an organization not only permits understanding of complex segregation and inte­ gration during high cognitive processes but also allows assessing connectivity alterations underly­ ing neurodegenerative diseases and the potential effects of restorative therapies (Ciccarelli et al., 2008; Guye et al., 2008; Hirano et al., 2008; Huang et al., 2008; Smith et al., 2009). Functional imaging can be also used to explore the possible role of host inflammatory reaction on outcomes following transplantation. Detailed experimental and human postmortem studies have shown that activation of microglia, the brain’s resident tissue macrophages, has a particu­ larly close association with active brain pathology. Microglia constitute 10–20% of glial cells and are ontogenetically related to cells of mononuclear phagocyte lineage. They are known to respond to a variety of pathological stimuli by expressing de novo numerous immunologically relevant mole­ cules including HLA antigens, chemokines, and cytokines. The transformation from a resting to an activated state is rapid and often takes place before any other sign of tissue damage or cell death can be seen. The presence of activated microglia is, therefore, a useful indicator of ongoing neuronal injury and reflects disease activ­ ity rather than a specific etiology. One of the molecules expressed de novo during the “activa­ tion” of microglia is the translocator protein (TSPO). 11C-PK11195 is a selective ligand for the TSPO. The role of neuroinflammation in influen­ cing the outcome of transplantation procedures in PD is not known. Finally the proportion of different cells within the graft particularly the ratio between dopamine and serotonin cells and their role in the develop­ ment of GID could be assessed in vivo by using PET ligands targeting presynaptic dopaminergic and serotoninergic terminals.

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202 Lindvall, O., & Hagell, P. (2000). Clinical observations after neural transplantation in Parkinson’s disease. Progress in Brain Research, 127, 299–320. Lindvall, O., Sawle, G., Widner, H., Rothwell, J. C., Björklund, A., Brooks, D., Brundin, P., et al. (1994). Evidence for long-term survival and function of dopaminergic grafts in progressive Parkinson’s disease. Annals of Neurology, 35, 172–180. Ma, Y., Feigin, A., Dhawan, V., Fukuda, M., Shi, Q., Greene, P., et al. (2002). Dyskinesia after fetal cell transplantation for parkinsonism: A PET study. Annals of Neurology, 52, 628–634. Ma, Y., Tang, C., Chaly, T., Greene, P., Breeze, R., Fahn, S., et al. (2010). Dopamine cell implantation in Parkinson’s disease: Long-term clinical and (18)F-FDOPA PET out­ comes. Journal of Nuclear Medicine, 51, 7–15. Mendez, I., Dagher, A., Hong, M., Gaudet, P., Weerasinghe, S., McAlister, V., et al. (2002). Simultaneous intrastriatal and intranigral fetal dopaminergic grafts in patients with Parkin­ son disease: A pilot study. Report of three cases. Journal of Neurosurgery, 96, 589–596. Mendez, I., Dagher, A., Hong, M., Hebb, A., Gaudet, P., Law, A., et al. (2000). Enhancement of survival of stored dopami­ nergic cells and promotion of graft survival by exposure of human fetal nigral tissue to glial cell line-derived neuro­ trophic factor in patients with Parkinson’s disease. Report of two cases and technical considerations. Journal of Neuro­ surgery, 92, 863–869. Mendez, I., Viñuela, A., Astradsson, A., Mukhida, K., Hallett, P., Robertson, H., et al. (2008). Dopamine neurons implanted into people with Parkinson’s disease survive with­ out pathology for 14 years. Nature Medicine, 14, 507–509. Miletich, R. S., Chan, T., & Gillespie, M. (1988). Contralateral basal ganglia metabolism is abnormal in hemiparkinsonian patients. An FDG-PET study. Neurology, 38, S260. Morris, E. D., Fisher, R. E., Alpert, N. M., Rauch, S. L., & Fischman, A. J. (1995). In vivo imaging of neuromodulation using positron emission tomography: Optimal ligand charac­ teristics and task length for detection of activation. Human Brain Mapping, 3, 35–55. Morrish, P. K., Rakshi, J. S., Bailey, D. L., Sawle, G. V., & Brooks, D. J. (1998). Measuring the rate of progression and estimating the preclinical period of Parkinson’s disease with [18F]dopa PET. Journal of Neurology, Neurosurgery and Psychiatry, 64, 314–319. Morrish, P. K., Sawle, G. V., & Brooks, D. J. (1996). An [18F] dopa-PET and clinical study of the rate of progression in Parkinson’s disease. Brain, 119, 585–591. Muñoz, A., Li, Q., Gardoni, F., Marcello, E., Qin, C., Carlsson, T., et al. (2008). Combined 5-HT1A and 5-HT1B receptor agonists for the treatment of L-DOPA-induced dyskinesia. Brain, 131, 3380–3394. Olanow, C. W., Goetz, C. G., Kordower, J. H., Stoessl, A. J., Sossi, V., Brin, M. F., et al. (2003). A double-blind controlled

trial of bilateral fetal nigral transplantation in Parkinson’s disease. Annals of Neurology, 54, 403–414. Pate, B. D., Kawamata, T., Yamada, T., McGeer, E. G., Hewitt, K. A., Snow, B. J., et al. (1993). Correlation of striatal fluorodopa uptake in the MPTP monkey with dopa­ minergic indices. Annals of Neurology, 34, 331–338. Peschanski, M., Defer, G., N’Guyen, J. P., Ricolfi, F., Monfort, J. C., Remy, P., et al. (1994) Bilateral motor improvement and alteration of L-dopa effect in two patients with Parkin­ son’s disease following intrastriatal transplantation of foetal ventral mesencephalon. Brain, 117, 487–499. Piccini, P., Brooks, D. J., Björklund, A., Gunn, R. N., Grasby, P. M., Rimoldi, O., et al. (1999). Dopamine release from nigral transplants visualized in vivo in a Parkinson’s patient. Nature Neuroscience, 2, 1137–1140. Piccini, P., Lindvall, O., Björklund, A., Brundin, P., Hagell, P., Ceravolo, R., et al. (2000). Delayed recovery of movementrelated cortical function in Parkinson’s disease after striatal dopaminergic grafts. Annals of Neurology, 48, 689–695. Piccini, P., Pavese, N., & Brooks, D. J. (2003). Endogenous dopamine release after pharmacological challenges in Par­ kinson’s disease. Annals of Neurology, 53, 647–653. Piccini, P., Pavese, N., Hagell, P., Reimer, J., Björklund, A., Oertel, W. H., et al. (2005). Factors affecting the clinical outcome after neural transplantation in Parkinson’s disease. Brain, 128, 2977–2986. Playford, E. D., & Brooks, D. J. (1992). In vivo and in vitro studies of the dopaminergic system in movement disorders. Cerebrovascular and Brain Metabolism Reviews, 4, 144–171. Playford, E. D., Jenkins, I. H., Passingham, R. E., Nutt, J., Frackowiak, R. S., & Brooks, D. J. (1992). Impaired mesial frontal and putamen activation in Parkinson’s disease: A positron emission tomography study. Annals of Neurology, 32, 151–161. Politis, M., Wu, K., Loane, C., Quinn, N. P., Brooks, D. J., Rehncrona, S., Bjorklund, A., Lindvall O., & Piccini, P. (2010). Serotonergic neurons mediate dyskinesia side effects in Parkinson’s patients with neural transplants. Science Translational Medicine, 2, 38–46. Rascol, O., Sabatini, U., Chollet, F., Fabre, N., Senard, J. M., Montastruc, J. L., et al. (1994). Normal activation of the supplementary motor area in patients with Parkinson’s disease undergoing long-term treatment with levodopa. Journal of Neurology, Neurosurgery and Psychiatry, 57, 567–571. Remy, P., Samson, Y., Hantraye, P., Fontaine, A., Defer, G., Mangin, J. F., et al. (1995). Clinical correlates of [18F]fluor­ odopa uptake in five grafted parkinsonian patients. Annals of Neurology, 38, 580–588. Rinne, J. O., Laihinen, A., Rinne, U. K., Någren, K., Bergman, J., & Ruotsalainen U. (1993). PET study on striatal dopa­ mine D2 receptor changes during the progression of early Parkinson’s disease. Movement Disorder 8, 134–138.

203 Sawle, G. V., Bloomfield, P. M., Björklund, A., Brooks, D. J., Brundin, P., Leenders, K. L., et al. (1992). Transplantation of fetal dopamine neurons in Parkinson’s disease: PET [18F]6-L-fluorodopa studies in two patients with putaminal implants. Annals of Neurology, 31, 166–173. Smith, S. M., Fox, P. T., Miller, K. L., Glahn, D. C., Fox, P. M., Mackay, C. E., et al. (2009). Correspondence of the brain’s functional architecture during activation and rest. Proceed­ ings of the National Academy of Sciences of the United States of America, 106, 13040–13045. Snow, B. J., Tooyama, I., McGeer, E. G., Yamada, T., Calne, D. B., Takahashi, H., et al. (1993). Human positron emission tomographic [18F]fluorodopa studies correlate with dopamine cell counts and levels. Annals of Neurology, 34, 324–330. Sullivan, A. M., Pohl, J., & Blunt, S. B. (1998). Growth/differ­ entiation factor 5 and glial cell line-derived neurotrophic factor enhance survival and function of dopaminergic grafts in a rat model of Parkinson’s disease. European Journal of Neuroscience, 10, 3681–3688.

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 11

Imaging non-motor aspects of Parkinson’s disease David J. Brooks
,† and Nicola Pavese‡ †

Division of Experimental Medicine, Imperial College London, Hammersmith Hospital, London, UK MRC Clinical Sciences Centre and Department of Medicine, Imperial College London, London, UK

‡

Abstract: In this chapter the imaging changes associated with non-motor aspects of Parkinson’s disease (PD) are reviewed. The relationship between reduced monoaminergic and cholinergic function and cognitive difficulties, depression, fatigue, sleep disorders, and dysautonomia is discussed and the relevance of Alzheimer pathology to PD dementia debated. Finally the discordance between the development of functional changes in PD and Braak staging is highlighted. Keywords: Parkinson; Dementia; Depression; Sleep; Fatigue Autonomic

of patients who survive for 20 years (Emre 2003; Hely et al., 2008). One third of PD patients will develop significant depression (Burn 2002) while the majority experience daytime somnalence and nocturnal insomnia later in the course of their disease (Rye et al., 2000). The pathological hallmark of PD is the formation of ubiquitin-positive intraneuronal inclusions (Lewy bodies and neurites) containing alpha­ synuclein. Neuronal loss is greatest in the substan­ tia nigra pars compacta (SNc) but other brainstem nuclei and cortical areas are also targeted. Loss of nigrostriatal projections results in profound dopamine depletion and, when 80% of striatal dopamine and 50% of the nigra compacta cells have been lost, the classical motor signs of PD become evident (Fearnley and Lees, 1991). Along with nigral degeneration in PD, noradrenergic, seroto­ ninergic, and cholinergic neurotransmission are

Introduction Parkinson’s disease (PD) is clinically characterized by the presence of asymmetrical limb bradykinesia, resting tremor, rigidity, and later postural instability. However, non-motor symptoms are also common and can have a greater impact on patient’s quality of life. The spectrum of non-motor symptoms in PD is wide and includes hyposomia, autonomic dysfunction, chronic fatigue, sleep disorders, mood disorders, psychosis, impaired cognition, and dementia (Chaudhuri et al., 2006). These symptoms can precede motor symptoms and signs though more often complicate advanced stages of the disease. The overall prevalence of dementia in PD is two to six times that of aged matched controls and affects 80%


Corresponding author. Tel.: þ44-20-83833172; E-mail: david. [email protected]

DOI: 10.1016/S0079-6123(10)84011-7

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also disrupted (Agid et al., 1987). Braak and col­ leagues have reported that Lewy body pathology evolves in an ascending manner, beginning in the medulla oblongata and olfactory structures in stage 1, spreading to the pons in stage 2, and the SNc and midbrain brain nuclei in stage 3, by which point motor symptoms appear (Braak et al., 2004). By stage 4 limbic areas become affected and in stages 5–6, the inclusions appear in the association and primary neocortex. Braak staging has, how­ ever, been challenged as these workers selected cases that were all known to have medullary Lewy bodies present (Kalaitzakis et al., 2008). As the locus ceruleus and median raphe are in the pons, dysfunction of noradrenergic and serotoninergic transmission would be predicted by stage 2, while cholinergic involvement could occur in stage 3 or 4. Both sleep control and mood are regulated by the locus ceruleus and median raphe while the cholinergic system is important for memory and attention. Their involvement by Lewy body pathology could, therefore, result in non-motor problems for PD patients. Imaging can reveal the structural and functional changes associated with pathology. T1-weighted magnetic resonance imaging (MRI) reveals reduc­ tions in whole and regional brain volume (atrophy) which can be quantitated while T2-weighted MRI is sensitive to white matter changes. Water normally flows along neural tracts in the brain. Diffusion-weighted (DWI) or diffusion tensor imaging (DTI) can be used to quantify loss of anisotropy (directionality) or increases in ampli­ tude of water diffusion and thus demonstrate dis­ ruption of neural tracts. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) are radiotracerbased modalities and allow one to investigate in vivo the function of basal ganglia, their cortical projection areas, and other brainstem nuclei. In PD, PET and SPECT have been principally used to evaluate (1) dysfunction of presynaptic dopa­ minergic terminals and levels of synaptic dopa­ mine; (2) associated patterns of change in cerebral metabolism; and (3) the contribution of

non-dopaminergic neurotransmitters to motor and non-motor complications of PD. It is now also possible to image amyloid deposition in the brain allowing the contribution of Alzheimer pathology to dementia in PD to be assessed. This chapter will discuss the role that imaging has played in revealing the mechanisms underlying non-motor symptoms in PD. We will primarily focus on dementia, depression, and sleep disorders, as they are the most common symptoms reported by patients. However, other non-motor symptoms, including hyposmia and cardiac sympathetic denervation, will also be discussed.

Imaging dementia in Parkinson’s disease Glucose metabolism In PD, dementia can result from cortical Lewy body disease, concommitant Alzheimer pathol­ ogy, small vessel disease, and disruption of neuro­ transmitter systems. If the dementia occurs more than 1 year after motor problems, the syndrome is termed Parkinson’s disease dementia (PDD) but if patients present with dementia prior to or con­ commitantly with parkinsonism they are labeled as having dementia with Lewy bodies (DLB) (McKeith et al., 2005). Like Alzheimer’s disease (AD), DLB and PDD are associated with impaired recall, speech, and perceptual difficulties but in addition are characterized by rigidity, fluctuating confusion, psychosis, and visual hallucinosis. At postmortem the majority of DLB cases have mixed pathology with cortical Lewy body inclusions, amyloid plaques, and neurofibrillary tangles. However, in contrast to AD cases, DLB patients lose dopami­ nergic function due to Lewy body degeneration of nigrostriatal projections. In PDD the presence of Alzheimer pathology is less common (Aarsland et al., 2005). Functional imaging can detect the presence of dopaminergic dysfunction, cortical hypometabolism, and amyloid pathology

207

in these dementia syndromes, thus aiding their classification. 18 F-2-fluoro-2-deoxyglucose (FDG) PET is a marker of hexokinase activity and reveals resting patterns of regional cerebral glucose metabolism (rCMRGlc) in humans. AD cases show a charac­ teristic pattern of reduced rCMRGlc which starts in posterior cingulate, then parietal and temporal association regions, and subsequently spreads to involve prefrontal cortex (Hoffman et al., 2000). In PDD patients FDG PET shows a similar pat­ tern of reduced glucose metabolism targeting pos­ terior cingulate and temporoparietal association areas. Yong and colleagues have compared pat­ terns of glucose metabolism in PD, PDD, and DLB patients (Yong et al., 2007). Statistical com­ parisons between groups were performed at a voxel level using statistical parametric mapping (SPM). Compared with normal controls, both PDD and DLB patients showed significant decreases of rCMRGlc in the parietal lobe, occipital lobe, temporal lobe, frontal lobe, and anterior cingulate. Compared with PD, DLB and PDD patients showed relative reductions of glucose metabolism in inferior and medial frontal lobes bilaterally. A direct comparison between DLB and PDD showed a relative metabolic decrease in anterior cingulate in patients with DLB. These findings support the concept that PD with later dementia and DLB have a similar underlying pattern of cortical dysfunction reminiscent of AD, although the ante­ rior cingulate and occipital lobe are more involved in DLB. Reduced temporoparietal rCMRGlc can be detected even in non-demented PD patients with established disease. Hu and colleagues were able to localize with SPM significant decreases in posterior parietal rCMRGlc in one third of PD patients (Hu et al., 2000). This hypometabolism is likely to reflect the presence of occult primary cortical pathology but it remains to be determined whether the observed glucose hypometabolism in these subjects is a predictive factor for later onset of dementia.

Dopaminergic function 123

I-N-3-fluoropropyl-2b-carbomethoxy-3b-(4­ iodophenyl)tropane (123I-FP-CIT) SPECT, an in vivo marker of dopamine transporter (DAT) binding, will discriminate PDD and DLB from AD during life based on the detection of striatal dopamine terminal dysfunction. Walker and colleagues assessed the integrity of nigrostriatal metabolism in PD, AD, and DLB patients and reported that DLB and PD patients showed similar levels of reduced striatal uptake of 123 I-FP-CIT while patients with AD had normal DAT binding (Walker et al., 2002). In a follow-up clinicopathological study where autopsy material was available for 20 dementia cases previously imaged, these authors concluded that 123I-FP-CIT SPECT had a sensitivity of 88% and specificity of 100% for DLB compared with a sensitivity of 75% and specificity of 42% for clinical impression(Walker et al., 2007). DAT imaging, therefore, significantly increases the specificity for a diagnosis of DLB in dementia cases. O’Brien and colleagues used 123I-FP-CIT SPECT to assess the extent and pattern of DAT loss in patients with clinically probable DLB, PDD, and AD (O’Brien et al., 2009). Again, AD patients showed similar striatal 123I-FP-CIT uptake to healthy controls while transporter loss in patients with DLB was of similar magnitude to that seen in PD. Compared with PD patients, where selective targeting of putamen DAT binding was seen, patients with DLB and PDD showed a more global striatal reduction in 123I-FP-CIT binding with loss of the caudate–putamen gradient present in PD. There was a significant correlation between Mini Mental State Examination scores and striatal 123 I-FP-CIT binding in PD patients with dementia, supporting the hypothesis that striatal dopaminer­ gic loss may contribute to the cognitive impairment of these patients. No such correlation, however, was found in patients with DLB. While striatal dopamine terminal dysfunction is present in DLB and PDD and discriminates them both from AD, it is unlikely to explain fully the

208

presence of the dementia. Function of mesolimbic and mesocortical dopaminergic projections in PD dementia has been investigated with 18F-dopa PET. Regional 18F-dopa uptake is determined by rates of presynaptic decarboxylation of the radio­ tracer by aromatic amino acid decarboxylase (AADC) and the density of the axonal terminal plexus. Measurements of 18F-dopa uptake in dopaminergic areas in PD, usually determined as an influx constant, Ki, therefore, reflect the func­ tion of remaining dopamine terminals. Using 18F­ dopa PET, Ito and colleagues examined changes in dopaminergic function in non-demented and demented PD patients matched for age, disease duration, and disease severity (Ito et al., 2002). Compared with the PD patients without dementia, the PDD patients showed 18F-dopa Ki reductions in the right caudate and bilaterally in the ventral striatum and the anterior cingulate. These findings therefore add support to the concept that demen­ tia in PD is associated with impaired mesocortical and caudate dopaminergic function. Cholinergic function 123

I-iodobenzovesamicol (123I-BVM) SPECT is a marker of acetylcholine vesicle transporters and has been used to detect cholinergic deficiency in PD (Kuhl et al., 1996). Patients without dementia have reduced parietal and occipital cortex 123I-BVM bind­ ing, whereas PDD patients show globally reduced cortical binding. The PET ligands 11C-methyl-4­ piperidyl autate (11C-MP4A) and 1- [11C] methylpi­ periden-4-yl propionate(11C-PMP) are substrates for acetylcholinesterase (AChE). Global cortical 11 C-MP4A binding was reduced by 30% in PDD but by only 11% in PD (Fig. 1) (Hilker et al., 2005). Individual levels of frontal and temporo-par­ ietal 11C-MP4A binding correlated with levels of striatal 18F-dopa uptake across the combined PD and PDD cohort. The authors concluded that a par­ allel reduction in dopaminergic and cholinergic func­ tion occurs in PD and PDD. Using 11C-PMP PET, a significant correlation was reported between cortical

18F-dopa

11C-NM4PA

Normal

PD

PDD

Fig. 1. Imaging dopamine storage capacity (18F-dopa) and acetylcholine esterase activity (11C-NMP4A) in Parkinson’s disease with (PDD) and without dementia (PD). Striatal dopaminergic function and cortical acetylcholine esterase activity are both reduced in PD and PDD (Hilker et al., 2005).

AChE activity and performance on tests of atten­ tional and executive functions in a combined group of PD and PDD patients (Bohnen et al., 2005). Interestingly, cortical AChE deficiency did not cor­ relate with motor symptoms. In conclusion, signifi­ cant cognitive decline in PD is associated with dysfunction of the cholinergic system. PET imaging findings provide a rationale for the use of central AChE inhibitors in PD dementia. Muscarinic M1 acetylcholine receptor (AChR) availability has also been assessed in PD. [11C]­ N-methyl-4-piperidylbenzilate (11C-NMPB) PET has shown raised frontal cortex M1 availability in PD compatible with the presence of depleted ACh levels (Asahina et al., 1998). A PDD case had the highest 11C-NMPB binding in frontal and temporal areas. The authors suggested that the increased availability of muscarinic receptor in the frontal cor­ tex in PD reflected denervation hypersensitivity caused by loss of ascending cholinergic input to the cortex. However, in contrast, when muscarinic AChr receptor binding was measured with 123I-iodo-quinu­ clidinyl-benzilate (QNB) SPECT, PDD patients showed significantly reduced tracer uptake in frontal regions and temporal lobes bilaterally (Colloby et al., 2006). It is possible that local cortical Lewy body disease led to loss of M1 expression in these cases.

209

Amyloid load

Imaging PD depression

Over the last few years, PET radiotracers have been developed to image b-amyloid plaque load in dementia. Pittsburgh Compound B (11C-PIB) is a neutral derivative of the histological dye thiofla­ vin T that in AD brain slices shows nanomolar affinity for neuritic b-amyloid plaques but low affi­ nity toward amorphous diffuse b-amyloid deposits and intracellular neurofibrillary tangles and Lewy bodies. PET studies have shown a twofold increase in 11C-PIB retention in association cortex and cin­ gulate of patients with AD compared with healthy controls (Edison et al., 2006; Klunk et al., 2004). Interestingly, around 70–80% of DLB cases have been reported to show increased cortical 11C-PIB uptake but this is true of fewer PDD cases despite the presence of reduced tempero-parietal glucose metabolism (Fig. 2) (Edison et al., 2008; Maetzler et al., 2009). These findings suggest that b-amyloid deposition does not play a major role in the patho­ genesis of late dementia in PD. In DLB levels of amyloid do not appear to correlate with the pattern of symptomatology (Gomperts et al., 2008). It has been suggested that a high amyloid load may act to trigger early dementia in DLB but that the nature of the dementia is determined by local Lewy body disease.

Around one third of PD patients will experience significant depression depending on the criteria used for diagnosis and depression can precede the onset of motor symptoms (Burn, 2002). The etiology of affective symptoms in PD is multifocal: a reaction to the presence of disability, serotoner­ gic and noradrenergic dysfunction, and loss of limbic dopaminergic neurotransmission could all be contributors. In PD patients with depression, Mayberg and colleagues (1989) reported selective glucose hypo­ metabolism in the head of caudate and the inferior orbital region of the frontal lobe compared with non-depressed PD patients and control subjects. Depressed PD patients have also been reported to have decreased DAT binding involving the whole striatum, the left head of caudate nucleus, and the left anterior putamen (Weintraub et al., 2005). Taken together, these findings suggest that dys­ function of basal ganglia circuits projecting to the inferior frontal lobe may cause aberrant regula­ tion of mood. Function of the serotonergic system can be inves­ tigated in PD with both PET and SPECT ligands. [11C]-3-amino-4-(2-dimethylaminomethyl-phe­ nylsulfanyl)-benzonitrile (11C-DASB) PET is a

11C-PIB

Control

PDD

No significant uptake

PET Dementia with Lewy bodies

Increased uptake in 70%

Fig. 2. Amyloid load in a case of Parkinson’s disease with later dementia (PDD) and two cases of dementia with Lewy bodies (DLB) detected with 11C-PIB PET. The two DLB cases but not the PDD case show amyloid deposition. Courtesy of Paul Edison.

210

postsynaptically on cortical pyramidal neurons and glial cells. HT1A sites are most highly expressed in the hippocampus and are absent in the cerebel­ lum. A 25% reduction of 11C-WAY100635 bind­ ing has been reported in the midbrain raphe of PD patients though the magnitude of reduction was no different in those cases with and without depression (Fig. 3) (Doder et al., 2000). As with 123 I-b-CIT SPECT findings, these 11C-WAY100635 PET results do not support the view that seroto­ nergic loss plays a major role in causing depres­ sion in PD. Having said that, none of these PD cases had severe depression and further investiga­ tions are required to establish whether serotonin neurotransmission in the neocortex is decreased in PD patients with a major depressive disorder. 11 C-RTI 32 PET is a marker of both noradre­ nergic and dopaminergic functions (Guttman et al., 1997). In PD there is reduced binding of 11 C-RTI 32 in both striatal and extrastriatal areas. When 11C-RTI 32 PET findings were compared in PD patients with and without depression matched for age, disability, disease duration, and doses of anti-parkinsonian medications, the patients with depression showed a relative reduction of 11 C-RTI 32 binding in the noradrenergic locus

marker of the brain serotonin transporter (SERT) and in advanced PD patients without overt depression, SERT binding levels have been reported to be reduced by 22–30% in stria­ tal and cortical brain areas (Guttman et al., 2007). 123I-b-CIT is a tropane analogue which binds with nanomolar affinity to DAT, noradre­ naline trans porter, and SERT. Striatal binding of the tracer 24–after intravenous injection reflects DAT binding, whereas midbrain binding 1-4­ after intravenous injection reflects SERT availabil­ ity (Berding et al., 2003). With 123I-b-CIT SPECT, Kim and colleagues reported reduced striatal DAT binding in early PD but found no reduction of 123I-b-CIT binding in their brainstem (Kim et al., 2003). PD patients with and without depres­ sion showed similar midbrain uptake of the radi­ oligand at 1- and there was no correlation between radiotracer binding in this region and Hamilton depression rating scale scores. 11 C-WAY100635 PET is a marker of serotonin 5-hydroxytryptophan-1A (5-HT1A) receptor avail­ ability. These receptors not only act as autorecep­ tors when localized on 5-HT cell bodies in the midbrain raphe nuclei, inhibiting serotonin release, but are also widely distributed 11

C-WAY 100635 PET HT1A binding in PD depression 5 4.5

Healthy Volunteer

4

Raphe BP

3.5

p = 0.001

3 2.5

p = 0.002 Raphe

n=9

2

n = 12

n = 12

PD

1.5 1 0.5 0

Healthy volunteers

PD euthymic

PD depression

Fig. 3. Imaging serotonin HT1A sites in PD with and without depression with 11C-WAY100635 PET. Median raphe tracer uptake is reduced in PD to a similar extent whether depression is a problem or not. Courtesy of M. Doder.

211

ceruleus, the thalamus, and several regions of the limbic system, including amygdala, ventral stria­ tum, and anterior cingulate (Remy et al., 2005). Severity of anxiety in PD was inversely correlated with 11C-RTI 32 binding in these regions, whereas apathy was inversely correlated with the radiotra­ cer binding in the ventral striatum. These results suggest that depression and anxiety in PD are associated with both loss of noradrenaline inner­ vation and selective loss of dopaminergic projec­ tions to the limbic system.

Chronic fatigue in PD Around one third of PD patients complain of chronic fatigue and this symptom can antedate the development of motor disability (Abe et al., 2000; Karlsen et al., 1999). Patients typically com­ plain of a feeling of constant exhaustion but its severity does not correlate well with physical dis­ ability or suggesting that the two conditions may arise from independent mechanisms. Addition­ ally, no direct relationship between presence of fatigue and type, dosage, and duration of antiparkinsonian medication has been found (Abe et al., 2000; Hagell and Brundin, 2009). Fatigue can be influenced by co-existent depression, sleep disturbance, and autonomic dysfunction but can also be a complaint in non-depressed patients and those with normal sleep hygiene. 11C-DASB

Healthy volunteer

Basal ganglia dysfunction may play a role in generating fatigue as it has been associated with reduced glucose metabolism and blood flow in the putamen and supplementary motor areas (Hitten et al. 1993). However, levodopa-naïve PD patients with fatigue have similar striatal DAT availability to patients without fatigue, suggesting that striatal dopaminergic dysfunction is not directly involved (Schifitto et al., 2008). It would, therefore, seem likely that extrastriatal dopaminergic pathways or non-dopaminergic neurotransmitters underlie the pathogenesis of fatigue in PD. As SERTs have been reported to be reduced in chronic fatigue syndrome (Yamamoto et al., 2004), we have investigated their availability in eight fatigued and eight non-fatigued PD cases with 11 C-DASB PET (Fig. 4, Table 1). The presence of fatigue was defined as a score  8 on the Parkinson Fatigue Scale (PFS-16). The fatigued and nonfatigued PD groups of patients were matched for disability and drug exposure and were not depressed or cognitively impaired. PET scans were performed using an ECAT EXACT HR þ (CTI/Siemens 962) camera. All patients had their medication stopped for at least 12th before PET. Scans were acquired in 3D mode over 90 min fol­ lowing a bolus injection of 450 MBq of 11C-DASB. Parametric images of specific 11C-DASB binding potentials (BPs) were generated on a voxel-wise basis for the whole brain using a basis function implementation of the simplified reference region PET

PD without fatigue

PD with fatigue

Fig. 4. Measuring serotonin transporter (SERT) availability with 11C-DASB PET in Parkinson’s disease with and without fatigue. The fatigued cases show a significant reduction in striatal and cortical SERT binding.

212 Table 1. Demography of PD patients having 11C-DASB PET Age (yrs)

Disease duration

Levodopa equivalent Units mg

PFS-16 score

UPDRS off

UPDRS on

PD-NF patients M M F F M M M F

53 71 46 76 52 77 70 69

7 4 3 4 2 6 4 4

450 200 750 915 835 715 550 250

1 1 3 5 5 6 6 7

30 42 33 36 26 40 36 28

15 25 15 27 19 25 20 17

PD-F patients M F F M F M M M

69 57 66 55 58 77 70 66

5 3 7 3 6 8 3 11

750 785 1480 280 635 600 1000 1135

10 10 11 13 14 14 15 16

35 22 42 32 36 38 34 38

25 14 22 17 17 21 23 31

PD-NF, with no fatigue; PD-F, Partinson’s disease with fatigue; PFS-16, Parkins on Fatigue Scale; UPDRS Parkins disease Unified Parkinson’s Disease Rating Scale.

compartmental model with the cerebellum providing the reference tissue input function. Parametric images of 11C-DASB BP were then normalized, using SPM2 software, to a smoothed 11C-DASB ADD image template in standard Montreal Neurologicval Institute (MNI) space created in-house from healthy control subjects. Integrated ADD images of

11

C-DASB uptake were also created as it was a standard object map to extract region of interest (ROI) data for caudate nucleus, putamen, ventral striatum, thalamus, and median raphe. ROIs were free-hand traced, using ANALYZE 6.0 software, onto the single subject MRI in MNI space available in SPM2. The standard object map was applied to

Table 2. Regional mean 11C-DASB binding potential values (+ Standard Deviation) in normal subjects and in Parkinson’s disease patients with (PD-F) and without fatigue (PD-NF)

Caudate Putamen Ventral striatum Thalamus


Healthy subjects (n = 10)

PD-NF (n = 8)

1.68 + 2.10 + 1.86 + 1.49 +

0.80 + 1.09 + 1.06 + 1.13 +

p < 0.05 compared to healthy subjects; p < 0.01 compared to healthy subjects; p < 0.001 compared to healthy subjects; direct comparison PD-NF and PD-F: § p < 0.05; §§ p < 0.01 ; §§§ p < 0.001.










0.87 0.9 0.63 0.37

0.17

0.15

1.17

0.21


PD-F (n = 7) 0.28 + 0.16


§§§ 0.51 + 0.26


§§§ 0.48 + 0.34


§§ 0.5 + 029

§§

213

each spatially normalized image of 11C-DASB BP and correspondent ADD images. Visual inspec­ tion of each plane for both images was made to ensure correct placement of the object regions over the correspondent structures. After applying target regions to structures, 11C-DASB BP values were quantified using ANALYZE 6.0. Both PD groups had reduced 11C-DASB bind­ ing in the caudate, putamen, ventral striatum, and thalamus compared with healthy controls subjects (see Table 2). However, in all these areas SERT binding was significantly lower in fatigued than non-fatigued PD cases and, in addition, cingulate 11 C-DASB uptake was relatively reduced. Within the PD cohort, individual 11C-DASB BPs inver­ sely correlated with PFS-16 fatigue scores in all the examined ROIs. These findings would suggest that serotonergic function, particularly in limbic areas, may well underlie this disabling symptom when present in PD.

Imaging sleep disorders in PD Sleep disorders affect up to 90% of PD patients and disrupt patients’ working and social life (Chaudhuri et al., 2006). They include nocturnal

insomnia, daytime somnolence, and parasomnias such as vivid dreams and nightmares. The pro­ blem is likely to arise from an interaction between degeneration of central sleep regulatory centers which modulate wakefulness and rapid eye move­ ment (REM) sleep and exposure to dopaminergic replacement therapy. Sleep regulatory centers include the noradrenergic locus ceruleus, seroto­ ninergic dorsal raphe, histaminergic tuberomam­ milary nucleus, dopaminergic ventral tegmental area, and the hypothalamus. Dopaminergic function has been studied in PD cases with sleep problems. An inverse correlation between excessive daytime sleepiness, rated with the Epworth sleepiness scale (ESS), and striatal DAT binding measured with 123I-FP-CIT SPECT has been reported in early PD (Happe, et al., 2007). No correlation of the EES score with age, disease duration, UPDRS motor score, or depression score was found. These data suggest that dopaminergic nigrostriatal degeneration plays a role in daytime sleepiness in early PD. 18 F-dopa PET, which is commonly used as an in vivo marker of dopaminergic function in PD, can also be employed to evaluate the distribution and function of the serotonergic and noradrenergic systems in the brain. 18F-dopa uptake reflects the

18F-dopa

uptake in PD patients with REM sleep behavior disorder Patients with early-stage PD (n = 10) FDOPA Ki contralat. mesopont. (10−3 × min−1)

60 r = –0.65 p < 0.05

50 40 30 20 10 0 0

5

10

15

20

25

REM-sleep duration [%SPT]

Fig. 5. Correlating dopamine storage capacity measured with 18F-dopa PET with duration of REM sleep in PD. Reduced midbrain 18 F-dopa uptake was associated with shorter REM sleep duration (Hilker et al. 2003).

214

activity of the enzyme AADC which is also found in serotonergic and noradrenergic terminals. Hilker and colleagues measured striatal and mid­ brain 18F-dopa uptake in PD patients with a his­ tory of sleep disorders (Fig. 5) (Hilker et al., 2003). They found a significant inverse correlation between mesopontine 18F-dopa uptake and REM sleep duration, as measured by polysomnography. The authors suggested that increased monoami­ nergic activity in the rostral brainstem might result in the suppression of nocturnal REM sleep in PD though whether this involves dopmainergic or non-dopaminergic mechanisms remains uncertain.

Imaging cardiac sympathetic denervation A majority of patients with idiopathic PD show a significant loss of sympathetic innervation of the heart whereas this is not seen in atypical parkinso­ nian variants (Braune, 2001). Decreased myocardial uptake of the sympathetic markers 123I-metaiodo­ benzylguanidine (MIBG) and 18F-fluorodopamine has been reported in PD patients even at early stages of the disease when cardiovascular reflexes are still intact (Oka et al., 2007). However, MIBG SPECT is not as sensitive a marker of early PD as striatal DAT loss, as 50% of Hoehn and Yahr stage 1 cases still show normal tracer binding (Sawada et al., 2009). Both 18F-dopamine and MIBG use the same metabolic pathway as nore­ pinephrine and their myocardial uptake reflects not only the density of postganglionic sympathetic neurons but also their functional integrity. The association between myocardial 123I-MIBG uptake and orthostatic hypotension, pulse and blood pressure changes during the Valsalva maneuvre, and erect and supine plasma norepinephrine concentrations in PD patients has been studied (Oka et al., 2007). Mean myocardial 123I-MIBG uptake was significantly lower in PD patients with orthostatic hypotension and an abnormal Valsalva response. However, no association was found between the fall in supine BP on head-up

tilt and the baroreflex sensitivity or plasma nor­ epinephrine concentrations. These results suggest that cardiac sympathetic dysfunction is a primary cause of impaired cardiovascular reflexes in PD.

Olfactory function in PD Hyposmia affects up to 60% of PD patients and can precede the motor symptoms by years, provid­ ing a potential marker of subclinical disease. Using DWI, Scherfler and colleagues have detected abnormal olfactory tract signals in PD cases compatible with axonal loss (Scherfler et al., 2006). In contrast, olfaction is normal in atypical PD and so olfactory function tests may be a useful tool for the differential diagnosis of parkinsonian syndromes. PET and SPECT have been used to correlate the presence of olfactory and dopaminergic dysfunction in subjects with PD. Bohnen and colleagues investi­ gated the relationship between selective deficits in smell identification and loss of striatal DAT binding measured with 11C-CFT PET (Bohnen et al., 2008). Total University of Pennsylvania Smell Identifica­ tion Test (UPSIT) scores were significantly reduced in the PD patients and there was a significant corre­ lation between dorsal striatal 11C-CFT uptake and UPSIT scores (Fig. 6). A second study has evaluated the relationship between integrity of striatal DAT binding, measured with [99mTc] [2-[[2-[[[3-(4-chloro­ phenyl)-8-methyl-8-azabicyclo[3,2,1]oct-2-yl]meth­ yl](2-mercaptoethyl)amino]ethyl]amino]ethanethio­ lato(3-)-N2,N20 ,S2,S20 ]oxo-[1R-(exo-exo)] (TRO­ DAT-1) (TRODAT) SPECT, odor identification skills, and motor function in patients with early PD (Siderowf et al., 2005). UPSIT scores were strongly correlated with TRODAT uptake in the putamen. Haehner and colleagues followed patients with late-onset idiopathic hyposmia for 4 years (Haehner et al., 2007). Olfactory testing was combined with transcranial sonography of the substantia nigra and 123I-FP-CIT SPECT. Seven percent of indivi­ duals with idiopathic hyposmia developed clinical

215 11C-CFT

PET

45

Normal

UPSIT-40

35 25 15 5 0

PD

0.05 0.1 0.15 0.2 Hippocampal dat

Fig. 6. The relationship between level of hyposmia in PD and hippocampal dopamine transporter (DAT) availability measured with 11 C-CFT PET. Hippocampal levels of DAT binding correlate with scores on the UPSIT (Bohnen et al., 2008).

PD and another 6% had soft motor abnormalities. Reduction of striatal 123I-beta-CIT binding has been reported in 7 out of 40 (17.5%) hyposmic relatives of PD patients who had no parkinsonian symptoms. Four out of these seven (57%) sub­ jects with reduced striatal 123I-beta-CIT binding converted to clinical PD over a 2-year follow-up. These findings suggest that a combination of olfactory testing and neuroimaging techniques may provide a screening tool for the risk of developing PD, although the sensitivity of these modalities is relatively low and will only become of real value when neuroprotective agents are available for PD.

to explore other non-dopaminergic brain pathways involved in the pathology of non-motor symptoms of PD. For example, PET radioligands to image the cerebral cannabinoid receptor CB1 have now become available which could throw light on mechanisms of psychosis in PD. Further neuroima­ ging studies are also warranted to detect early neu­ rotransmitter changes that could be predictive factors for later onset of depression, dementia, or sleep disorders in non-complicated PD patients.

Abbreviations CFT

Conclusions

DASB

Functional imaging is now able to reveal pharma­ cological changes underlying non-motor complica­ tions of PD. In particular, it has demonstrated loss of extra-striatal dopaminergic function in patients with depression and dementia and detected reduced serotonergic transmission in cases with chronic fatigue. Loss of cholinergic function corre­ lates with disability on attentional tests. In the future development of new radiotracers is needed

DAT DLB DTI DWI FDG IBVM MIBG

2-carbomethoxy-3-(4-[18F]­ fluorophenyl)tropane 3-amino-4-[2-[(di(methyl) amino)methyl]phenyl] sulfanylbenzonitrile Dopamine transporter Dementia with Lewy bodies Diffusion tensor imaging Diffusion weighted imaging 2-Fluoro-2-deoxy-D-glucose 123 I-iodobenzovesamicol Iodine-131-meta­ iodobenzylguanidine

216

NMP4A PD PDD PET PMP RTI-32

SERT SNc SPECT UPDRS UPSIT WAY100635

N-11C-methyl-4-piperidyl acetate Parkinson’s disease Parkinson’s disease with later dementia Positron emission tomography 1-[11C]Methylpiperidin-4-yl propionate 11 C-Methyl (1R-2-exo-3-exo)­ 8-methyl-3-(4-methylphenyl)­ 8-azabicyclo[3.2.1]octane-2­ carboxylate Serotonin transporter Substantia nigra compacta Single-photon emission computed tomography Unified Parkinson’s disease rating scale University of Pennsylvania Smell Identification Test N-[2-[4-(2-methoxyphenyl)-1­ piperazinyl]ethyl]- N­ (2-pyridyl) cyclohexanecarboxamide

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217 binding in non-depressed patients with Parkinson’s disease. Eur J Neurol, 14(5), 523–528. Guttman, M., Burkholder, J., Kish, S. J., Hussey, D., Wilson, A., DaSilva, J., et al. (1997). [11C]RTI-32 PET studies of the dopamine transporter in early dopa-naive Parkinson’s dis­ ease: Implications for the symptomatic threshold. Neurology, 48, 1578–1583. Haehner, A., Hummel, T., Hummel, C., Sommer, U., Junghanns, S., & Reichmann, H. (2007). Olfactory loss may be a first sign of idiopathic Parkinson’s disease. Mov Disord, 22(6), 839–842. Hagell, P., & Brundin, L. (2009). Towards an understanding of fatigue in Parkinson disease. J Neurol Neurosurg Psychiatry, 80(5), 489–492. Happe, S., Baier, P. C., Helmschmied, K., Meller, J., Tatsch, K., & Paulus, W. (2007). Association of daytime sleepiness with nigrostriatal dopaminergic degeneration in early Parkinson’s disease. J Neurol, 254(8), 1037–1043. Hely, M. A., Reid, W. G., Adena, M. A., Halliday, G. M., & Morris, J. G. (2008). The Sydney multicenter study of Par­ kinson’s disease: the inevitability of dementia at 20 years. Mov Disord, 23(6), 837–844. Hilker, R., Razai, N., Ghaemi, M., Weisenbach, S., Rudolf, J., Szelies, B., et al. (2003). [18F]fluorodopa uptake in the upper brainstem measured with positron emission tomogra­ phy correlates with decreased REM sleep duration in early Parkinson’s disease. Clin Neurol Neurosurg, 105(4), 262–269. Hilker, R., Thomas, A. V., Klein, J. C., Weisenbach, S., Kalbe, E., Burghaus, L., et al. (2005). Dementia in Parkinson dis­ ease: functional imaging of cholinergic and dopaminergic pathways. Neurology, 65(11), 1716–1722. Hitten, J. J., van Hoogland, G., van der Velde, E. A., et al. (1993). Diurnal effects of motor activity and fatigue in Parkinson’s disease. J Neurol Neurosurg Psychiatry, 56, 874–877. Hoffman, J. M., Welsh-Bohmer, K. A., Hanson, M., Crain, B., Hulette, C., Earl, N., et al. (2000). FDG PET imaging in patients with pathologically verified dementia. J Nucl Med, 41(11), 1920–1928. Au, M. T.M., Taylor-Robinson, S. D., Chaudhuri, K. R., Bell, J. D., Labbé, C., Cunningham, V. J., et al. (2000). Cortical dysfunction in non-demented Parkinson’s disease patients: A combined 31Phosphorus MRS and 18FDG PET study. Brain, 123, 340–352. Ito, K., Nagano-Saito, A., Kato, T., Arahata, Y., Nakamura, A., Kawasumi, Y., et al. (2002 Jun). Striatal and extrastriatal dysfunction in Parkinson’s disease with dementia: a 6-[18F]fluoro-L-dopa PET study. Brain, 125(Pt 6), 1358–1365. Kalaitzakis, M. E., Graeber, M. B., Gentleman, S. M., & Pearce, R. K. (2008). Controversies over the staging of

alpha-synuclein pathology in Parkinson’s disease. Acta Neu­ ropathol, 116(1), 125–128; author reply 129-131. Karlsen, K., Larsen, J. P., Tandberg, E., & Jorgensen, K. (1999). Fatigue in patients with Parkinson’s disease. Mov Disord, 14(2), 237–241. Kim, S. E., Choi, J. Y., Choe, Y. S., Choi, Y., & Lee, W. Y. (2003). Serotonin transporters in the midbrain of Parkinson’s disease patients: a study with 123I-beta-CIT SPECT. J Nucl Med, 44(6), 870–876. Klunk, W. E., Engler, H., Nordberg, A., Wang, Y., Blomqvist, G., Holt, D. P., et al. (2004). Imaging brain amyloid in Alzheimer’s disease with Pittsburgh Compound-B. Ann Neu­ rol, 55(3), 306–319. Kuhl, D. E., Minoshima, S., Fessler, J. A., Frey, K. A., Foster, N. L., Ficaro, E. P., et al. (1996). In vivo mapping of choli­ nergic terminals in normal aging, Alzheimer’s disease, and Parkinson’s disease. Ann Neurol, 40(3), 399–410. Maetzler, W., Liepelt, I., Reimold, M., Reischl, G., Solbach, C., Becker, C., et al. (2009). Cortical PIB binding in Lewy body disease is associated with Alzheimer-like characteristics. Neurobiol Dis, 34(1), 107–112. Mayberg, H., Starkstein, S., Preziosi, S., Bolduc, P., Robinson, R., Sadzot, B., et al. (1989). Frontal lobe hypometabolism is associated with depression in Parkinson’s disease. Neurol­ ogy., 39 suppl, 274. McKeith, I. G., Dickson, D. W., Lowe, J., Emre, M., O’Brien, J. T., Feldman, H., et al. (2005). Diagnosis and management of dementia with Lewy bodies: third report of the DLB Consortium. Neurology, 65(12), 1863–1872. O’Brien, J. T., McKeith, I. G., Walker, Z., Tatsch, K., Booij, J., Darcourt, J., et al. (2009). Diagnostic accuracy of 123I-FP­ CIT SPECT in possible dementia with Lewy bodies. Br J Psychiatry, 194(1), 34–39. Oka, H., Yoshioka, M., Onouchi, K., Morita, M., Mochio, S., Suzuki, M., et al. (2007). Characteristics of orthostatic hypotension in Parkinson’s disease. Brain, 130(Pt 9), 2425–2432. Remy, P., Doder, M., Lees, A. J., Turjanski, N., Brooks, D. J. (2005). Depression in Parkinson’s disease: loss of dopamine and noradrenaline innervation in the limbic system. Brain, 128, 1314–1322 Rye, D. B., Bliwise, D. L., Dihenia, B., & Gurecki, P. (2000 Mar). Daytime sleepiness in Parkinson’s disease. Journal of Sleep Research, 9(1), 63–69. Sawada, H., Oeda, T., Yamamoto, K., Kitagawa, N., Mizuta, E., Hosokawa, R., et al. (2009). Diagnostic accuracy of cardiac metaiodobenzylguanidine scintigraphy in Parkinson disease. Eur J Neurol, 16(2), 174–182. Scherfler, C., Schocke, M. F., Seppi, K., Esterhammer, R., Bren­ neis, C., Jaschke, W., et al. (2006). Voxel-wise analysis of diffusion weighted imaging reveals disruption of the olfactory tract in Parkinson’s disease. Brain, 129(Pt 2), 538–542.

218 Schifitto, G., Friedman, J. H., Oakes, D., Shulman, L., Comella, C. L., Marek, K., et al. (2008). Fatigue in levodopa-naive subjects with Parkinson disease. Neurology, 71(7), 481–485. Siderowf, A., Newberg, A., Chou, K. L., Lloyd, M., Colcher, A., Hurtig, H. I., et al. (2005). [99mTc]TRODAT-1 SPECT imaging correlates with odor identification in early Parkin­ son disease. Neurology, 64(10), 1716–1720. Walker, Z., Costa, D. C., Walker, R. W., Shaw, K., Gacinovic, S., Stevens, T., et al. (2002 Aug). Differentiation of dementia with Lewy bodies from Alzheimer’s disease using a dopami­ nergic presynaptic ligand. J Neurol Neurosurg Psychiatry, 73 (2), 134–140. Walker, Z., Jaros, E., Walker, R. W., Lee, L., Costa, D. C., Livingston, G., et al. (2007). Dementia with lewy bodies: A comparison of clinical diagnosis, FP-CIT SPECT

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SECTION III

Frontiers in PD treatment

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 12

Gene therapy for dopamine replacement Tomas Björklund
, Erik Ahlm Cederfjäll and Deniz Kirik Brain Repair and Imaging in Neural Systems, Department of Experimental Medical Science, Lund University,

Lund, Sweden

Abstract: Dopamine replacement for Parkinson’s disease (PD) have seen three major iterations of improvements since the introduction of L-3,4-dihydroxyphenylalanine (L-DOPA) pharmacotherapy: dopamine receptor agonists, ex vivo gene transfer for cell transplantation and most recently in vivo gene therapy. In this chapter, we describe the principles behind viral vector-mediated enzyme replacement in PD. We focus on the enzymes involved in the dopamine synthesis and their internal regulation, the early experimental work on gene therapy using different viral vector types and selection of transgenes, and finally discuss the recently completed early phase clinical trials in PD patients Keywords: gene therapy; Parkinson’s disease; continuous DOPA delivery; adeno-associated viral vectors; clinical trials

complex enzymatic machinery for dopamine pro­ duction requires not only a thorough understanding of the enzymes involved but also the necessary co-factors and intrinsic mechanisms regulating the synthesis. Thus, false assumptions can have severe detrimental effects on the functional outcome of the approach. Furthermore, the mechanisms and vehicles for gene delivery have also seen dramatic improvements in the last decade. Today, highpurity vector preparations with low immunogeni­ city can be produced at very high titers not only in skilled academic laboratories for experimental studies but also in commercial facilities under good manufacturing practices standard suitable for use in early clinical trials. In this chapter we first give a brief summary of the enzymatic

Introduction The concept of reinstating neurotransmitter synthesis in the dopamine-depleted human striatum through gene therapy has made great strides in recent years toward a real treatment alternative for Parkinson’s disease (PD). In fact, attempts to introduce a new pool of L-3,4-dihydroxyphenylalanine (L-DOPA)-producing cells in the rodent striatum go back to the late 1980s. There are a number of reasons why this particular concept has taken close to 20 years to refine: Reinstating the
Corresponding author. Tel.: þ46-46-2224557; Fax: þ46-46-2223436; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84012-9

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processes required for dopamine synthesis and release. This is followed by a unified outline of the early development of enzyme replacement in PD and the final part of the chapter describes the ongoing activities to bring a number of approaches to the clinic.

Dopamine synthesis machinery Dopamine synthesized in the brain originates from the essential amino acid (aa) L-tyrosine that is processed in a two-step reaction. Tyrosine is first converted into (L-DOPA) by hydroxylation at the position 3 of the aromatic ring via the tyr­ osine hydroxylase (TH) enzyme. This enzyme is expressed by dopaminergic, noradrenergic, and adrenergic neurons in the central nervous system (Björklund and Dunnett, 2007; Hökfelt et al., 1984). The enzymatic conversion into DOPA is catalyzed through a process involving, besides the TH enzyme, the co-factor 5,6,7,8-tetrahydro­ 2þ L-biopterin (BH4), ferrous iron (Fe ), and mole­ cular oxygen (Fitzpatrick, 1989; Haavik et al., 1991). BH4 is generated from guanosine 50 -tripho­ sphate (GTP) through a three-step enzymatic reaction, where the first enzyme, GTP cyclohydro­ lase 1 (GCH1), is rate-limiting in the biosynthesis. The latter two, 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase, are ubiqui­ tously expressed (Milstien and Kaufman, 1989; Pfleiderer, 1975; Tobin et al., 2007). The second enzyme in the dopamine synthesis is the aromatic L-amino acid decarboxylase (AADC) that is expressed in diverse cell popula­ tions in the forebrain. Studies in rodents have shown that the majority of AADC enzyme in the striatum (~95%) is found in dopaminergic and serotonergic terminals (Arai et al., 1994; Tison et al., 1991). The remaining activity is thought to come from one or multiple cellular compartments, including striatal neurons, astrocytes, and the endothelial wall (Ikemoto et al., 1997; Juorio et al., 1993; Kang et al., 1992; Mura et al., 1995). On a molar basis, the enzymatic efficiency of AADC is around three orders of magnitude

higher than TH (Waymire and Haycock, 2002) and converts DOPA to dopamine in a reaction that appears not to be as dynamically regulated as the tyrosine hydroxylation (Cumming et al., 1995; Hadjiconstantinou et al., 1993). Thus, under phy­ siological conditions, all newly synthesized DOPA can be readily converted to dopamine in the cell. In dopamine neurons, dopamine is rapidly seques­ tered into a specialized vesicular compartment, which prevents its breakdown into 3,4-dihydroxy­ phenylacetic acid (DOPAC) in the cytosol by monoamine oxidase type A (MAO-A). Dopamine is transported into synaptic vesicles through a pro­ ton antiporter, the vesicular monoamine transpor­ ter 2 (VMAT2) (Kanner and Schuldiner, 1987). Vesicular dopamine constitutes the vast majority of the readily releasable pool and is an essential part of the normal dopamine neurotransmission. Cytosolic dopamine can in fact induce oxidative stress through the formation of dopamine qui­ nones, which could become detrimental for the cell if vesicular storage sites were not available (Chen et al., 2008). Therefore, the dopamine levels in the cytosol are tightly controlled by the feedback inhibition of the first step in the synthesis reaction, the TH enzyme. The TH enzyme consists of two functional domains, a 158–165 aa N-terminal regu­ latory domain and a 296 aa C-terminal catalytic region (Abate et al., 1988). In the brain, the TH enzyme combines into functional homo- or hetero­ tetramer structures (Grima et al., 1985). The N-terminal domain exerts a tonic inhibition over the catalytic domain. This inhibition can be modu­ lated through either a truncation or a site-specific mutation of serine residues within the regulatory domain. The four relevant sites are clustered in the first 40 aa’s of the N-terminal domain and are denoted ser8, ser19, ser31, and ser40 in the hTH1 isoform (Campbell et al., 1986; Haycock, 1990). The TH enzyme is subject to two forms of inhi­ bition: classic kinetic-mediated, readily reversible, substrate inhibition (i.e., as a consequence of high local catecholamine concentrations) and longterm inactivation. Long-term inactivation can be caused via a formation of stable, inactivating com­ plexes formed by ferric iron (Fe3þ) and dopamine

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within the catalytic site (Haavik et al., 1990; Okuno and Fujisawa, 1985). Phosphorylation can increase the activity of the TH enzyme through alleviation of both long-term inactivation and sub­ strate inhibition. Phosphorylation at ser40 has the most marked highest effect, by reducing the Michaelis–Menten constant (Km) of TH for BH4 and at the same time decreasing the affinity to dopamine, thus increasing the inhibitory constant (Ki) for dopamine feedback inhibition (Haavik et al., 1990; Ramsey and Fitzpatrick, 2000). The substrate inhibition is achieved through a steric occupation at the catalytic site (Dunkley et al., 2004). Both DOPA and dopamine can inhi­ bit de novo tyrosine hydroxylation but dopamine is a vastly more efficient inhibitor as it has a Ki of around 9 mM while that of DOPA is almost 20 times higher (Mann and Gordon, 1979; Nagatsu et al., 1964). The Ki of catecholamines on the TH is in turn dependent on the site-specific phosphor­ ylation state of the enzyme and the availability of BH4 (Ames et al., 1978; Mann and Gordon, 1979). Truncation of parts of the N-terminal domain appear to mimic the full phosphorylation in regard to increasing TH enzyme activity and increasing the Ki of dopamine (Kuczenski, 1973; Musacchio et al., 1971; Vigny and Henry, 1981).

Viral vector-mediated dopamine replacement Early developments The idea to restore striatal dopamine production by gene transfer of the dopamine synthetic enzymes goes back to the late 1980s, constituting a very productive new line of research where the TH gene was overexpressed in cultured cells (Coker et al., 1988; Horellou et al., 1989). In majority of the studies, the cell type of choice was cultured fibroblasts (Horellou et al., 1990a,b; Uchida et al., 1992; Wolff et al., 1989). When supplied with BH4, these cells produced stable levels of DOPA in vitro and when transplanted into the striatum of unilat­ eral 6-OHDA-lesioned rats, they reduced turning behavior by up to 50% (in almost all cases induced

by injection of apomorphine to the animals). How­ ever, at least in some cases, it remained ambiguous if the decrease seen in the rotational response to apo­ morphine was, at least partly, due to non-specific damage of the striatal parenchyma caused by graft overgrowth (Horellou et al., 1990b). To further improve the efficacy of this approach, expression of the AADC gene was also introduced in the same cells (Horellou et al., 1990a; Kang et al., 1993; Wachtel et al., 1998). However, this appeared to have a detrimental effect on the behavioral recov­ ery and the extracellular dopamine levels, which was documented both in vitro and in vivo. This effect was most likely due to the negative feedback inhibition of cytosolic dopamine on the TH enzyme as it could be mitigated, either by co-culturing individually TH or AADC-transfected fibroblasts, or through a truncation of the N-terminal regulatory domain of the TH enzyme (Moffat et al., 1997). Although the ex vivo gene transfer approach has served as an important step in the develop­ ment of gene-based therapeutics for DOPA deliv­ ery, it did not yield the magnitude and longevity of functional effects to trigger development of a clin­ ical program. Instead, the alternative in vivo gene transfer to host brain cells gained momentum and showed the potential for efficacy and spread of transgene delivery necessary for clinical applica­ tion. As in many other cases, identification of clinically competitive candidates emerged after several attempts and with continuous improve­ ment of the vector preparations. In the first attempts of in vivo gene transfer, defective herpes simplex virus type one (HSV-1) was used to transduce striatal neurons in cell cul­ ture with the human TH gene (Geller et al., 1995). When these vectors were injected in the striatum, 6-OHDA-lesioned rats showed surprising longterm reduction in apomorphine rotation, even though the transduction appeared to be limited to a very small number of cells (During et al., 1994). Another study published shortly thereafter showed that these early vector preparations were indeed toxic and caused degeneration at the injec­ tion site, which might have caused the reduction in the rotation response (Isacson, 1995).

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Another caveat of the early in vivo studies was that in essentially none of them the BH4 co-factor was added; the investigators instead relied on the avail­ ability of endogenous co-factor to support the activity of the ectopically expressed TH enzyme. This was based on the assumption that BH4 levels would be sufficient, even in the parkinsonian brain, and that it could be available to the transduced cells in a para­ crine manner. However, no evidence exists to sup­ port these assumptions. On the contrary, brain tissue levels of BH4 are decreased by 75% after a 6-OHDA lesion in the rat (Levine et al., 1981), and studies in culture suggest that the level of BH4 in striatal neu­ rons is too low to support efficient DOPA synthesis in the absence of added co-factor (Geller et al., 1995). Evidence showing the requirement for addition of BH4 in vivo came from three studies. In the first study, Kang and collaborators showed that fibro­ blasts, transfected with both TH and the GCH1 gene, could sustain high levels of DOPA synthesis without addition of BH4, proving that the GCH1 enzyme indeed is required for ectopic BH4 synthesis (Bencsics et al., 1996; Pfleiderer, 1975). Parallel stu­ dies in 6-OHDA-lesioned rats showed that grafted fibroblasts, co-expressing TH and GCH1 enzymes, could reduce amphetamine-induced rotation whereas those transduced only with the TH gene had no effect (Bencsics et al., 1996). In two other studies, in vivo gene transfer, either recombinant adenoviral (Ad) or adeno-associated viral (AAV) vectors, were utilized to deliver the TH gene to the striatum. Corti and colleagues showed that animals that received Ad-TH vector displayed behavioral recovery linked to DOPA synthesis only after admin­ istration of significant amounts of BH4 peripherally (Corti et al., 1999), while Leff and collaborators used AAV vectors to co-express TH and GCH1 and achieved similar degree of recovery without admin­ istration of exogenous BH4 (Leff et al., 1998). Clinical enzyme replacement The early enzyme replacement studies resulted in three major approaches for dopamine replacement in PD that are all based on gene therapy to render

striatal neurons into dopamine- and DOPAproducing cells (Fig. 1). This is achieved by the delivery of one, two or three of the genes involved in dopamine biosynthesis. To date, two of these approaches have reached clinical trials. The dopa­ mine replacement strategy aims at making striatal neurons fully capable of producing dopamine. The potential strength of this approach is that it minimizes the need for endogenous expression of enzymes necessary for dopamine production. While attempts have been made to utilize only the two genes, TH and AADC, for this approach (During et al., 1994; Sun et al., 2003), the most convincing results have been achieved with a triple enzyme transfer approach delivering also the GCH1 gene (Azzouz et al., 2002; Muramatsu et al., 2002; Shen et al., 2000). A four-gene deliv­ ery has also been proposed with the addition of the VMAT2 transporter to address the important issue of release of ectopic synthesis of dopamine in cells that lack storage and release mechanisms for this neurotransmitter (Sun et al., 2004). For the dopamine delivery principle, AAV, HSV-1, and the equine infectious anemia virus (EIAV, a lentiviral vector of non-human origin) have been used. Whereas the HSV-1 and EIAV vectors have a loading capacity large enough to encompass a single tri-cistronic expression cassette (Azzouz et al., 2002; Sun et al., 2004), the use of AAV has required a mix of three separate vectors, each coding for one of the transgenes (Muramatsu et al., 2002; Shen et al., 2000). Nevertheless, triple AAV transduction in the rat brain confirmed ear­ lier results, i.e., the dopamine production reduced apomorphine-induced rotation in 6-OHDA-lesioned rats (Shen et al., 2000), and studies in 1-methyl-4­ phenyl-l-1,2,5,6-tetrahydropyridine (MPTP)-treated non-human primates showed increased dopamine synthesis accompanied by functional recovery as assessed by a primate parkinsonian rating scale (PPRS) (Muramatsu et al., 2002). Using the EIAV vector, Azzouz and collaborators have reported partial recovery in apomorphine-induced rotation in 6-OHDA-lesioned rats (Azzouz et al., 2002) and more recently also long-lasting behavioral recovery in MPTP-lesioned monkeys (Jarraya

A Normal DA synthesis DA neuron Tyr

TH

DOPA

AADC

DA

DOPA

AADC

DA

AADC

DA

BH4

GCH1

B Continuous DA delivery Striatal neuron Tyr

TH

BH4

GCH1

C Pro-drug approach Striatal neuron L-DOPA

D Continuous DOPA delivery Striatal neuron Tyr

GCH1

5HT/DA neuron TH

DOPA

AADC

DA

BH4

Fig. 1. Enzyme replacement strategies for dopamine replacement in Parkinson’s disease. (a) Schematic overview of the dopamine (DA) synthesis pathway. Dietary tyrosine (Tyr) passes the blood–brain barrier, illustrated on the left-hand side of the neuronal compartment, and is taken up and processed by the dopaminergic neuron. Tyr is converted by tyrosine hydroxylase (TH) into L-3,4-dihydroxyphenylalanine (DOPA), which is in turn converted by the aromatic amino acid decarboxylase (AADC) into dopamine. TH is the rate-limiting enzyme in the dopamine synthesis pathway and its activity is highly dependent on the presence of tetrahydrobiopterin (BH4) which acts as a cofactor. BH4 itself is synthesized from GTP in a three-enzyme reaction where the activity of the GTP cyclohydrolase 1 (GCH1) enzyme is rate limiting. (b–d) The three approaches of dopamine replacement with gene therapy all aim at reconstituting the dopaminergic tone in the striatum. The continuous dopamine delivery approach (b) renders transduced striatal neurons fully capable of producing dopamine by delivery of the TH, AADC, and GCH1 genes. The pro-drug approach (c) relies on peripheral administration of L-DOPA and focuses on increasing the local conversion to dopamine in the striatum by transduction of solely the AADC gene. Finally, the continuous DOPA delivery approach (d) where striatal neurons are transduced with the TH and GCH1 genes which gives them the ability to produce DOPA. This approach relies on residual AADC activity in residual dopaminergic or serotonergic (DA/5HT) terminals to further convert de novo DOPA into dopamine. Gray arrows represent endogenous enzymes and black arrows represent ectopic enzymes induced by transgene expression.

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et al., 2009). (See also Bjorklund et al., 2009a for commentary.) This vector, now called Prosavin as a commercial product, includes the three neces­ sary genes for dopamine production in one EIAV vector where the TH gene lacks the coding region for the regulatory N-terminal part of the enzyme to avoid negative feedback from cytosolic dopa­ mine. Based on these two pre-clinical studies, a phase I/II clinical trial using this vector was initiated in 2007 by the French team and sup­ ported by Oxford BioMedica (2007). The second dopamine replacement strategy that has reached clinical trials is based on the AAV– AADC delivery, where the aim is to make striatal neurons capable of generating dopamine from peripherally administered L-DOPA. This approach builds on the clinical finding that striatal AADC activity is significantly decreased in patients with advanced PD. In post-mortem analyses of brains, the AADC enzyme concentration was found to be reduced, relative to that of brains from control individuals (Lloyd and Hornykiewicz, 1970; Nagatsu et al., 1979). By targeted transduction of cells in the striatum with the AADC gene, the local conversion rate of L-DOPA into dopamine can be enhanced, which potentially decreases the required dose for symptomatic relief. Thus, this treatment may decrease the effect of overstimulat­ ing other parts of the brain where the degeneration is less severe. As this strategy still relies on peripheral administration of L-DOPA, it has a potential safety mechanism. The transgene expression should yield an active neurotransmitter production only after the patient takes his/her L-DOPA medication (defined as “on” state). The­ oretically, it should also be possible to regulate the magnitude of the functional effect by adjustment of the oral L-DOPA dose. However, there is also a risk that the conversion of the delivered L-DOPA into dopamine in the local tissue becomes too rapid. This could make the dosing of the peripheral medica­ tion difficult and require decreased intervals between the oral supplies of L-DOPA to avoid severe fluctuations in the brain dopamine levels. The AAV–AADC delivery approach has been

explored in non-human primates by Bankiewicz and collaborators (Bankiewicz et al., 2006a, b; Forsayeth et al., 2006). In these studies, AAV2 vectors expressing the AADC gene have been injected into the putamen of MPTP-lesioned rhe­ sus macaques. These studies reported stable longterm expression of the AADC gene in the brain up to 6 years after AAV injection as assessed using [18F]fluoro-L-m-tyrosine (FMT) positron emis­ sion tomography (PET). This was coupled with long-term improvement in clinical rating scores, significantly lowered L-DOPA requirements, and a reduction in L-DOPA-induced side effects. This treatment strategy reached the clinic as a phase I clinical trial in the late 2005. This safety trial included five patients with PD that received injections of an AAV vector to deliver the AADC gene delivered where a total of 9E9 vector gen­ omes were injected bilaterally in the striatum (Eberling et al., 2008). The reported efficacy after 6 months was modest and since it was an open-label trial with only five patients and no controls, no real conclusions can be drawn. An interesting finding is that the only significant decrease for the patients compared with baseline was in “off” state. This finding is not trivial to interpret, since the approach itself is suggested, of course, to mainly be active in “on” state. Whether University of California San Francisco (UCSF)/Genzyme continues with further studies on the AADC approach is yet to be determined. In a follow-up paper, a second cohort of patients, who received a high dose (3E11 vector genomes), was included and also evaluated after 6 months (Christine et al., 2009). While there was a dosedependent increase in FMT binding in these patients, no difference in the improvement in the unified Parkinson’s disease rating scale (UPDRS) was observed between the two doses.

The continuous DOPA delivery approach One drawback of the two approaches for enzyme replacement for treatment of PD patients that

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have reached the clinic is that the vectors used so far—EIAV or AAV—transduce almost exclu­ sively the intrinsic striatal neurons, i.e., cells that do not normally produce dopamine. Since these cells lack the vesicular storage and release mechanisms normally present in dopamine neu­ rons, ectopic synthesis of the neurotransmitter will result in high cytosolic concentrations of dopamine and its end products, which may have negative consequences for the transduced cells. It is conceivable that the accumulation of cytosolic dopamine will expose the transduced cells to excessive oxidative stress, possibly leading to toxic damage or neurodegeneration (Chen et al., 2008). In addition, data obtained by Bankiewics and co-workers in MPTP-treated monkeys have raised the concern that non-regulated dopamine production induced by overexpression of AADC may trigger or aggravate dyskinesias (Bankiewicz et al., 2006a). This phenomenon was also reported in the first clinical safety trial where one of the five patients reported “mild subjective increase in pre­ existing dyskinesias” (Eberling et al., 2008). These above issues can be avoided in the con­ tinuous L-DOPA delivery strategy, which aims to deliver TH and GCH1, but do not interfere with the endogenous AADC activity. In this approach, the end product is DOPA and the synthesis of dopamine relies on entopic AADC activity in the host brain to provide symptomatic relief. Clinical findings have shown that the therapeutic dose, i.e., the minimal concentration of L-DOPA to provide symptomatic relief, does not increase with disease progression (Mouradian et al., 1987). The compli­ cation is instead that the dose required for induc­ tion of adverse events, e.g., dyskinesias and fluctuations, is steadily decreasing until it is over­ lapping with the dose giving symptomatic relief, essentially eliminating the therapeutic window. Furthermore, while the pulsatile delivery of highdose L-DOPA at intermittent intervals requires rapid conversion into dopamine as the precursor is available only transiently, the continuous DOPA delivery will have less requirements on the rate of the conversion per unit time. Thus,

even if the striatal AADC activity is too low to efficiently provide sufficient dopamine synthesis from peripheral L-DOPA, it may still be enough in a continuous DOPA supply setting. Although studies in rodents suggest that com­ plete reversal of the motor deficits may be achieved more readily if the endogenous dopa­ mine innervation is partially spared (Kirik et al., 2002), significant symptomatic improvement can be obtained also in animals with complete lesions of the nigrostriatal dopamine system (Björklund et al., 2010). As noted above, AADC is present also outside the dopamine neurons in the striatum, particularly in the spared serotonin innervation, which is able to convert DOPA to dopamine and to store and release the newly synthesized neuro­ transmitter in an impulse-dependent manner (Carta et al., 2007). The separation of DOPA production (in the transduced striatal neurons) from the site of dopamine synthesis and release (in spared dopamine and serotonin terminals) is advantageous in that it avoids the potential com­ plications associated with non-regulated dopa­ mine production within the transduced striatal neurons. There is increasing evidence showing that invo­ luntary movements, dyskinesias, develop at least partly due to the intermittent oral administration of L-DOPA that causes fluctuations in dopamine levels at the synaptic site (de la Fuente-Fernández et al., 2004). Recent studies have shown that one important neuronal compartment that contributes to these fluctuations is the striatal serotonin inner­ vation (Carta et al., 2007; Muñoz et al., 2008, 2009). As described above, these terminals express both AADC and VMAT2. Thus they can efficiently convert exogenous L-DOPA to dopa­ mine and release it as a false neurotransmitter (Arai et al., 1994, 1995; Zhou et al., 2005). How­ ever, due to the lack of dopamine receptor type 2 (D2) autoreceptors and the dopamine transporter (DAT), these terminals cannot regulate dopamine release correctly. We argue that the swings in the release of L-DOPA-derived dopamine from serotonin

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terminals could be avoided if L-DOPA was sup­ plied at a non-fluctuating, constant rate. In fact, in clinical cases of severe peak-dose dyskinesias, with fluctuations, continuous, intravenous, L-DOPA administration has a dramatic effect in decreasing both the severity of dyskinesias and the time spent in “off” state (Mouradian et al., 1987, 1990) (For a review on this topic see, e.g., Olanow et al., 2006). This principle has now been developed into a clinical product for controlling severe fluctuations. The product, with the brand name Duodopa, acts through direct infusion of L-DOPA into the duodenum (Nyholm et al., 2003). While this approach provides improved therapeutic efficacy, systemic administration does not allow for concen­ tration gradients in the brain. Thus, brain regions less affected by dopamine denervation are still at risk of being constantly over-stimulated at the doses required for alleviation of motor symptoms. In addition, the pump infusion device is cumber­ some and requires continuous maintenance to avoid technical failures, local irritation and infec­ tions which make it difficult to apply to a wider patient population. In recent years, a number of studies have been conducted to improve the efficacy of AAVmediated continuous DOPA delivery and to show the therapeutic potential of this approach. In early gene therapy studies, little attention was given toward the relative expression levels of the individual genes and how they contribute to the final synthesis of DOPA and dopamine. In a recent study, we compared a number of AAV5­ GCH1 dilutions delivered in vivo together with a fixed amount of AAV5-TH and found that opti­ mal TH enzyme function was obtained when the TH:GCH1 ratio was between 3:1 and 7:1 (Bjork­ lund et al., 2009b). In a follow-up study, we con­ ducted a series of experiments to investigate the therapeutic potential of AAV5 vectors delivered at a TH:GCH1 ratio of 5:1 (Björklund et al., 2010). This experiment aimed to answer three longstanding questions: first, if the optimized AAV5­ mediated DOPA delivery could restore advanced motor function in the rat model of end-stage

disease; second, if the continuous DOPA supply would protect against the induction of L-DOPA­ induced dyskinesias when the animals are chal­ lenged with daily pulsatile, systemic L-DOPA administration; third, if the therapeutic potential of the AAV-mediated DOPA supply is solely depen­ dent on a spared striatal serotonin innervation (Fig. 2a). In the stepping test, the animals in the treatment group progressively improved from severe baseline impairment over the first 9 weeks and then reached a plateau at a level comparable to the performance of their intact paw. This performance was maintained over the following 6 months. The animals recovered to the same degree in other behavior tests as well. Following a 16-day treatment period with L-DOPA, the animals in the treatment group developed no or mild dyski­ nesias, whereas most animals in the control groups developed moderate-to-severe abnormal involun­ tary movements (AIMs) resembling peak-dose dys­ kinesias (Fig. 2c). Removal of the serotonergic system by a 5,7-dihydroxytryptophan (5,7-DHT) lesion did not have any measurable effect on the performance of the animals in the stepping test, as they remained fully recovered also after the second lesion. In the cylinder test, on the other hand, we found that the functional benefits seen in the TH þ GCH1 group were completely abolished 2 weeks after the second lesion. The 5,7-DHT lesion did not have any effect on the behavior of the Les– Sham animals. AAV-mediated DOPA delivery not only prevents against the induction of LIDs, but is also highly effi­ cient in reversing already established dyskinesias in L­ DOPA-primed rats. Carlsson and collaborators found that the reversal of pre-manifested dyskinesias in these rats was coupled with a normalization of post-synaptic signaling cascades as demonstrated by reversal of abnormal d-FosB, prodynorphin, and preproenke­ phalin mRNA levels (Fig. 2d) (Carlsson et al., 2005). The normalization of post-synaptic markers indicated a physiological action of de novo synthe­ sized dopamine after AAV-mediated continuous DOPA delivery. However, none of these studies were designed to show that the novel pool of

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dopamine generated by gene therapy reached the relevant receptors and could normalize dopami­ nergic neurotransmission. Toward this goal, we applied a novel approach where D2 receptor occu­ pancy and total receptor quantities could be cal­ culated from a single PET scanning session where the ligand, [11C]raclopride, was injected at partial saturation (Leriche et al., 2009). In this study we found that a medial forebrain bundle (MFB) 6-OHDA lesion induces a 50% increase in binding potential (BP) of [11C]raclopride in the striatum

a

ipsilateral to the lesion. This increase is normal­ ized by the AAV5-mediated DOPA delivery. The estimation of total number of receptors available for binding (Bmax) and dissociation constant (KdVr) revealed that the increase in BP was not due to an increased number of receptors but instead decreased competition of binding. The competition was nearly totally restored after the gene therapy without affecting the Bmax. In line with these observations, the change in KdVr was a much better predictor of the therapeutic efficacy

Assessment in the cylinder test after modulation of the nigrostriatal DA system

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Fig. 2. Collected results from behavioral and functional assessment of AAV-mediated continuous DOPA delivery. (a) Metaanalysis of results collected using the cylinder test. Forelimb asymmetry in animals with a unilateral 6-OHDA lesion to the nigrostriatal pathway combined with pharmacological challenge with L-DOPA, AAV-vector injection and/or lesion to the forebrain serotonin system. This data is collected from five studies (Björklund et al., 2010; Carlsson et al., 2005; Carta et al., 2007; Kirik et al., 2002; Leriche et al., 2009). The striatal lesion is achieved through injection of 6-OHDA along three injection tracts in the striatum to produce a partial lesion model (Carlsson et al., 2005; Carta et al., 2007; Kirik et al., 2002) and the complete lesion through an injection into the medial forebrain bundle (MFB) (Björklund et al., 2010; Carta et al., 2007; Kirik et al., 2002; Leriche et al., 2009). Lesion to the serotonin system was achieved through either intracerebroventricular (ICV) (Carta et al., 2007) or intraparenchymal injection into the MFB (Björklund et al., 2010) of the neurotoxin 5,7-DHT. In the striatal lesion model, both peripheral L-DOPA and de novo DOPA synthesis achieved through AAV2 injection are sufficient to induce full recovery in the cylinder test (Carlsson et al., 2005; Carta et al., 2007; Kirik et al., 2002). In the complete lesion model however, these interventions provide only partial symptomatic relief (Björklund et al., 2010; Kirik et al., 2002). An optimized delivery of TH and GCH1 using AAV5 vectors however, was able to sustain full recovery even in completely dopamine depleted animals (Björklund et al., 2010; Leriche et al., 2009). In addition, the requirement of an intact striatal serotonin innervation for functional recovery differs between the two 6-OHDA lesion models. In the partial lesion model, the 5,7-DHT lesion has no impact on the therapeutic effect of peripheral L-DOPA (Carta et al., 2007), whereas in the complete lesion model, the functional effect of both AAV5-mediated and peripheral DOPA supply is significantly reduced (Björklund et al., 2010; Carta et al., 2007). (b) Reversal of dyskinesias (Carlsson et al., 2005). Animals with a striatal 6-OHDA lesion were rendered moderate to severely dyskinetic through daily peripheral L-DOPA injections. Thereafter the animals received intrastriatal injection of either AAV2-TH þ GCH1 or AAV2-GCH1 only and were maintained on twice weekly peripheral L-DOPA injections.
, Significantly different from L-DOPA­ treated sham controls and rAAV-GCH1 control vector-injected animals;

, not different from drug-naive lesion. (c) Prevention of dyskinesias (Björklund et al., 2010). Animals with an MFB 6-OHDA lesion received AAV5 vectors for DOPA synthesis or a control vector. Fifteen weeks post AAV injection, the animals were challenged with daily peripheral L-DOPA injections for 16 consecutive days and abnormal involuntary movements were scored. Graph shows the time course after the daily L-DOPA injection on the last day.
, Different from Les–Sham group; #, different from GCH1-only group. (d) Normalization of striatal gene expression by in vivo gene transfer (Carlsson et al., 2005). Transduction with AAV2-TH þ GCH1 vectors reverses all of the changes in Prodynorphin mRNA expression and number of FosB-positive in the striatum induced by peripheral L-DOPA treatment. The dashed line in d represents Prodynorphin gene expression on the contralateral non-lesioned side.
, Significantly from the drug-naive control group and the AAV-TH þ GCH1 vector group. (e) Recovery of motor function in the stepping test of forelimb use (Björklund et al., 2010). Near complete lesion of the ascending dopamine innervation by injection of 6-OHDA in the MFB unilaterally caused a severe impairment in sidestepping ability in the contralateral limb. After striatal injection of AAV vectors, the affected-limb use in the TH þ GCH1-treated animals gradually improved over the first 9 weeks, to reach the level of use on the intact side at 12 weeks post-injection (illustrated by the dashed line).
, Different from Les– Sham group; #, different from GCH1-only group.

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than the BP parameter, as seen by the correlation to improvement in the cylinder test and the synthe­ sized dopamine levels in the denervated striatum.

Concluding remarks In this chapter we gave a concise summary of the field of viral vector-mediated dopamine replace­ ment in PD. This is not only a field of rapid scientific development but also shows signs of gra­ dual maturation toward the clinics. Over the years, three conceptually different approaches, with unique theoretical merits, have emerged. How­ ever, there are still a number of points that remain to be resolved. What combination of genes will provide the best functional recovery without the burden of adverse effects? Will the therapeutic efficacy of viral vector-mediated dopamine repla­ cement trump the efficacy of deep brain stimula­ tion by sufficient margin to warrant the invasive and (at this point) irreversible nature of in vivo gene therapy? These questions will hopefully be answered in the coming years within the realms of the ongoing and planned clinical trials. The future of gene therapy is bright with emerging strategies reaching initial clinical testing in a number of areas. The promise of a single surgical event leading to a life-long symptomatic relief is certainly great. The scientific community should now work closely with leading clinical experts to ensure suc­ cessful translation of the gene therapy approaches to widely applicable treatments for PD patients.

Abbreviations AADC AAV Ad AIMs BH4 Bmax BP D2 DAT DOPA DOPAC EIAV Fe2þ Fe3þ FMT GCH1 GTP HSV-1 KdVr Ki Km MAO-A MPTP PD PPRS TH VMAT2

aromatic L-amino acid decarboxylase adeno-associated virus adenovirus abnormal involuntary movements 5,6,7,8-tetrahydro-L-biopterin number of receptors available for binding binding potential dopamine receptor type 2 dopamine transporter L-3,4-dihydroxyphenylalanine 3,4-dihydroxyphenylacetic acid equine infectious anemia virus ferrous iron ferric iron [18F]fluoro-L-m-tyrosine GTP cyclohydrolase 1 guanosine 50 -triphosphate herpes simplex virus type one dissociation constant inhibitory coefficient Michaelis–Menten constant monoamine oxidase type A 1-methyl-4-phenyl-l-1,2,5,6­ tetrahydropyridine Parkinson’s disease primate parkinsonian rating scale tyrosine hydroxylase vesicular monoamine transporter 2

Acknowledgments The authors acknowledge financial support from the Swedish Research council (K2008-75SX­ 20860-01-3, K2009-61P-20945-03-1) and the Neu­ gene-focused research program of European Community’s Seventh Framework Program (HEALTH-F5-2008-222925). TB is supported by the Bagadilico Linnaeus program of the Swedish Research Council.

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234 Moffat, M., Harmon, S., Haycock, J., & O’Malley, K. L. (1997). L-dopa and dopamine-producing gene cassettes for gene therapy approaches to Parkinson’s disease. Experimental Neurology, 144(1), 69–73. Mouradian, M. M., Heuser, I., Baronti, F., & Chase, T. N. (1990). Modification of central dopaminergic mechanisms by continuous levodopa therapy for advanced Parkinson’s disease. Annals of Neurology, 27(1), 18–23. Mouradian, M. M., Juncos, J. L., Fabbrini, G., & Chase, T. N. (1987). Motor fluctuations in Parkinson’s disease: Pathoge­ netic and therapeutic studies.Annals of Neurology, 22(4), 475–479. Muñoz, A., Carlsson, T., Tronci, E., Kirik, D., Bjorklund, A., & Carta, M. (2009). Serotonin neuron-dependent and -inde­ pendent reduction of dyskinesia by 5-HT1A and 5-HT1B receptor agonists in the rat Parkinson model.Experimental Neurology, 219(1), 298–307. Muñoz, A., Li, Q., Gardoni, F., Marcello, E., Qin, C., Carlsson, T., et al. (2008). Combined 5-HT1A and 5-HT1B receptor agonists for the treatment of L-DOPA-induced dyskinesia. Brain, 131(Pt 12), 3380–3394. Mura, A., Jackson, D., Manley, M., Young, S., & Groves, P. (1995). Aromatic L-amino acid decarboxylase immunoreac­ tive cells in the rat striatum: A possible site for the conver­ sion of exogenous L-DOPA to dopamine. Brain Research, 704(1), 51–60. Muramatsu, S.-I., Fujimoto, K.-I., Ikeguchi, K., Shizuma, N., Kawasaki, K., Ono, F., et al. (2002). Behavioral recovery in a primate model of Parkinson’s disease by triple transduction of striatal cells with adeno-associated viral vectors expressing dopamine-synthesizing enzymes. Human Gene Therapy, 13 (3), 345–354. Musacchio, J. M., Wurzburger, R. J., & D’Angelo, G. L. (1971). Different molecular forms of bovine adrenal tyrosine hydro­ xylase.Molecular Pharmacology, 7(2), 136–146. Nagatsu, T., Levitt, M., & Udenfriend, S. (1964). Tyrosine hydroxylase. The initial step in norepinephrine biosynthesis. Journal of Biological Chemistry, 239, 2910–2917. Nagatsu, T., Yamamoto, T., & Kato, T. (1979).A new and highly sensitive voltammetric assay for aromatic L-amino acid decarboxylase activity by high-performance liquid chromatography. Analytical Biochemistry, 100(1), 160–165. Nyholm, D., Askmark, H., Gomes-Trolin, C., Knutson, T., Lennernäs, H., Nyström, C., et al. (2003). Optimizing levodopa pharmacokinetics: Intestinal infusion versus oral sustained-release tablets. Clinical Neuropharmacology, 26(3), 156–163. Okuno, S., & Fujisawa, H. (1985). A new mechanism for reg­ ulation of tyrosine 3-monooxygenase by end product and cyclic AMP-dependent protein kinase. Journal of Biological Chemistry, 260(5), 2633–2635. Olanow, C. W., Obeso, J. A., & Stocchi, F. (2006). Continuous dopamine-receptor treatment of Parkinson’s disease:

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 13

Neurotrophic factor therapy for Parkinson’s disease Suresh Babu Rangasamy, Katherine Soderstrom, Roy A.E. Bakay and Jeffrey H. Kordower
Departments of Neurological Sciences and Neurosurgery, Rush University Medical Center, Chicago, IL, USA

Abstract: Parkinson’s disease (PD) is a chronic, progressive neurodegenerative movement disorder for which there is currently no effective therapy. Over the past several decades, there has been a considerable interest in neuroprotective therapies using trophic factors to alleviate the symptoms of PD. Neurotrophic factors (NTFs) are a class of molecules that influence a number of neuronal functions, including cell survival and axonal growth. Experimental studies in animal models suggest that members of neurotrophin family and GDNF family of ligands (GFLs) have the potent ability to protect degenerating dopamine neurons as well as promote regeneration of the nigrostriatal dopamine system. In clinical trials, although no serious adverse events related to the NTF therapy has been reported in patients, they remain inconclusive. In this chapter, we attempt to give a brief overview on several different growth factors that have been explored for use in animal models of PD and those already used in PD patients.

of DA. A significant loss of DA results in the cardinal motor symptoms of tremor, rigidity, bra­ dykinesia, and postural instability (Bernheimer et al., 1973; Hornykiewicz and Kish, 1987; Parkin­ son’s Disease Foundation, 2009). While the pathol­ ogy of PD is not limited to the nigrostriatal circuit, it has, to date, been the focus of most therapeutic interventions. The motor symptoms of PD do not typically appear until 50% of the nigral DA neurons have been lost. Initially symptoms are mild and can be effectively treated with levodopa. However, as the disease progresses many patients show a gradual loss of levodopa efficacy, with oscillations in motor performance and the emergence of

Introduction Parkinson’s disease (PD) is a common, progressive neurodegenerative disorder that affects approximately 1,000,000 Americans. PD is characterized by a gradual loss of dopamine (DA) neurons in the pars compacta region of the substantia nigra. In the healthy brain, these neurons send out axonal fibers which widely innervate neurons in the caudate and putamen and modulate basal ganglia activity via the synaptic release
Corresponding author. Tel.: 312-563-3585; Fax: 312-563-3571;

E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84013-0

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levodopa-induced dyskinesias. In an effort to impede or avoid these drug-related side-effects, the administration of levodopa is often delayed until later in the disease process with early symp­ toms being treated with DA agonists such as pra­ mipexole, ropinirole, and pergolide. When the effectiveness of these drugs has diminished, levodopa treatment is then initiated with the hope that it will be effective in a more advanced patient. Alternative DA replacement strategies have demonstrated some efficacy, but most fail to address the progressive nature of DA neuronal loss and thus provide limited benefit. Surgical procedures, such as pallidotomy and deep brain stimulation, have also provided significant clinical benefits; however, like most means of DA repla­ cement, none of these approaches have addressed the progressive nature of PD. Indeed, to date the available surgical or pharmacological therapies have not shown the ability to impede the progres­ sive loss of DA neurons observed in PD. There­ fore, there is a need for more effective long-lasting neuroprotective agents or restorative strategies to prevent degeneration of nigrostriatal neurons and axons and slow disease progression by preventing further DA loss. In recent years, one strategy that has gained interest has been the application of trophic factors to affected regions in PD. Trophic factors are a class of neuroprotective compounds that can promote development, influence neuronal survi­ val and axon growth, and modulate neuronal function. In 1951, Rita Levi-Montalcini and col­ leagues characterized the first nervous system growth factor, nerve growth factor (NGF) (Cohen and Levi-Montalcini, 1957; Levi-Montalcini and Hamburger, 1951). This discovery has been followed by decades of research resulting in the discovery and characterization of multiple differ­ ent trophic factors. Currently, several putative nervous system growth factors have been identi­ fied and used to treat human diseases (e.g., Tuszynski, 1999). These factors possess a wide range of structures, receptor signaling mechan­ isms, target neurons, and biological effects.

In response to injury, many trophic factors and their receptors have been shown to increase in concentration, suggesting an endogenous regen­ erative response by these molecules (Hughes et al., 1999). Specific factors have been demon­ strated to be potent neuroprotective agents for specific populations of neurons selectively affected in neurodegenerative diseases. Furthermore, these factors serve as neuroprotectant molecules against cytotoxic cell damage. They can act as anti-excito­ toxins and antioxidants and, as such, have the capa­ city to enhance mitochondrial function. They have also been shown to upregulate calcium buffering proteins, antioxidant enzymes, and anti-apoptotic factors (Mattson, 1998). Based upon these proper­ ties, different trophic factors may be useful for treating a variety of neurological diseases. Many studies have demonstrated the therapeu­ tic effects of trophic factor delivery in PD. Multi­ ple trophic factors have been shown to provide neuroprotection, neurorestoration, and functional improvement in a range of various PD animal models, as well as occasionally in clinical studies. In the following section, we address several differ­ ent trophic factors that have been explored for use in PD, both in animal models and patients.

The neurotrophin family The first family of growth factors to be identified was the “classic neurotrophin family. While some employ the term “neurotrophin” as a synonym for neurotrophic factor”, it can be used to specifically denote four types of structurally related factors: nerve growth factor (NGF) (Levi-Montalcini, 1987; Levi-Montalcini and Hamburger, 1953), brainderived neurotrophic factor (BDNF) (Barde et al., 1982), neurotrophin-3 (NT-3) (Maisonpierre et al., 1990; Rosenthal et al., 1990), and neurotro­ phin-4/5 (NT-4/5) (Berkmeier et al., 1991; Ip et al., 1992). Neurotrophins are found in both lower vertebrates and mammals, and the neurotrophin homologs neurotrophin-6 (NT-6) (Gotz et al., 1994) and neurotrophin-7 (NT-7) (Nilsson et al.,

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1988) have been cloned in fish (Gotz et al., 1994; Lai et al., 1998). All members of the neurotrophin family are structurally similar, sharing approxi­ mately 50% sequence homology (Ibanez, 1994; Timm et al., 1994). Members of the neurotrophin family interact with two cell surface receptors, the Trk family of tyrosine kinase receptors and the p75 pan neurotrophin receptor (p75NTR).

Nerve growth factor Nerve Growth Factor (NGF) was the first known neurotrophic factor, discovered serendipitously 55 years ago by Levi-Montalcini and colleagues (Levi-Montalcini and Hamburger, 1951). NGF supports sympathetic and sensory neurons in the peripheral nervous system and also functions in the development and maintenance of cholinergic neurons in the basal forebrain (Ebendal, 1989). In humans, the NGF gene is located on the short arm of chromosome 1. It has two known receptors, tyrosine kinase A (TrkA) and p75NTR (Kaplan and Miller, 1997). Early studies suggest that NGF plays a role in axonal guidance by increasing the expression of cell adhesion molecules on nerve cells (Weidner et al., 1999). Considerable evidence now suggests that NGF also protects neurons fol­ lowing tissue injury and facilitates re-growth and repair. For instance, NGF signaling provides neuroprotection and promotes the survival and differentiation of sensory and sympathetic neurons (Sofroniew et al., 2001). Exogenous application of NGF has been shown to increase the level of antioxidant enzymes, heme oxygenase-1, and prevent apoptosis by inducing anti-apoptotic gene signaling pathways following exposure to the neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Salinas et al., 2003). A series of studies that may support the use of NGF for the treatment of PD have demonstrated that NGF levels are decreased in PD patients and parkinsonian rats (Lorigados Pedre et al., 2002). One study found the level of NGF in serum

samples from parkinsonian rat and monkey reduced when compared with normal animals (Lorigados et al., 1992, 1996). Similarly, clinical studies reported decreased NGF levels in the sera of PD patients when compared to normal individuals (Lorigados et al., 1992). These findings of NGF level alterations in the parkinsonian brain may suggest a relationship between NGF and the neurodegenerative changes observed in PD. Historically, the intrastriatal grafting of adrenal chromaffin cells was investigated as a therapy for PD. In these studies, NGF was shown to promote the differentiation of grafted cells and processes, and increase chromaffin graft survival (Stromberg et al., 1990). In rodent models of PD, NGF treat­ ment resulted in a longer graft survival and increased functional benefit (Date et al, 1997; Silani et al., 1990). While one adrenal transplant study in a human patient found NGF administered intraputaminally prolonged graft survival (Olson et al., 1991), overall benefits have not translated to studies in primates and PD patients. In most human studies, adrenal graft survival has been poor and behavioral recovery minimal (Hurtig et al., 1989; Peterson et al., 1989). Many studies have also looked at the potential beneficial effects of NGF administration in tandem with ventral mesencephalic cell transplantation (Salinas et al., 2003; Shimoke and Chiba, 2001; Stromberg et al., 1990). In a recent study, Chaturvedi and colleagues found that NGF administration at the time of transplantation enhanced graft survival, protected remaining host nigral DA neurons, and increased functional restoration in rats receiving a 6-OHDA lesion (Chaturvedi et al., 2006).

Brain-derived neurotrophic factor The second neurotrophic factor to be identified, brain-derived neurotrophic factor (BDNF), was isolated in 1982 from pooled extracts of porcine brain (Barde et al., 1982). Like NGF, BDNF sup­ ports the survival and differentiation of cultured cholinergic neurons (Alterson et al., 1990; Knusel

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et al., 1991). The BDNF gene is located on chromosome 11 (Jones and Reichardt, 1990; Maisonpierre et al., 1991) and codes for a large prepromolecule with a secretory signal peptide that presents BDNF as an extracellular factor. This factor binds at least two receptors, tyrosine kinase B (TrkB) and low-affinity NGF receptor (LNGFR or p75) (Patapoutian and Reichardt, 2001). BDNF has been shown to play a critical role in neurogenesis (Benraiss et al., 2001; Zigova et al., 1998). Indeed, mice deficient in BDNF show defects in neural development and soon die (Ernfors et al., 1995). BDNF also provides support for developing and adult DA neurons (Hyman et al., 1991; Lindsay et al., 1993; Mufson et al., 1999). Further, BDNF is necessary for the proper number of developing nigral DA neurons (Baquet et al., 2005). Indeed, a knock-down of TrkB, and related NT-3 receptor, TrkC, results in decreased nigral DA neurons, as well as increased accumulation of a-synuclein in remaining DA neurons, and reduced striatal DA (von Bohlen et al., 2005). BDNF has also been shown to alter synaptic transmission both by regu­ lating synaptogenesis and by modulating synaptic plasticity and efficacy (Fumagalli et al., 2006; Murer et al., 2001; Zhang et al., 2006). Analysis of nigral tissue has revealed a consid­ erable reduction in BDNF and BDNF mRNA in the brains of PD patients compared to controls, suggesting the possibility that there may be a link between reduced levels of BDNF and PD (Howells et al., 2000; Mogi et al., 1999). Addition­ ally, Kohno and colleagues have shown that pathogenic a-synuclein mutations in PD may be linked to a loss in BDNF production (Kohno et al., 2004). Polymorphic variations in the BDNF gene have been linked to familial (Parsian et al., 2004) and idiopathic (Momose et al., 2002) PD, and an early age of symptomatic onset in familial PD (Karamohamed et al., 2005). BDNF has also been shown to control expression of the DA receptor 3 (D3), which is abnormally expressed in PD (Guillin et al., 2001). The localization of BDNF receptors on nigral DA neurons, its trophic effects on DA neurons, and its potential link to

genetic mutations associated with PD have all implicated BDNF in the pathophysiology of PD (Fumagalli et al., 2006; Murer et al., 2001). Several studies have demonstrated the effects of BDNF on DA neurons both in culture (Feng et al., 1999; Hyman et al., 1991; Yoshimoto et al., 1995) and in whole tissue following exposure to the neurotoxins 6-OHDA and MPTP (Hung and Lee, 1996; Levivier et al., 1995). A recent inves­ tigation demonstrated a neurotrophic effect on DA neurons and increased behavioral improve­ ment following a 6-OHDA lesion in BDNFtreated young and aged rats (Singh et al., 2006). Additionally, intrastriatal injection of BDNF in rats prior to a unilateral 6-OHDA lesion decreased DA neuron loss and reduced apomor­ phine-induced rotations (Shults et al., 1995). Infu­ sions of BDNF to the nigra before and after nigrostriatal injury have been shown to reverse behavioral and neurochemical deficits, either by attenuating DA neurodegeneration or by poten­ tiating the function of surviving DA neurons (Altar et al., 1992). However, BDNF protein infused into the nigra failed to prevent degenera­ tion following a medial forebrain bundle (MFB) lesion (Lapchak et al., 1993). These neuroprotective effects may be mediated by an antioxidant-promoting capacity, as BDNF has been shown to increase the glutathione reduc­ tase activity of DA SH-SY5Y neuroblastoma cells twofold and prevents the fivefold elevation of oxi­ dized glutathione normally associated with 6-OHDA toxicity (Spina et al., 1992). BDNF also stimulates DA activity and turnover and is hypothesized to play a role in the compensatory actions of surviving DA neurons in early-stage PD (Blochl and Sirrenberg, 1996; Murer et al., 2001). Indeed, Bustos and colleagues have recently stu­ died the coupling reaction between glutamate release, NMDA receptor activation, and nigral BDNF expression triggered in response to partial DA loss in a rat model of presymptomatic PD (Bustos et al., 2009). Some studies have additionally looked at the combination of a BDNF therapy with neural

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transplantation. Yurek and colleagues found BDNF infusion greatly enhanced the reinnervation of grafted DA neurons within the host striatum and increased amphetamine-induced locomotor behavior (Yurek et al., 1996). In a similar study, BDNF applied to grafted fetal DA neurons reduced amphetamine-induced rotational beha­ vior but did not enhance graft survival or neurite outgrowth in grafted rats (Sauer et al., 1993). Additional research in rats has shown that implan­ tation of genetically modified fibroblasts secreting BDNF near the nigra prior to striatal MPPþ infusions reduced DA neuron loss by 86% (Frim et al., 1994). However, some studies have found no behavioral recovery following implantation of BDNF-expressing fibroblasts in rats receiving striatal 6-OHDA lesions (Lucidi-Phillipi et al., 1995). Although BDNF has been found to protect DA neurons in cell culture and animal models of PD, the large molecular size of BDNF may prevents its delivery to DA neurons. It has been suggested that alternative strategies for BDNF delivery may be more effective, such as using endogenous factors to induce BDNF expression. Nigral DA neurons can produce BDNF via either autocrine or paracrine mechanisms. So the application of low molecular weight compounds, such as neuro­ peptides, intracellular signaling agents, neuro­ transmitters, could increase the production of BDNF in DA neurons (Chun et al., 2000). Another strategy employed to deliver BDNF to the parkinsonian brain has been the transplantation of ex vivo transfected cells. In one study astrocytes transduced by a retrovirus to produce BDNF cDNA injected into the striatum after a 6-OHDA lesion resulted in a reversal of rotational behavior but no significant increase of DA neuronal density or a detectable expression of BDNF 42 days postinjection (Yoshimoto et al., 1995). Another inves­ tigation transplanting BDNF-expressing primary fibroblasts to the 6-OHDA-treated nigra found significant neuroprotection of DA cell bodies but limited protection of DA fibers (Frim et al., 1994). In rats that underwent MPPþ toxic insult, infusion of immortalized fibroblasts expressing human BDNF

increased neuronal survival and increased nigral DA levels (Frim et al., 1994; Galpren et al., 1996). Another strategy utilizing BDNF is in vivo gene transfer. In one study, recombinant adeno­ associated viral vector (AAV) expressing BDNF to transduce nigral neurons (Klein et al., 1999) resulted in the block of amphetamine-induced, ipsiversive, turning behavior, but did not show considerable effect on the number of TH-labeled neurons in SN of rats that underwent 6-OHDA lesions. In a similar study, Sun and colleagues used herpes viral vector expressing BDNF to examine the effects of long-term expression of BDNF in a rat model of PD. Despite long-term BDNF expres­ sion in the rat striatum post-injection, no significant neural protection was observed (Sun et al., 2005). While BDNF treatment has been partially effec­ tive in alleviating behavioral deficits and protecting DA neurons in animal models, findings have not been sufficiently encouraging to call for clinical trials. Indeed, the use of BDNF in clinical trials may require the combination of several therapeutic approaches, both in endogenous and exogenous form, to rescue DA neurons and promote beha­ vioral recovery. Finally, despite BDNF’s neuropro­ tective effects, there is no current data suggesting it has any restorative effects on the DA system.

Neurotrophin-3 and neurotrophin-4/5 Neurotrophin-3 (NT-3) is the third neurotrophic factor in NGF family of neurotrophins. NT-3 has been shown to support the survival and differen­ tiation of existing neurons and to encourage the growth and differentiation of new neurons and synapses (Snider, 1994; Tessarollo, 1998). In humans, NT-3 is encoded by the NTF3 gene (Maisonpierre et al., 1991). NT-3 binds the TrkC receptor with the greatest affinity (Lamballe et al., 1991; Tessarollo et al., 1993) and TrkB and LNGFR with lesser affinity. Studies have demon­ strated the importance of NT-3 in the develop­ ment of the central nervous system; for example, mice deficient in NT-3 lack proprioceptive and

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subsets of mechanoreceptive sensory neurons (Klein et al., 1994; Tessarollo et al., 1994). NT-3 has been shown to promote neuronal survival, differentiation, and neurite growth (Coppola et al., 2001; Zhou et al., 2003). It has also been shown to protect DA cells following toxic insult. Recently, Gu and colleagues demon­ strated that injections of neural stem cells expres­ sing NT-3 has significantly reduced apomorphineinduced rotational asymmetry, improved spatial learning ability, and protected nigral DA neurons compared to controls (Gu et al., 2009). These results suggest the efficacy of NT-3 as a potent trophic factor and potential therapy for PD. Based on its similar amino acid sequence, neurotrophin-4/5 (NT-4/5) is also identified as member of the neurotrophin family of neuronal growth factors (Alterson et al., 1993; Hyman et al., 1994). NT-4/5 selectively binds the TrkB receptor (Glass et al., 1991; Squinto et al., 1991). Both in vitro and in vivo studies have demon­ strated the potent neuroprotective effects of NT-4/5. For instance, in vivo studies revealed that NT-4/5 attenuated the loss of developing motor neurons (Henderson et al., 1993) and enhanced the survival of axotomized retinal ganglion cells (Clarke et al., 1994). In vitro studies have indicated that NT-4/5 supports the phenotypic expression and survival of cultured embryonic hippocampal (Ip et al., 1993), cholinergic (Alterson et al., 1993), noradrenergic (Friedman et al., 1993), striatal (Ardelt et al., 1993), and DA neurons (Hyman et al., 1991, 1994). One study has examined and compared the neuroprotective actions of NGF, BDNF, NT-3, and NT-4/5 on the survival and differentiation of embryonic rat striatal neurons. Results found that while NGF had no effect, BDNF treatment resulted in a 40% increase in overall neuronal survival, a 3–5-fold increase in the number of calbindin­ expressing neurons and an 80% increase in GABA neurons. NT-3 and NT-4/5 treatment produced a slightly smaller 2–3-fold increase in the number of calbindin-expressing neurons and an increase in GABA neurons similar to that induced by BDNF (Ventimiglia et al, 1995). In another study, the

growth of striatal cultures in the presence of NT-4/5 resulted in increased cell survival and increased GABA expression, indicating a trophic action on striatal GABA neurons (Widmer and Hefti, 1994). Another study comparing the effects of BDNF and NT-4/5 on neuronal protection found that noradrenergic neuronal survival was considerably promoted by NT-4/5 but not by BDNF (Friedman et al., 1993). NT-4/5 has been shown to promote the survival of twice as many cultured DA neu­ rons compared to BDNF despite an increase in DA transporter activity in BDNF-treated cultures (Hyman et al., 1994). Chronic supranigral infusions of BDNF and NT-3 have been shown to augment behavioral and electrophysiological DA function (Altar et al., 1994a; Morse et al., 1993) and increase the turnover of DA in the caudate–putamen; however, NT-3 was found to be considerably less potent than BDNF (Altar et al., 1994a). In vivo observations suggest that supranigral infusions of NT-4/5 also augment basal ganglia function (Altar et al., 1994b). Interestingly, BDNF, NT-3, and NT-4/5 have all been reported to exacerbate cell death in cortical cell cultures when exposed to either glutamate N-methyl-D-aspartate (NMDA) receptor activa­ tion or oxygen–glucose deprivation despite showing neuroprotective activity following insults of either serum deprivation or calcium channel antagonism (Koh et al., 1995).

The GDNF family of ligands The glial cell-derived neurotrophic factor (GDNF) family of ligands (GFL) is distantly related to the transforming growth factor (TGF) superfamily and includes four structurally related trophic fac­ tors: GDNF, neurturin (NTN), artemin (ARTN), and persephin (PSPN; Baloh et al., 1998; Ibanez, 1998; Krieglstein et al., 1995; Milbrandt et al., 1998). The GFL family has been shown to play a pivotal role in a number of biological processes including cell survival, neurite outgrowth, cell dif­ ferentiation, and cell migration.

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Glial cell line-derived neurotrophic factor GDNF, the first identified member of the GFL, was originally isolated from the rat B49 glial cell line. It supports the survival of several different neuronal populations in the central and peripheral nervous system. GDNF binds to a multi-subunit receptor system comprised of GFRa-1 and Ret subunits, which function as ligand binding and signaling components, respectively. Ret is a trans­ membrane receptor tyrosine kinase, encoded by the proto-oncogene c-ret, that triggers many intracellular signaling pathways including the Ras-MAPK (Worby et al., 1996), phosphoinositol 3-kinase (PI3-K), Jun N-terminal kinase (JNK; Van Weering and Bos, 1998), and PLCg-depen­ dent pathway (Borello et al., 1996). Suvanto and colleagues have demonstrated that GDNF has a very distinct expression pattern in rat embryos (Suvanto et al., 1996). As Ret expression largely overlaps that of GDNF, it was proposed that Ret may function as a receptor for GDNF (Norsat et al., 1997). Several studies have supported this idea by showing that mice lacking GDNF have an essen­ tially similar phenotype to Ret-null mice (Moore et al., 1996; Sanchez et al., 1996). GDNF has also been shown to induce intracellular signaling via a Ret-independent mechanism (Trupp et al., 1999). Reports have suggested that overall GDNF levels do not differ between parkinsonian and control patients (Mogi et al., 2001). However, GDNF mRNA expression was increased in the putamen of PD patients when compared to con­ trols. Beckman and coworkers have found a sig­ nificant upregulation of GDNF mRNA levels in the putamen of PD patients with a marked loss of nigral neurons and no significant changes in the expression of GDNF receptor subunits in the same patients (Beckman et al., 2006). The potential therapeutic value of GDNF on PD was first recognized when it was found to increase DA neurite length, cell size, cell number, and DA uptake in vitro (Lin et al., 1993). In vivo experiments have since confirmed GDNF’s trophic role on DA neurons (Tomac et al., 1995). Unlike

BDNF, there is evidence of functional restoration following toxic insult with GDNF treatment (Kirik et al., 2000a). However, these restorative actions are not as striking as GDNF’s protective ones (Tomac et al., 1995). GDNF has also been shown to have direct effects on DA neurons by modulat­ ing excitability via changes in A-type potassium channels (Yang et al., 2001), a possible mechanism by which GDNF acutely increases DA release (Hebert et al., 1996). Efficacy in animal models of PD: GDNF has been shown to be neuroprotective, to encourage neuronal fiber outgrowth, and to improve motor function when delivered into the cerebral ventri­ cles or directly into the striatum or nigra in both rodent and primate models of PD (Bjorklund et al., 2000 Chiocco et al., 2007; Peterson and Nutt, 2008; Siegel and Chauhan, 2000). In control rats, GDNF augments nigrostriatal DA function (Hudson et al., 1995) and prevents the functional and structural consequences of nigrostriatal degeneration in animal models of PD including axotomy (Beck et al., 1995), 6-OHDA lesion (Bjorklund et al., 2000; Rosenblad et al., 2000), MPTP toxicity (Kordower et al., 2000; Zhang et al., 1997), and the weaver mouse (Broom et al., 1999). In one study, administration of GDNF to the region just above the nigra 1 week post-lesion resulted in a partial but substantial protection of nigral neurons (Sauer et al., 1995). However, the neurons that remained appeared significantly atrophied, indicating that GDNF administered farther from the site of lesion may not preserve neural function. In mice receiving a partial MPTP lesion, a single dose of GDNF admi­ nistered 24 h prior to MPTP was effective in pro­ tecting nigral neurons (Tomac et al., 1995). Administration of GDNF 9 weeks following a 6-OHDA lesion resulted in a reduction in apomor­ phine-induced rotational behavior for up to 20 weeks following GDNF administration (Lapchak et al., 1997). The results of this study revealed a partial recovery of DA activity in the nigra, but not in the striatum. Likewise, Hoffer et al. (1994) reported that a single intranigral dose of GDNF

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administered 4 weeks after a 6-OHDA lesion induced a 3–4-fold increase in nigral DA 5 weeks post GDNF surgery. The site of GDNF’s administration has been shown to have differential effects on its therapeutic outcomes (Kearns and Gash, 1995). In a seminal study, Kirik and colleagues injected GDNF or vehi­ cle into the striatum, nigra, or lateral ventricles of rats prior to a 6-OHDA lesion. They found that intrastriatal GDNF injections resulted in preser­ vation of striatal terminals, nigral cell bodies, and, notably, preservation of motor function as measured by stepping and drug-induced rotation asymmetry tests. However, while rats receiving intranigral injections showed a protective effect on nigral cell bodies, DA axons were not protected and these rats did not exhibit behavioral recovery. Intraventricular GDNF injection did not have any appreciable restorative effect on nigrostriatal integrity, nor did it aid in functional recovery (Kirik et al., 2000a). Similar to rodent studies, the positive effects of GDNF have also been demonstrated in nonhuman primate PD models. It was initially shown that GDNF increases DA in control rhesus monkeys (Gash et al., 1995). These authors also demon­ strated that a single dose of GDNF (150–450 mg) administered into the monkey striatum, nigra, or lateral ventricle 3 months post-lesion significantly improved a variety of motor deficits in parkinso­ nian monkeys; however, the recovery of nigral DA neurons was modest (Gash et al., 1996). Simi­ larly, intracerebroventricular GDNF delivery at monthly intervals to MPTP-treated monkeys resulted in diminished parkinsonism in a doseresponsive manner (Zhang et al., 1997). Another primate study reported that GDNF also attenuated levodopa-induced dyskinesias in MPTP-treated monkeys (Miyoshi et al., 1997). The neuroprotec­ tive action of GDNF in MPTP-treated monkeys was correlated with increased levels of the DA metabolites: 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA; Gerhardt et al., 1999). Costa and colleagues reported that intraventricular administration of GDNF

promoted the recovery of the injured nigrostriatal DA system and improved both locomotor activity and disability in common marmosets that under­ went MPTP treatment (Costa et al., 2001). Efficacy in PD patients: Based on evidence demonstrating the therapeutic effects of GDNF in rodent and nonhuman primate models, GDNF began to be used in clinical trials in 1996. The first of these trials was a randomized, double-blinded study administering recombinant GDNF protein into the lateral ventricles using mechanical pumps (Nutt et al., 2003). Fifty patients, ranging in age from 35 years to 75 years and suffering from mod­ erate to advanced idiopathic PD were selected for investigation. Patients received either placebo or GDNF in doses varying between 25 and 4000 mg into the ventricles once a month over 8 months. Sixteen of these patients then received 4000 mg of GDNF for an additional 20 months in an openlabeled trial. When the study was un-blinded after 8 months, results were disappointing. Not only was there no evidence of improvement, but patients also experienced several adverse side-effects including nausea, vomiting, and anorexia for sev­ eral days following GDNF administration. Further­ more, patients who received higher doses of GDNF experienced weight loss and depressive symptoms. Even patients who received 4000 mg of GDNF in the 20-month open-label continuation of the trial showed no improvements in either their “on” or “off” Unified Parkinson’s Disease Rating Scale (UPDRS) scores. The trial initiated by AMGEN was halted in September 2004 (Slevin et al., 2007). The lack of symptomatic relief seen in these patients may have been attributed to the inadequate penetration of GDNF from the cerebrospinal fluid into the adjacent striatum. In a study describing a PD patient from the above clinical trial receiving intracerebroventricular injections of GDNF, Kor­ dower and colleagues found only slight GDNF expression in the caudate and putamen, the major­ ity of GDNF cells restricted to the ependymal lining of the lateral ventricles and the overlying cortex (Kordower et al., 1999a). These results suggested that intraventricularly administered GDNF did not

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diffuse sufficiently to reach the degenerating nigros­ triatal fibers and neurons. In an attempt to improve GDNF’s efficacy, researchers began to explore ways of delivering GDNF more effectively to the target regions affected in PD. Studies have shown that intraputa­ mental injection of GDNF in rhesus monkeys causes a variable distribution with only 2–9% of the area receiving GDNF (Salvatore et al., 2006). Furthermore, it has been shown that even slowly diffusing GDNF using convection-enhanced deliv­ ery results in a great deal of variability (Gash et al., 2005). In MPTP-treated monkeys convectionenhanced delivery resulted in a volume of GDNF ranging anywhere from 59 to 325 mm3 in the puta­ men. This may have been due to GDNF binding to receptor sites in the extracellular matrix, impeding its even distribution (Hamilton et al., 2001). Despite these varying results in animal models using intraputamenal infusion, AMGEN con­ ducted an initial Phase I open-labeled trial using this method of delivery in five PD patients (Gill et al., 2003). All but one of the patients received bilateral infusions of GDNF into the posterior putamen for 43 months. The doses of GDNF were increased from 14.4 to 28.8 mg/putamen/day to optimize the benefit. This increased dose resulted in a sustained and progressive improvement (Patel et al., 2005), and no considerable adverse side-effects were reported after 1 year of treat­ ment. In fact, significant decreases were reported in both “on” and “off” UPDRS scores. Additionally, there was a 39% decrease in the off-medication motor score, a 61% improvement in the activities of daily living sub-score, a 20% decrease in severe immobility, a decrease in medication-induced dyskinesias, and a 28% increase in fluoro-dopa (18F-dopa) uptake in the posterior putamen. Postmortem analyses found a more than five­ fold increase in tyrosine hydroxylase (TH) immu­ noreactivity in the right versus left putamen. There was also a higher level of TH immunoreac­ tivity and a greater numbers of THþ neurons in the left versus the right substantia nigra; however, these may have been due to the asymmetrical

pathology seen in most PD patients. Interestingly, there was an increase in growth-associated protein 43 (GAP-43) staining in the right putamen, indicat­ ing that GDNF induced sprouting in nigral fibers. These exciting results prompted a double-blinded, placebo-controlled study using 34 subjects, half of whom received placebo and half receiving 15 mg/ putamen/day of GDNF. Unfortunately, in this more rigorously controlled study GDNF did not significantly reduce UPDRS scores even after 6 months of treatment, although there was a 23% increase in 18F-dopa uptake in the posterior puta­ men. The discrepancy between the increase in 18 F-dopa uptake and a lack of clinical benefit might indicate that although there is an increase in DA in the putamen as a result of GDNF treat­ ment, it was not being efficiently released. Another attempt to improve GDNF delivery has been the use of catheters to infuse GDNF over prolonged periods of time using pumps. In one of the experimental study, a catheter was inserted into the putamen of aged rhesus monkeys to continuously infuse GDNF into the putamen over 8 weeks (Ai et al., 2003). This delivery method effectively distributed the trophic factor up to 11 mm away from the site of catheter insertion, diffusing GDNF to the rostral putamen, internal capsule, external capsule, caudate nucleus, and globus pallidus. Additionally, retrograde transport of GDNF was seen in nigral cell bodies. This transport of GDNF into adjacent areas translated into a significant improvement in the overall motor performance of these aged monkeys in the last 3 weeks of the study compared to con­ trols (Maswood et al., 2002). Additionally, there was a 50% increase in DA levels in the ipsilateral caudate nucleus and a 390% increase in DA in the ipsilateral globus pallidus. Similarly encouraging results using continuous infusion of GDNF were seen in a study infusing GDNF to the lateral ventricles and putamen of MPTP-treated monkeys (Grondin et al., 2002). GDNF promoted a signif­ icant anti-parkinsonian effect in monkeys that received the trophic factor to both the ventricle and the striatum. This positive motor effect was

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brought on by a very modest increase in overall THþ fiber density throughout the striatum. How­ ever, THþ fiber density was increased fivefold only in the immediate area surrounding the lat­ eral ventricles, indicating that small areas that are efficiently delivered GDNF can experience robust trophic effects. Ex-vivo cell therapy: One successful GDNF delivery method has been to have GDNF secreted from ex vivo transfected cells that are encased in polymer capsules (e.g., Apfel, 1997). Genetically engineered cells are encapsulated in semi-perme­ able matrices, and then implanted at selected sites. The polymer allows the free exchange of nutrients and waste products, and also allows the release of large molecules, such as neurotrophic factors, from the encapsulated cells into the surrounding tissue. At the same time, the cells are protected from the host’s immune system and therefore are more likely to survive implantation. Encapsulated PC12 cells have been used suc­ cessfully in parkinsonian rats (Tresco et al., 1992) and primates (Aebischer et al., 1994) where trans­ planted cells have survived, produced DA, and reduced abnormal motor behaviors. Tseng and coworkers demonstrated that polymer-encapsulated GDNF-secreting hamster kidney cells implanted unilaterally at a site lateral to the MFB and rostral to the nigra doubled the number of surviving nigral neurons following a medial forebrain axotomy (Tseng et al., 1997). Sautter and colleagues revealed that the implantation of polymer-encapsulated cells genetically engineered to secrete GDNF into the adult rat striatum improves DA graft survival in 6-OHDA-treated rats (Sautter et al., 1998). Researchers have transplanted neural stem cells that stably express transgenes and locally deliver soluble molecules such as GDNF (Behrstock et al., 2006; Yasuhara et al., 2006). These cells engineered to release GDNF-engrafted parkinso­ nian striatum have given rise to neurons, astro­ cytes, and oligodendrocytes, maintained high levels of GDNF expression for at least 4 months, prevented the loss of DA neurons in the nigra, and reduced behavioral impairment in a mouse model

of PD (Akerud et al., 2001). GDNF-producing astrocytes have been shown to protect adult nigrostriatal DA neurons (Ericson et al., 2005). In an ex vivo study (Cunningham and Su, 2002), genetically modified primary astrocytes expres­ sing GDNF were tested for their ability to protect DA neurons in a 6-OHDA mouse model of PD. Mice that received striatal or nigral implants of GDNF-producing astrocytes displayed elevated levels of GDNF compared to mice that received control non-transduced astrocytes. GDNF-produ­ cing astrocytes provided marked protection of nigral DA cells, and partial protection of striatal DA fibers, when implanted into the midbrain 6 days prior to a 6-OHDA lesion. Additionally, GDNF-producing astrocytes have attenuated amphetamine-induced rotational behavior in 6-OHDA-treated mice and completely prevented DA depletion within the nigra. Other cells such as fetal kidney cells (Chiang et al., 2005), bone marrow cells (Park et al., 2001), or adrenal chro­ maffin cells (Espejo et al., 2001) that express GDNF have also been shown to improve parkin­ sonism in animal models of PD. In vivo gene therapy: Another approach to expressing trophic factors continuously is in vivo gene therapy. In vivo gene therapy has great poten­ tial for delivering trophic factors continuously to focal regions of the CNS. This procedure uses an in vitro construction of a viral vector that harbors a cassette of a specific gene of interest. The vector genome is usually completely gutted, leaving no viral genes, rendering the virus incapable of repli­ cation. The modified virus can then be injected directly into the brain. Once delivered, the gene is then transcribed and translated into a functional therapeutic protein. Various viral vectors have been engineered to carry out therapeutic genes into the CNS, including herpes simplex virus (HSV), adenovirus (Ad), adeno-associated virus (AAV), and lentivirus (LV). Viral vectors are attractive candidates for suc­ cessfully delivering GDNF to the PD brain. They provide a safe and robust way to deliver GDNF uniformly over very long periods of time. Since

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PD is a chronic, progressive disease, it requires continuous therapy over the course of months and years in order to sustain DA neuron survival and function (Bjorklund et al., 2000). The first vehicle tested for the administration of GDNF was the adenoviral (Ad) vector. Working in rats, Bohn and coworkers delivered a single injection of an adenovirus containing the gene for GDNF (Ad-GDNF) near the nigra 1 week prior to a striatal 6-OHDA lesion. They found a 79% survival of nigral cells compared to 31% for controls. However, it did not alter TH expression in the striatum, indicating that there may not have been any therapeutic consequences from solely treating the nigra (Choi-Lundberg et al., 1997). A subsequent study examining the effects of Ad-GDNF delivery to the striatum found a 40% protection of cells in the nigra but no effect in the striatum. Interestingly, Ad-GDNF administration to the striatum improved motor performance in treated rats, indicating that either a preservation of striatal TH fibers was not necessary for beha­ vioral improvement or TH levels were increased to a level undetected by the methods used (Choi-Lundberg et al., 1998). Using a more severe 6-OHDA lesion, Bilang-Bleuel and colleagues showed that Ad-GDNF administered at nine sites throughout the striatum was able to protect both nigral cells and striatal fibers (Bilang-Bleuel et al., 1997). They found a 60–62% survival of DA nigral neurons in rats treated with Ad-GDNF compared to only 30% in controls as well as an attenuation of amphetamine-induced rotational behavior. In another study, Connor and colleagues prevented the death of DA nigral neurons and enhanced motor function using Ad-GDNF in aged rats with progressive 6-OHDA lesions (Conner et al., 1999). Similar studies have been conducted in parkinsonian mice. For example, Kojima and colleagues found that Ad-GDNF delivered to the striatum of MPTP-treated mice was able to increase striatal DA levels 1 week post-lesion when compared to controls (Kojima et al., 1997). While previous studies had utilized the firstgeneration E1/E3 defective recombinant virus to

deliver GDNF to the parkinsonian brain, in 2004 studies began to assess the efficacy of the E1, E3/E4 defective recombinant Ad-GDNF (Do et al., 2004), which was shown to be less toxic and elicit a lower inflammatory response (Dedieu et al., 1997). Using this construct, GDNF was targeted to striatal astrocytes in the parkinsonian striatum of rats resulting in DA cell survival and improved druginduced rotational behavior (Conner et al., 1999; Cui et al., 2001; Do et al., 2007; Nicole et al., 2001). Zheng and colleagues recently investigated the efficacy of Ad-GDNF administered to the stria­ tum 4 weeks following 6-OHDA lesion, a state more closely resembling advanced PD. Results showed a significant increase in THþ nigral neu­ rons, increased levels DA levels, and an ameliora­ tion of rotational behavior in the GDNF-treated group when compared with untreated controls (Zheng et al., 2005). This study illustrated that Ad-GDNF could be efficacious even in late stages of 6-OHDA-induced PD. Adenoviral vectors are advantageous because they can accommodate large pieces of DNA, can infect both dividing and non-dividing cells, and can be generated free of contaminant replicationcompetent viruses at high titers. Despite the abun­ dance of positive structural and functional data using Ad-GDNF in rodent models of PD, the use of the adenovirus has been associated with cellular toxicity and inflammatory response, which increases with larger titer volumes injected (e.g., Bjorklund et al., 2000). Thus, there is a need for the development and testing of alternative lessimmunogenic vectors, before this technology can be used in clinical trials. Another viral vector system that is under wide­ spread use is the adeno-associated viral vector (AAV). Due to the inflammatory properties of adenoviruses, AAV vectors have been developed that have close to 95% of the viral genome removed. To date, no studies have reported toxi­ city or an inflammatory reaction in response to injections of AAV. AAV is known to be safe to humans, many of whom already harbor the virus. These viruses have the ability to integrate with the

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host genome and express the gene of interest over long periods of time. Like adenovirus vectors, AAV vectors can integrate into both dividing and non-dividing cells. However, unlike adeno­ virus, the absence of nearly all viral genes mini­ mizes the likelihood of an immune response. Studies using the AAV-GDNF construct in ani­ mal models of PD have yielded promising results. Mandel and coworkers found that perinigral injec­ tions of AAV-GDNF administered 3 weeks prior to an intrastriatal 6-OHDA lesion were able to significantly protect nigral neurons compared to rats treated with control vector. These authors found a 94% protection of nigral neurons in AAV-GDNF-treated rats when compared to a 50% loss in controls (Mandel et al., 1997). They also showed constant expression of GDNF using the AAV vector for up to 10 weeks. Importantly, however, they did not observe any functional recovery using AAV-GDNF. Like with infused GDNF, Kirik and colleagues have found that the site of GDNF delivery is crucial to functional outcome. They have shown that stable GDNF expression can be achieved for up to 6 months with a single injection of AAV­ GDNF in rats. AAV-GDNF administered to both the nigra and striatum is capable of providing complete protection of nigral neurons from 6-OHDA-induced toxicity (Kirik et al., 2000b). Nevertheless, only AAV-GDNF administered to the striatum can provide functional recovery mediated by a regeneration of striatal THþ fibers. This striatal fiber regeneration was gradual and occurred over 4–5 months. Many of the aforementioned studies adminis­ tered GDNF prior to performing a lesion in order to determine GDNF neuroprotective effects. While these studies are necessary for test­ ing the effectiveness of GDNF, a study in which the trophic factor is administered following a lesion has greater clinical relevance. For example, intrastriatal administration of AAV-GDNF 4–5 weeks following a 6-OHDA lesion in rats was found to prevent DA neuron degeneration and promoted significant recovery of behavioral

functions (McGrath et al., 2002; Wang et al., 2002). AAV-GDNF-treated rats showed a higher density of DA striatal fibers and an increased number of nigral neurons compared to untreated controls. Additionally, DA and DA metabolite levels were higher in the GDNF-treated striatum. These neuroprotective effects correlated with a significant behavioral recovery that began 4 weeks following AAV-GDNF treatment. Ultimately, these findings indicate that AAV-GDNF treatment is effective even when administered after the neu­ rodegenerative process is initiated. The promising outcomes in GDNF-based gene therapy for PD in rat models paved the way for the evaluation of AAV-GDNF therapy in nonhuman parkinsonian primates. Bankiewicz and colleagues have demonstrated, using other transgenes, that AAV can transfect striatal neurons and demon­ strate non-toxic long-term gene expression in non­ human primates (Bankiewicz et al., 2000). In a 6-OHDA-induced marmoset monkey model, AAV-GDNF was injected into the striatum and the nigra 4 weeks prior to a unilateral lesion (Eslamboli et al., 2003). GDNF treatment pro­ tected 40% of the THþ cells in the nigra com­ pared to 21% of cells that remained in untreated monkeys. Close observation additionally revealed some remaining DA fibers in the GDNF-treated striatum 5 weeks following 6-OHDA lesion. This finding suggests AAV-GDNF partially prevented the loss of DA innervation of the striatum, which may have been responsible for the amelioration of behavioral deficits in amphetamine- and apomor­ phine-induced rotations, and PD deficits observed in treated monkeys (Eslamboli et al., 2003). Two recent studies have illustrated the use of AAV2-GDNF delivered by a convection-enhanced method into the putamen of two rhesus monkeys models. These authors reported clinical improve­ ment, an absence of adverse effects, and prolonged GDNF expression over a 12-month period in MPTP-treated monkeys. They also found that GDNF vector distribution is not limited to the site of delivery but rather occupies a large tissue volume. Indeed, the authors also reported that the

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treatment has remarkably increased the levels of serotonin and norepinephrine in the putamen and nigra (Eberling et al, 2009; Johnston et al., 2009). Despite these positive findings, some questions remain whether long-term GDNF-induced eleva­ tion of neurotransmitter levels is beneficial or may become a source of therapy-related complications. Despite the successful completion of experimental studies using animal PD models, clinical trials to assess the efficacy of AAV-GDNF treatment have yet to be conducted. Recombinant lentiviral vectors (rLV) containing gene constructs for GDNF have also been devel­ oped to protect nigrostriatal neurons. Lentiviral vectors are retroviruses that contain a large clon­ ing capacity (at least 9 kb), integrate with the host genome, produce viable proteins for long periods of time, and can integrate into non-dividing cells (Bjorklund et al., 2000). After infection, the vector RNA is transcribed into DNA. The DNA then forms a preintegration complex with the accessory protein VP, the enzyme integrase, and a protein matrix. The localization sequence of these pro­ teins enables the preintegration complex to cross the nuclear membrane. This is unlike vectors from other viruses that require a breakdown of the nuclear membrane during cell division before they can access the host DNA. Once inside, the DNA then inserts into the host genome by inte­ grase. This version of lentivirus deletes almost 90% of the genome including virtually all genes needed to form human immunodeficiency virus. Thus, there is almost no risk of recombination (Kordower, 2003). In an early study, Naldini and coworkers (1996) established the transfer, integra­ tion, and long-term expression of the marker gene, bGal, in the adult rat striatum and hippo­ campus using a lentiviral vector. This study showed that the lentiviral vector was able to integrate with the host genome and produce a functional protein without causing an immune response up to 3 months post injection. Many studies have shown that lenti-GDNF injected into the striatum or nigra prior to lesion in rodent models of PD models can protect DA

neurons and provide behavioral recovery. For example, in a study by Deglon and colleagues lenti-GDNF was injected unilaterally just above the nigra 1 week prior to an ipsilateral severing of the MFB. In LV-GDNF-treated rats there was a 56% preservation of THþ nigral neurons com­ pared to 24% in controls (Deglon et al., 2000). In the other study conducted by Bensadoun and col­ leagues, LV-GDNF injected into mice just dorsal to the SN 3 weeks prior to 6-OHDA was able to protect 69.5% of THþ nigral cells compared to 33.1% in controls (Bensadoun et al., 2000). Impor­ tantly, apomorphine-induced rotational behavior was substantially decreased in the LV-GDNF-treated group. LV-GDNF injected into both the nigra and striatum 1 week prior to 6-OHDA lesion pro­ tected nigral neurons as well with a dual adminis­ tration being even more potent than single-site treatments (81% of the THþ cells compared to 17–24% in controls; Rosenblad et al., 2000a). Georgievska and colleagues found that LV-GDNF injected into the striatum in a 6-OHDA model of PD was successfully transported to nigral neurons where it protected 65–77% of these cells. This neuroprotection was dose-dependent and rats receiving a higher dose of GDNF showed a greater magnitude of cellular protection. However, fibers in the striatum were not significantly protected. Despite this lack of striatal fiber preservation, deficits in amphetamine-induced rotational beha­ vior were prevented in LV-GDNF-treated rats (Georgievska et al., 2002a). In a subsequent study, the authors found that a longer period of GDNF expression (9 months) failed to provide DA fiber protection (Georgievska et al., 2002b). To assess the efficacy of lentiviral vectors in the primate brain, Kordower and colleagues (1999b) injected LV-ßGal into the caudate and putamen of one hemisphere and the nigra of the contralateral hemisphere of three young adult monkeys to investigate the level of transduction that could be achieved. Monkeys were sacrificed at either 1 or 3 months post LV-ßGal injections. Results from this study found that the virus was successfully able to transduce between 1 and 1.5 million striatal cells

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and produced robust amounts of marker protein that, over time, filled more of the cellular com­ partment. Double labeling of the striatal neurons indicated that 80–87% of the nigral neurons that were ßGalþ also stained for the neuronal marker NeuN supporting the concept that LV preferen­ tially transduces neurons. Injection into the nigra also produced a significant number of ßGalþ cells. Importantly, no inflammatory response or cellular toxicity was observed. Kordower and colleagues have found that intercerebrally injected LV-GDNF provides almost complete protection and functional recovery in the primate MPTP model of PD (Kordower et al., 2000). In this study, the efficacy of LV-GDNF was tested both in aged rhesus monkeys and in monkeys 1 week prior to MPTP treatment. Aged monkeys receiving LV-GDNF treatment to the striatum showed an enhanced 18F-dopa uptake ipsilaterally. These monkeys had an increase in TH in the striatum, an 85% increase in the num­ ber of THþ neurons within the nigra, and a 35% increase in the volume of these neurons. In MPTPtreated monkeys, LV-GDNF reversed functional deficits and completely prevented nigrostriatal degeneration. Monkeys receiving striatal LV-GDNF showed significant improvements in clinical rating scale scores during the 3-month period following GDNF treatment. Additionally, LV-GDNF treat­ ment reversed motor deficits in an operant handreach task. LV-GDNF-treated monkeys also showed robust increases in 18F-dopa uptake on the impaired side compared to untreated controls. All LV-GDNF-treated monkeys displayed enhanced striatal TH levels and 32% more THþ nigral neu­ rons compared to the intact side. Also, LV-GDNF treatment did not induce a significant immune response, as evidenced by only minor staining for the immune markers CD54, CD3, and CD8. Moreover, the number of DA cells within the striatum, which normally increases following DA depletion, was enhanced in both aged and par­ kinsonian monkeys following LV-GDNF delivery (Palfi et al., 2002). Therefore, the use of LVs to deliver GDNF in PD patients appears most

encouraging, given these positive results in a non­ human primate model as well as demonstration of continued long-term expression of GDNF 8 months after initial administration of the LV (Kordower, 2003). Even though gene delivery of GDNF has been convincingly recognized for its potential therapeu­ tic value to protect against toxin-induced models of PD, it has been found ineffective in protecting neurons in the novel, disease-relevant transgenic model. A relatively new genetic model of PD attempts to mimic one of the major pathological hallmarks of the disease, a-synucleinþ Lewy bodies. In 2002, Aebischer and colleagues created a genetic rat model of PD using a LV vector to administer mutated a-synuclein to the nigra (Lo et al., 2002). This group administered the LV-a­ synuclein vector to the nigra 2 weeks after LV­ GDNF treatment (Lo et al., 2004). They found robust expression of GDNF but no beneficial neuroprotective effects on nigral neurons. The findings of this study may present a major chal­ lenge to the use of GDNF for the treatment of PD, as it has been proposed that nigral degeneration may result from a-synuclein toxicity (Lo et al., 2002, 2004). If true, then the use of GDNF to protect nigral neurons may need to be reconsid­ ered. However, it is also important to consider the shortcomings of the LV-a-synuclein model. The levels of a-synuclein achieved from LV vector administration are supraphysiological. Therefore, although GDNF may not be able to overcome the toxic effects induced by the high level of a-synu­ clein expressed in the LV-a-synuclein rat PD model, it may not need to render such vigorous trophism in the clinic. Overall, the use of LV vectors to administer GDNF in animal models of PD has been quite successful.

Neurturin Neurturin (NTN), the second member of the GFL, is 40% identical to GDNF and was origin­ ally isolated from Chinese hamster ovary cells

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based on its ability to support the survival of sympathetic neurons (Kotzbauer et al., 1996). While NTN clearly supports the survival of variety of peripheral neurons, the complete scope of its actions has not yet been determined. Like GDNF, NTN acts through a receptor complex composed of ligand-binding subunits. NTN binding to GDNF family receptors alpha-1 and alpha-2 (GFRa1 and GFRa2) induces Ret-tyrosine phosphorylation, activation of MAP-K and PI3K signaling pathways, and promotes DA neuron survival (Creedon et al., 1997). While both GDNF and NTN bind GFRa1 and GFRa2, GDNF binds GFRa1 with more effi­ ciency, and NTN binds GFRa2 preferentially (Klein et al., 1998; Sanicola et al., 1997). The dis­ tribution of GFR receptors reveals overlapping but distinct patterns of expression (Horger et al., 1998; Klein et al., 1998; Widenfalk et al., 1997), with GFRa1 mRNA expressed primarily in the nigra and ventral tegmental area with only modest and diffuse GFRa2 expressed in these regions (Horger et al., 1998; Klein et al., 1998). The devel­ opmental expression of GFR receptors also dif­ fers. At E11 GFRa1 transcript expression first appears, begins to decrease at E15, and increases again dramatically at E17. Conversely, GFRa2 transcript expression is scarcely evident at E11 and increases gradually through E17 (Jing et al., 1997). These differences in the receptor distribu­ tions may suggest that the in vivo actions of GDNF and NTN are dissimilar (Hoane et al., 1999). Endogenously, NTN binds GFRa2; however, at the high level of expression following gene deliv­ ery, NTN is promiscuous and binds to GFRa1, which is abundant in the striatum (Burazin and Gundlach, 1999). Therefore, as GDNF is not available for clinical use at the present time, NTN could potentially be used in its place. Efficacy in animal models of PD: In one study, the delivery of NTN to the striatum every third day for 3 weeks following a 6-OHDA lesion resulted in a 72% protection of nigral DA neurons compared to controls, but failed to rescue the reduced TH expression of nigral neurons or the extent of striatal DA loss. No improvements were

seen when NTN was delivered intraventricularly (Rosenblad et al., 1999). Another study found that NTN administered 12 weeks following a 6-OHDA lesion failed to protect nigral neurons; however, an increase in striatal DA fibers was observed (Oiwa et al., 2002). Ex vivo cell methods have also been used to deliver NTN in animal models of PD. In one study, fibroblasts genetically engineered to deliver either GDNF or NTN were grafted supranigrally in a 6-OHDA rat model. Both trophic factors were effective at preventing nigral DA neuron loss. However, only GDNF was able to induce sprouting of nigral neurons. Another study admi­ nistered GDNF via polymer-encapsulated cells implanted near the nigra 1 week before a unilat­ eral MFB axotomy (Tseng et al., 1997, 1998). A week following axotomy, animals were sacrificed and postmortem analysis revealed a significant increase in nigral TH levels compared with con­ trols; however, this protection was not accompa­ nied by behavioral improvements. Liu and colleagues recently transplanted neural stem cells engineered to express NTN into striatum of rats receiving 6-OHDA lesions. They found significant protection of nigral neurons as well as attenuated apomorphine-induced rotational behavior for up to 10 months post-transplantation (Liu et al., 2007). In another recent study, transplantation of bone marrow stromal cells transduced with a recombinant Ad-NTN in the rat 6-OHDA PD model resulted in a reduction in amphetamineinduced rotational behavior 1 month following transplant (Ye et al., 2007). In vivo gene therapy has also been used to study the effects of NTN in animal models of PD. In one rodent study, Fjord-Larsen and coworkers found that LV-NTN-treated rats showed significant pro­ tection of nigral DA neurons following a 6-OHDA lesion (Fjord-Larsen et al., 2005). In a nonhuman primate model of PD, neuroprotective action of Escherichia coli-derived recombinant human-NTN injected into the cerebral ventricles 48 h prior to MPTP injections resulted in reduced PD symp­ tomology, complete protection of nigral DA

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neurons, and normalized DA and DA metabolite levels in NTN-treated monkeys (Li et al., 2003). A recombinant AAV2-based vector encoding for human neurturin known as CERE-120 has been developed by Ceregene Inc. When adminis­ tered to the brain, NTN expression has been shown to be rapid, increasing significantly up to 4 weeks and remaining stable for at least 1 year (Gasmi et al., 2007b). CERE-120 has many advan­ tages over other gene therapy vectors used pre­ viously. For instance, AAV2 is naturally replication-deficient, only weakly immunogenic, has not been associated with any human disease or symptoms, and does not easily integrate into the host chromosome. Further, CERE-120 only requires relatively small quantities to provide extensive coverage of the targeted putamen with NTN protein (Bartus et al., 2007). In rats, AAV2­ NTN delivery to the striatum has been shown to provide protection of nigral neurons following 6­ OHDA lesion in a dose-dependent manner (Gasmi et al., 2007a). In young, naive monkeys, striatally delivered CERE-120 produces sustained NTN expression in the striatum and nigra in a controlled dose-related fashion, leading to appro­ priate biological trophic signaling in the nigra (Herzog et al., 2008, 2009). Similarly, AAV-NTN was also tested in aged monkeys that received unilateral injections of AAV2-NTN into the stria­ tum (Herzog et al., 2007). Robust expression of NTN within the nigrostriatal system was observed for up to 8 months. PET scanning revealed increased 18F-dopa uptake in the NTN-treated striatum. Further, in AAV-NTN-treated monkeys, 8 months post administration, there was a signifi­ cant increase in THþ fibers in the striatum and an increase in the number of nigral THþ cells. In the other primate study, the neuroprotective effect of AAV2-NTN through CERE-120 administration was tested in MPTP-treated hemiparkinsonian monkeys. Here AAV2-NTN was delivered to five sites in the caudate, putamen, and nigra for 4 days following a MPTP lesion. In AAV2-NTN­ treated monkeys, CERE-120 produced long-lasting improvement in motor behavior beginning within

1–3 months and persisting for 10 months, the long­ est time point examined. Additionally, it provided protection against nigral neuron degeneration, enhanced TH and pERK activity in the preserved nigral neurons, and significantly protected against loss of striatal DA fibers (Kordower et al., 2006). Efficacy in PD patients: Based on the successes of CERE-120 in animal models, Ceregene Inc. launched a two-site open-label Phase I trial of CERE-120 in PD subjects. The primary goal of this study was to investigate the safety and toler­ ability of CERE-120, with a secondary goal of gaining preliminary evidence of possible efficacy. This study included 12 advanced PD patients showing significant motor fluctuations and Hoehn and Yahr stages in the “off” state of 3 or 4. All patients received bilateral injections of AAV2­ NTN into the putamen using either a low-dose (n = 6) or a high-dose (n = 6) paradigm (Marks et al., 2008). After 12 months, no serious adverse effects or clinically meaningful adverse events were reported and a significant reduction in PD symptoms was observed. This was reflected by an approximate 40% reduction in UPDRS-motor “off” scores, a substantial increase in time “on” without troubling dyskinesias, a reduction in “total off time” and improvements on a number of timed motor tasks. No differences between the two treatment doses were observed. Based on these findings, a Phase II doubleblinded trial was initiated including 58 patients, two-thirds of whom received CERE-120 and one-third placebo. Unfortunately, this study failed to provide beneficial results. However, technical issues concerning the delivery of CERE-120 to the human striatum likely underlie this failure and future clinical trials that will better test the hypothesis are currently being designed.

Novel neurotrophic factors for PD Two novel neurotrophic factors that were recently discovered are mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved

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dopamine neurotrophic factor (CDNF). The genes that code for MANF and CDNF are the argininerich, mutated in early-stage tumors gene (ARMET) and the arginine-rich, mutated in early-stage tumors­ like1 gene (ARMETL1), respectively. MANF and CDNF proteins form a novel MANF family of con­ served secreted factors with eight cysteine residues of similar spacing with a unique protein configura­ tion (Lindholm and Saarma, 2009; Lindholm et al., 2008; Palgi et al., 2009; Petrova et al., 2003 ). MANF is a 20-kDa human protein known as ARMET, because of its arginine-rich N-terminus (Shridhar et al., 1996). The expression of MANF is relatively higher in the hippocampus, cortex, and midbrain (Lindholm et al., 2008). In vitro studies indicate that MANF promotes the survival and sprouting of DA neurons in the ventral midbrain (Petrova et al., 2003, 2004). Electrophysiological studies have recently demonstrated MANF as a potent enhancer of GABAergic synapses on nigral DA neurons. The findings of this study suggest that the presynaptic enhancement of GABAergic inhibi­ tion may contribute to MANF’s protective action on DA cells (Zhou et al., 2006). MANF can also modify glutamatergic activity and provide neuro­ protection similar to that by glutamate receptor antagonists (Nicoletti et al., 1996; Sonsolla et al., 1998). Very recently, the neuroprotective and neuror­ estorative effect of MANF has been demonstrated in an experimental 6-OHDA rat model of PD (Voutilainen et al., 2009). In this study, the distri­ bution and transportation of intrastriatally injected MANF was compared with GDNF. Intrastriatal MANF injection either 6 h before or 4 weeks after 6-OHDA administration protected striatal DA fibers and significantly reduced amphetamineinduced rotational behavior. Also, striatal MANF expression was greater than that seen in GDNFtreated rats. Importantly, however, the authors found that intrastriatally delivered MANF was transported to the frontal cortex, whereas GDNF was transported to the nigra. Similar benefits have been reported in studies using CDNF treatment in the 6-OHDA rat model of PD (Lindholm et al., 2007).

Studies have also been previously conducted to understand the mechanism of MANF and CDNF action at a cellular level. Analyses of the structure of MANF and CDNF indicate that their neuro­ trophic activity may depend on their N-terminal domains and endoplasmic reticulum stress response in their C-terminal domains. While 6-OHDA toxicity is due in part to inhibition of mitochondrial complex I (Sachs et al., 1975) and the subsequent production of reactive oxygen species, it also encourages endoplasmic reticulum (ER) stress (Chen et al., 2004). MANF is a soluble protein that exists in ER and Golgi apparatus and may therefore exert its trophic actions through pre­ vention of ER stress (Apostolou et al., 2008; Mizobuchi et al., 2007). In one rodent study, MANF expression was upregulated following ischemic injury and inhibited the ER stress-induced cell death in HeLa cells in vitro (Apostolou et al., 2008). Accordingly, these data have thus indirectly suggested the neuroprotective effect of MANF against 6-OHDA-induced neurotoxicity that inhibits the ER-stress-induced reaction and thereby possibly creating a protection to the surviving DA neurons and preventing the progression of PD symptoms.

Conclusion In PD there is a progressive loss of DA neurons. As this loss continues and falls below a critical level, anti-parkinsonian drugs lose their efficacy. Therefore, the first step of an ideal therapy must be to prevent this cell death. Various neurotrophic factors have been studied for therapeutic interven­ tion of DA neuron loss in PD in both animal models and clinical trials. To date, the most pro­ mising of these appear to augment this cell loss associated with PD. Additionally, findings from neurotrophic factor studies suggest that a combi­ nation of therapeutic approaches using different delivery methods or endogenous induction may offer the most effective therapy against the devas­ tating neurodegenerative effects of PD.

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List of Abbreviations Ad AAV ARTN ARMETL1 BDNF CDNF DA DOPAC ER GAP-43 GDNF GFL GFRa1 GFRa2 HSV HVA JNK LNGFR LV MANF MFB MPTP MPPþ NGF NMDA NTF NTN NT-3 NT-4/5 NT-6 NT-7 6-OHDA p75NTR PD PI3-K PSPN

adenovirus adeno-associated virus artemin arginine-rich mutated in earlystage tumors-like1 brain-derived neurotrophic factor conserved dopamine neurotrophic factor dopamine 3,4-dihydroxyphenylacetic acid endoplasmic reticulum growth associated protein-43 glial cell-derived neurotrophic factor GDNF family of ligands GDNF family receptor alpha1 GDNF family receptor alpha2 herpes simplex virus homovanillic acid Jun N-terminal kinase low-affinity NGF receptor lentivirus mesencephalic astrocytederived neurotrophic factor medial forebrain bundle 1-methyl-4-phenyl-1,2,3,6­ tetrahydropyridine 1-methyl-4-phenylpyridinium nerve growth factor N-methyl-D-aspartate neurotrophic factor neurturin neurotrophin-3 neurotrophin-4/5 neurotrophin-6 neurotrophin-7 6-hydroxydopamine p75 pan neurotrophin receptor Parkinson’s disease phosphoinositol 3-kinase persephin

TGF TH Trk-A Trk-B Trk-C UPDRS

transforming growth factor tyrosine hydroxylase tyrosine kinase-A tyrosine kinase-B tyrosine kinase-C Unified Parkinson’s Disease Rating Scale

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

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

CHAPTER 14

Neural grafting in Parkinson’s disease: problems and possibilities Patrik Brundin†,�, Roger A. Barker‡ and Malin Parmar§ †

Neuronal Survival Unit, Wallenberg Neuroscience Center, Lund University, Lund, Sweden

‡ Cambridge Centre for Brain Repair, Robinson Way, Cambridge, UK

§ Neurobiology Unit, Wallenberg Neuroscience Center, Lund University, Lund, Sweden

Abstract: Neural transplantation has emerged as a possible therapy for Parkinson’s disease (PD). Clinical studies performed during the 1990s, where dopaminergic neurons derived from the human embryonic brain were transplanted into striatum of patients with PD, provided proof-of-principle that long-lasting therapeutic benefits can be achieved. Subsequent studies, in particular two that followed a double-blind, sham surgery, placebo-control design, showed variable and mostly negative results. They also revealed that some patients develop involuntary movements, so called graft-induced dyskinesias, as side effects. Thus, while nigral transplants clearly work well in select PD cases, the technique needs refinement before it can successfully be performed in a large series of patients. In this review, we describe the clinical neural transplantation trials in PD and the likely importance of factors such as patient selection, trial design, preparation of the donor tissue, and surgical techniques for successful outcome and avoiding unwanted side effects. We also highlight that it was recently found that neuropathological signs typical for PD can appear inside some of the grafted neurons over a decade after surgery. Finally, we discuss future possibilities offered by stem cells as potential sources of dopamine neurons that can be used for transplantation in PD. Keywords: Parkinson’s disease; Dopamine neuron; Transplantation; Dyskinesias; Lewy bodies; Stem cells

cells can survive for over 20 years and exert ben­ eficial effects in PD patients. Results obtained during the 1990s in open-label trials with grafted dopaminergic neurons derived from the human embryonic brain were very encouraging. The patients displayed impressive improvements of symptoms and restoration of dopaminergic

Introduction Over the past 30 years, neural transplantation has emerged as a possible therapy for Parkinson’s disease (PD). Today we know that grafted neural �

Corresponding author. E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84014-2

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neurotransmission. By contrast, two double-blind, sham surgery, placebo-controlled trials with nigral transplants in PD reported no improvement in grafted groups on primary endpoints. These trials also highlighted that some patients develop invo­ luntary movements, so called graft-induced dyski­ nesias (GIDs), as side effects. Thus, while nigral transplants clearly work well in select PD cases, the technique needs refinement and is difficult to successfully perform in a large series of patients. The aims of this review are to briefly review clinical neural transplantation trials in PD and describe factors that may influence likelihood of a successful outcome, such as patient selection, transplantation technique, and trial design. We underscore the problem of GIDs and how they might be avoided in the future. We describe other practical obstacles linked to fetal tissue transplantation that currently prevent transfer of the technology into an established treatment, and how they might be circumvented in a forthcoming multicenter trial that is sponsored by the Eur­ opean Commission. We highlight recent findings that neuropathological features typical for PD appear inside the grafted neurons over a decade after surgery. Finally, we discuss future possibili­ ties offered by stem cells as potential sources of dopamine neurons that can be used for transplan­ tation in PD.

Open-label transplantation trials in PD Clinical trials using fetal ventral mesencephalic (VM) tissue began in the late 1980s in Mexico (Madrazo et al., 1988) and Sweden (Lindvall et al., 1989) with tissue from 12–14–week- and 6–8-week-old fetuses/embryos, respectively. The tissue was transplanted into the striatum with minimal clinical improvement, at least in the patients from Sweden. However, by refining the techniques, several subsequent open-label clinical studies demonstrated that the patients can display significant improvements following implantation of fetal dopamine neurons. These trials took

place in different centres across Europe and the US and involved mainly patients with idiopathic PD, although three (two of whom which have been reported in the literature) patients with MPTP-induced parkinsonism were also grafted using this approach (Brundin et al., 2000; Freed et al., 1992; Hauser et al., 1999; Lindvall et al., 1990, 1994; Peschanski et al., 1994; Spencer et al., 1992; Wenning et al., 1997; Widner et al., 1992). Whilst many patients benefited from the proce­ dure, not everyone improved. The patients fell into three main categories: those exhibiting marked benefit of a clear therapeutic value and in some cases permitting withdrawal of anti-par­ kinsonian medication; those showing significant improvements, but to a modest degree and still needing continued medication; and finally, those who never displayed any measureable benefit. Nevertheless, even in those where marked effects were seen, the benefits were confined to certain aspects of motor control including improvements in the motor aspects of the Unified Parkinson’s Disease Rating Scale (UPDRS) in the defined off period as well as a range of timed motor tasks. This in turn had a positive impact on Activities of Daily Living (ADL) and health-related quality of life with reduced L-dopa requirements (Freed et al., 1992; Hauser et al., 1999; Lindvall et al., 1990, 1994; Peschanski et al., 1994; Spencer et al., 1992; Wenning et al., 1997; Widner et al., 1992). Importantly though, not all motor features of PD improved, in particular, features such as tremor and postural instability improved least, as one might expect given that these features of disease generally respond poorly to L-dopa therapy. Furthermore, non-motor features of PD were not investigated to any significant extent, mainly because the condition was seen as being primarily a movement disorder in the 1980s–1990s, with few if any non-motor features. This understanding of PD has, of course, been radically revised in recent years as the condition is now known to encompass as many non-motor as motor features (Foltynie et al., 2002; Lewis et al., 2005; Williams-Gray et al., 2009).

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The clinical improvements reported in these early open-label neural grafting trials have been, in many cases, long-lasting and associated with evidence of surviving dopaminergic cells within the grafts. The evidence for this has come from two main sources; firstly through functional ima­ ging in which it has been shown that the grafts are associated with increased fluorodopa (F-dopa) uptake on positron emission tomography (PET) scans, as well as regulated dopamine release and re-activation of motor cortical areas (Piccini et al., 1999, 2000). Secondly, post-mortem studies have been undertaken in patients that have died after grafting from unrelated causes. In these patients, there is evidence for the survival of grafted fetal VM dopaminergic neurons with local reinnerva­ tion of the striatum by these cells (Hauser et al., 1999; Kordower et al., 1996). Such studies have suggested that around 100,000 dopaminergic neu­ rons need to be present within the grafted striatum to achieve significant clinical benefit (Hagell and Brundin, 2001), and that those of nigral origin (as opposed to non-nigral dopaminergic midbrain neurons) are most able to innervate the striatum. These initial open-label clinical studies contin­ ued through the 1990s and were very important in showing that fetal VM allografts (transplants between genetically dissimilar individuals within the same species) could survive in patients with advanced PD, become functionally integrated, and produce sustained clinical benefits. However, it also soon became clear that transplants of this type produced very variable responses, with some patients showing only little improvement or transient benefits. The reason for this was not immediately clear, in that it was uncertain whether it was a technical issue to do with the tissue pre­ paration and implantation procedure or a more fundamental issue to do with patient selection and stage of disease (Bjorklund et al., 2003; Hagell and Brundin, 2001). Whilst this debate was being undertaken to try and explain this variability and how it could be minimized, the results from two double-blind, pla­ cebo-controlled trials of fetal VM transplantation

in PD were published which brought the issue centerstage (Freed et al., 2001; Olanow et al., 2003). This issue still causes heated debate today, especially as newer more effective therapies for the motor features of advanced PD have become available such as deep brain stimulation (DBS) and apomorphine/Duodopa� infusion therapies (Lewis et al., 2003).

Double-blind placebo-controlled transplantation trials in PD As the 1990s progressed, more data emerged from a number of open-label VM transplant studies in PD showing that these transplants were effective at treating advanced PD in some cases, although the emerging use of DBS caused some to question whether this was a better, more practical, solution in the management of advanced motor PD. How­ ever, with a change in administration in the US and with encouragement from the initial openlabel results, two double-blind placebo-controlled trials of fetal VM transplantation in PD were funded by the National Institutes of Health (NIH), USA. These studies sought to show that VM grafting in patients with PD was able to pro­ duce a real effect over and above any placebomediated benefits and to show that this technique was really working through a dopaminergic rein­ nervation mechanism and not some non-specific effect as had been the case in the 1980s with adrenal medullary transplants (Barker and Dunnett, 1993). The first of these controlled trials was published in 2001 by Freed et al. and involved 40 PD patients aged between 34 and 75 years (half under the age of 60 and half over) and with advanced disease (mean disease duration of 14 years) (Freed et al., 2001). Patients were randomly assigned to receive a transplant or imitation, sham surgery. The latter involved patients going to thea­ tre and having a burr hole but with no penetration of the dura, such that the patient and subsequently the assessing neurologist did not know whether

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the individual truly had a transplant or just imita­ tion surgery. Finally, 33 patients (some from the initial control group, after unblinding of the study) ended up receiving grafts (Ma et al., 2010). The VM tissue was obtained from aborted 7–8­ week-old embryos cultured in F12 medium con­ taining 5% human placental serum for 1–4 weeks prior to transplantation and unusually (compared to the other transplant trials) prepared into strands or noodles of tissue (Clarkson et al., 1998). The transplants took place under local anesthetic with the patient awake and tissue from two embryos was transplanted into the putamen on each side of the patient’s brain. Immunosuppression was not given, unlike the open-label studies that had all used standard immunotherapy (Freed et al., 2001). The primary outcome was a change in a subjec­ tive global rating of clinical improvement at 1 year post transplant. This revealed that there was no significant improvement in the transplanted patients compared to the sham surgery group (Freed et al., 2001). In further analysis, significant improvements were seen using more traditional measures such as the UPDRS motor scores in defined “off” times in grafted patients who were less than 60 years of age. Subsequent analysis suggested, however, that the main determinant of this correlation was the preoperative L-dopa responsiveness rather than the age of the patient, as even older patients with good preoperative L-dopa responsiveness showed improvement. In a recent report describing the long-term outcome (up to 4 years) in some of the patients from the same cohort, it now appears that a high residual preoperative level of dopamine in the anterior putamen, as determined by F-dopa PET, was asso­ ciated with better clinical outcome (Ma et al., 2010). Interestingly, the early claims that younger patients respond better to grafting do not hold up. The conclusion is that the older patients simply have a slower rate of recovery and eventually catch up with the younger ones (Ma et al., 2010). The main motor improvements were similar to those seen with dopaminergic medications and included rigidity and in the younger patients,

bradykinesia, while tremor showed no response. Despite the modest and variable clinical benefits, PET scanning showed significant increases in F-dopa uptake in the putamen of the transplant group compared to placebo and post-mortem examinations showed dopaminergic neuronal survival and fiber outgrowth in the grafts. However, the number of surviving grafted dopa­ mine neurons was less than that reported in patients showing greater response in open-label studies (e.g., �11,600–20,200 per graft at 7 months and �2100–22,800 at 3 years) (Freed et al., 2001). This failure to show benefit in the primary out­ come parameter was disappointing. In itself this would have been discouraging, but of greater con­ cern in this trial was the first clear description of the development of significant GIDs in 15% of the transplanted patients more than 1 year post trans­ plant—dyskinesias that occurred in the absence of medication, but presence of the graft. Several of these patients required further surgical interven­ tion with subthalamic DBS to relieve them of these GIDs (Olanow et al., 2001). The reason for these trials failing to achieve their primary outcomes coupled to the develop­ ment of GIDs is still debated and obviously is in need of resolution in order for the field to move forward. However in the first of the two NIHsponsored studies (Freed et al., 2001), a number of critical factors have been identified. In particu­ lar the following issues were problematic: • The primary endpoint was a subjective one. The patients mailed in to the clinic their impressions of how their clinical state had changed compared to 1 year earlier. This could give misleading data by virtue of the fact that patients expectations may be greater than that which a transplant can deliver at 1 year, and patients do not always perceive improvements in their PD as can be seen when medication is stopped as they feel the tablets are not working, only for them to get much worse. • The follow-up time was only 1 year, which is short especially considering that the grafted

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neurons are embryonic at the time of implantation. In some of the open-label studies the maximal benefit from the transplant was not seen until 3 years after grafting. Interestingly, the recent follow-up report of a subgroup from the same patient cohort showed that they continued to improve in the UPDRS motor score in “defined off” between 1 and 2 years after grafting (Ma et al., 2010). Indeed, there was a sustained 25% improvement in UPDRS motor score in the “defined off” at 4 years with a 45% increase in F-dopa signal in the grafted putamen over the same period of time. This suggests that a longer follow-up might have led to significant graft-induced improvement being detected also in the subjective global rating scale. • The amount of tissue used, in terms of number of donor fetuses, was less (two per putamen) than some other studies. • The preparation of the tissue involved prolonged incubation times which could have adversely affected the survival of the dopaminergic neurons, as suggested by the post-mortem findings in this study (Freed et al., 2001). • The absence of immunosuppression may have further compromised the viability of the dopaminergic cells in the transplants. • The neurosurgical approach was transfrontal with the tissue being placed as two long noodles into each putamen, which may have contributed to the development of dopaminergic hotspots in the grafted striatum (Ma et al., 2002). • The placebo arm of the trial was offered a transplant after a year and 13 out of the 20 patients were therefore subsequently grafted and by so doing the comparator control arm of the trial was lost. All of this may help explain why patients did not show significant improvements in their clinical state at the primary outcome parameter, and also why the transplants contained fewer dopaminergic cells at post-mortem compared to other histo­ pathological studies. As mentioned above, the two cases reported in the original paper contained

only 6800–38,400 grafted dopamine neurons per putamen (Freed et al., 2001). In addition, a followup PET study by Ma et al. showed dopaminergic hotspots, especially in patients developing GID (Ma et al., 2002). Although these hotspots were not described in the PET results from longer fol­ low-up times (Ma et al., 2010), the mode of cell preparation and graft implantation procedure may have contributed to the striatal complex initially being innervated unevenly by the transplants. For all these reasons, it could be argued that the first controlled neural grafting trial in PD pro­ duced negative results with side effects for meth­ odological reasons, rather than from a fundamental problem with VM transplants for PD per se. This concern, i.e., that the neural trans­ plantation technique in PD was not sufficiently well developed to merit a placebo-controlled trial, had been raised by European investigators already before the trial was initiated (Widner, 1994). In 2003, however, a second controlled study was published with a negative outcome that suggested that the original trial result could not be dismissed as a methodological aberration. This second NIH-sponsored study of Olanow et al. (2003) involved 34 patients with advanced PD, aged between 30 and 75 years. Patients were again randomized to receive either bilateral trans­ plants or sham surgery, but in this study patients were transplanted with either one or four donors in each putamen with a different surgical techni­ que and using immunosuppression. Solid pieces of VM tissue were obtained from 6–9-week fetuses, stored in a hibernation medium for 2 days prior to transplantation. The same group had used an identical tissue dissection and preparation pro­ tocol successfully in earlier open-label studies (Hauser et al., 1999). The surgery was performed under general anesthesia using a two-stage proce­ dure separated by a week, with the tissue from one or four embryos being transplanted into the puta­ men bilaterally. All patients received immunosup­ pression with cyclosporine monotherapy that was maintained for 6 months postoperatively (Olanow et al., 2003). As in the Freed et al. study, sham

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surgery consisted of partial burr holes only with no breach of the dura. In this trial, the primary outcome measure was a very standard one and involved a change in the motor component of the UPDRS in the practically defined “off” state, between the baseline and the final 24-month visit. Once again, no significant overall treatment effect was observed, although there was a clear trend for benefit as one moved from sham-operated patients to those receiving tissue from one and four embryos. However, this was only a trend and not statistically significant. Notably, the group sizes were small at 11–12 in each therapeutic arm. There were no changes in any of the secondary motor measures. Further­ more, the grafts caused significant motor benefits if the patients were divided by disease stage, with less advanced patients doing significantly better post grafting (Olanow et al., 2003). Importantly, patients in both the one- and four-donor transplant groups showed significant motor improvement compared to placebo at 6 and 9 months post trans­ plant, but not thereafter. The apparent loss of ben­ efit partially coincided with the discontinuation of their immunosuppressive therapy. Thus, it is possi­ ble that this triggered an immune response to the graft, compromising the function of the grafted dopaminergic neurons and contributing to an apparent loss of transplant function at 2 years. Indeed the magnitude and time course of the initial improvement (up to 6–9 months) was similar to that reported for previous open-label studies (Brundin et al., 2000; Freeman et al., 1995; Lindvall et al., 1990), which further supports this interpreta­ tion of the data. Despite a possible immune system-mediated impairment in graft function, PET scanning showed significant bilateral increases in striatal F-dopa uptake in both transplant groups, with the four-donor group showing the greatest increase. This fitted well with post-mortem data from this study showing that the dopaminergic neu­ rons survived in large numbers (around 100,000 per putamen in the four-donor group and 30,000 in the one-donor group) with marked reinnervation of the striatum (Olanow et al., 2003).

The results of the study by Olanow and cowor­ kers would have encouraged many in the field to see this therapy as having potential, but as in the Freed et al. study the development of significant “off-med­ ication” GIDs in 56.5% of the grafted patients 6–12 months after transplantation generated doubts about the safety of the surgery (Olanow et al., 2003). These GIDs typically consisted of stereotypic, rhythmic movements of one or both lower extremi­ ties, with three patients requiring further surgical intervention to reduce their severity. Thus, the results from these two double-blind placebo-controlled trials raised serious concerns about the utility and safety of fetal VM transplants in patients with PD. An anxiety that was magni­ fied by the relative safety and efficacy of DBS surgery in advanced PD and the change in presi­ dential administration in the US in 2001 to one that discouraged research involving human fetal tissue.

How does one reconcile differences in outcomes of open-label and double-blind trials? There are a number of possible interpretations. Firstly, one could take a very dogmatic approach, concluding that open-label studies are subject to patient and assessor bias with placebo effect and thus the double-blind studies reveal the true answer—namely neural grafting does “not” really work in PD. Secondly, one could take the opposite view and say that the double-blind studies were inadequately powered to see any benefit. Thus, they might be subject to Type II errors, namely the studies are so powered that there is a high risk that no difference will be seen when in reality there is one, and as such the only useful data we have is from individual patients and their responses in both the open-label and double-blind studies that suggest that neural grafting in PD works. A third interpretation would be that all trials used suboptimal donor tissue dissections, tissue preparation methods, and surgical techniques.

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Thus, the data so obtained tells us nothing signifi­ cant about the true potential of neural transplan­ tation in PD, and until the techniques are better developed no further trials should be done. A fourth and final way of approaching the data is to adopt a position of greater equipoise and say that all trials to date have their problems, but buried within the trial data are clues that this approach can work in some cases. Defining what is special about those cases would then enable VM transplants to be used in some PD patients again, albeit in a modified form. Today most people actively working with neural transplantation research would subscribe to this latter view. They accept that ultimately if cell transplantation is going to be useful and competi­ tive in PD then it will need to be subject to a properly powered double-blind study, which may not involve sham surgery but best alternative ther­ apy. If we accept this, then are there any clues from the studies to date as to what may be the critical methodological and neurobiological fac­ tors that govern whether PD patients will respond well to neural grafts. Perhaps the easiest way to identify the critical factors, short of a meta-analysis of all the trials which is not currently possible, is to look specifi­ cally at issues of patient selection, graft tissue pre­ paration and placement; the extent of immunosuppression; GIDs; and trial design. We will now deal with each of these items in turn. Patient selection It is now recognized that PD is not a single homo­ genous disorder, even if one excludes cases of Mendelian forms of Parkinsonism. Our own work has clearly defined patients with different cognitive profiles and disease courses (Foltynie et al., 2002; Lewis et al., 2005; Williams-Gray et al., 2009). It is highly likely that the transplant trials to date have been contaminated by this, and some of the grafted patients may not even have suffered from PD, given the diagnostic difficulties

that exist with PD (Hughes et al., 2002). Further­ more, it is not only the “type” of PD that is impor­ tant in predicting response to grafting, but also where in the course of the disease they lie. So, for example, the best results in the double-blind placebo-controlled trials were seen in patients with less severe disease (UPDRS < 49 at baseline), best preoperative response to L-dopa (Bjorklund et al., 2003), and least loss of dopamine in the anterior putamen (Ma et al., 2010). Older patients may have responded equally well, but the rate of improvement was slower (Ma et al., 2010). In addition, other open-label studies have suggested that preoperative sparing in the ventral putamen is an important predictor of a good graft response and patients with dopamine loss that extends out of the dorsal striatum might do less well post grafting (Piccini et al., 2005). This may reflect the fact that these patients either have a different type of PD or are at a different, possibly more advanced, stage of disease. Thus choosing younger patients with less advanced disease and dopamine loss restricted to the dorsal and poster­ ior putamen may produce better results with VM grafts. Graft tissue preparation and placement As has already been alluded to, the amount of tissue grafted, along with its preparation and mode of implantation, may all be important in graft survival and efficacy. This therefore includes issues of the number and age of embryos used; the way in which this fetal tissue is prepared and stored prior to implantation; and the actual tech­ nique used to implant that tissue. So, for example, in the two NIH-funded studies, Freed et al., in comparison to the previous open-label studies, used less tissue, stored for longer times, delivered by a transfrontal approach (Freed et al., 2001), whilst in the Olanow et al. study tissue pieces were implanted after being stored for short peri­ ods of time (Olanow et al., 2003). From studies in experimental animals, it is well known that

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immature dopaminergic neurons in the embryonic VM are very susceptible to damage and death when subjected to trauma or cell stress. As a result, many factors in the tissue preparation pro­ cess are known to adversely affect the survival of grafted dopaminergic neurons (Brundin et al., 2000; Laguna Goya et al., 2008). For example, prolonged storage times will adversely affect dopaminergic cell viability in VM grafts (Freeman and Brundin, 2006). Finally even in the highly controlled environment of neural allografting in experimental models of PD, graft variability in terms of dopaminergic cell survival is commonly seen for reasons that are not altogether clear. Thus, it must be assumed that tissue preparation will significantly impact graft survival in patients. Immunosuppression A fundamental question relates to whether it is necessary or not to immunosuppress a patient receiving an intracerebral neural allograft. As is well known, the brain is considered to be an immunologically privileged site by virtue of its blood–brain barrier (BBB), the absence of profes­ sional antigen-presenting cells and a properly developed lymphatic system (Barker and Widner, 2004; Sayles et al., 2004; Widner and Brundin, 1988). However, in the transplant situation this is significantly compromised as the BBB is breached by the grafting procedure. The trauma of the grafting itself triggers a local inflammatory response, with upregulation of major histocompat­ ibility complex (MHC) antigen expression on the cells within, and around, the fetal implant (Duan et al., 1995). This means that parts of the grafted material might be presented to the immune system locally by astrocytes/microglia acting as antigenpresenting cells and/or antigen from the graft might drain to the deep cervical lymph nodes resulting in a peripherally-induced immune allor­ ejection response. This immune rejection may only be partial and resolve over time, as we know that large VM grafts can survive in patients

who have not been immunosuppressed or in whom the immunosuppression was stopped years previously. In line with this, PET imaging does not reveal any changes in graft-mediated F-dopa uptake when immunosuppression is terminated (Piccini et al., 2005). However, a complete failure to give immunosuppression or a too short course of it might significantly compromise survival or function of grafted dopaminergic neurons, even though the graft is not completely rejected. This criticism could be levelled at some of the clinical transplant studies, most notably the two NIH-funded trials. In the Freed et al. study, no immunosuppression was given (Freed et al., 2001). By contrast, in the Olanow et al. trial immunosup­ pression with cyclosporine was given, albeit only for 6 months, and there was deterioration in clinical response 6–9 months after grafting, with post-mortem tissue evidence for activated micro­ glia and immune reactivity in and around the grafts (Olanow et al., 2003, 2009). Graft-induced dyskinesias (GIDs) Perhaps the single most important factor adversely affecting the development of cell-based therapies for PD has been the discovery that GIDs occur in a significant subset of grafted patients. Although mild in many cases, this side-effect has been disabling and in some instances requiring further neurosurgical intervention. Definitely, GIDs need to be better understood if we are to apply neural grafting to new cohorts of patients with PD. It must be recognized that GIDs (Cubo et al., 2001; Hagell et al., 2002) are different in nature to the typical dyskinesias seen in drug-trea­ ted PD (Cubo et al., 2001; Luquin et al., 1992). The reasons for, and the mechanism behind, the development of GID are still not fully understood, and a number of mechanisms have been suggested (Hagell and Cenci, 2005). Both clinical observa­ tions and recent studies in rodent models of GID have shed some light on the problem. An impor­ tant observation is that in all patients where GIDs

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have been seen, L-dopa-induced dyskinesias (LIDs) were present preoperatively. However, there was no correlation between the severity of the LIDS and the likelihood of the patient devel­ oping GIDs post-operatively (Hagell et al., 2002). Studies in animal models of GID have involved drug administration and not spontaneous GIDs and are therefore not the perfect model of what is seen in the patients. Nevertheless, they have also shown that prior priming with L-dopa-induced abnormal involuntary movements is necessary for the graft-induced abnormal movements to develop later (Lane et al., 2009; Steece-Collier et al., 2009). The relatively inhomogeneous graftmediated dopaminergic reinnervation of the host putamen (Ma et al., 2002) has also been suggested to promote GID by causing excessive release of dopamine in “hotspots”. Studies in grafted rats on amphetamine-induced abnormal involuntary movements have provided some tentative support for the “hotspot” hypothesis in that they have shown that amount of graft-derived innervation and the precise placement of the implant within the striatum influence the abnormal movements (Carlsson et al., 2006; Lane et al., 2006). The observation that GIDs typically develop after a delay in patients without immunosuppression or after immunosuppressive therapy has been dis­ continued (Freed et al., 2001; Olanow et al., 2003; Piccini et al., 2005), led to the suggestion that they may be due to a local neuroinflammatory response secondary to a low-grade immune rejec­ tion. In this case, however, animal studies have not provided unequivocal support for the hypothesis with different studies showing minor or no influ­ ence of inflammation on GID-like behaviors in rats (Lane et al., 2008; Soderstrom et al., 2008). Recent studies have shown that LIDS might involve serotonergic terminals that innervate the striatum (Carta et al., 2010). Specifically, these striatal serotonergic terminals can take up L-dopa and convert it to dopamine. Once the dopamine is released by the serotonergic terminal, it cannot be inactivated as these neurons lack presynaptic dopamine transporters (Carlsson et al., 2007;

Carta et al., 2007, 2010) which results in abnormal activation of dopamine receptors on striatal neurons. It is known that VM grafts can contain substantial numbers of 5-HT neurons, but con­ cerning GIDs, this mechanism of genesis seems less likely as some of the patients exhibit GIDs despite not taking L-dopa (Lane et al., 2006). Furthermore, some transplant patients showed an improvement in LID together with worsening GID (Hagell et al., 2002; Hauser et al., 1999). In animal models, GID-like movements can occur in the absence of serotonergic neurons in the implants and when the endogenous serotonin sys­ tem is lesioned, which further argues against an important role for serotonergic neurons in clinical GIDs (Lane et al., 2009). Trial design The design of the trials to investigate the efficacy of fetal VM grafts does not simply concern whether open-label or double-blind imitation sur­ gery trials are the best way to assess graft efficacy. An equally important issue is whether the latter trials were powered to show any significant trans­ plant-mediated effects. In similar studies in PD using GDNF infusions to treat the underlying dis­ ease, sample sizes of 17 were felt by some to be inadequate to show significant effects. Thus some commentators have argued that trials with such small numbers of cases are never going to be free of Type II errors and as such the trials are flawed in their design (Barker, 2006; Hutchinson et al., 2007; Matcham et al., 2007). Finally, it is important to remember that cell therapy approach, as discussed above, is only ever designed to replace the dopaminergic neurons that are lost within the nigrostriatal pathway, and PD is, of course, a disorder in which the pathology extends well beyond this system (Braak et al., 2004; Lang and Obeso, 2004). Whilst this is undoubtedly true, it is well known that dopaminergic drug therapies can radically improve the symptoms and signs of PD at all stages. Interestingly, the optimal control of the

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dopaminergic responsive elements of PD can indeed even improve some of the non-dopaminer­ gic features such as sleep disturbances, daytime somnolence, and fatigue (Honig et al., 2009). Thus using cells to replace the lost dopaminergic system is never going to cure patients of PD, but like dopaminergic drugs the cell therapy approach has the potential to dramatically improve their clinical state and quality of life (Honig et al., 2009).

Neuropathological changes in grafts raise new concerns As discussed above, the safety and efficacy of VM grafting for PD have become major points of dis­ cussion, but another important observation has further complicated the future of neural grafting in PD. This is the demonstration in post-mortem studies that neurons in fetal VM transplants con­ tain alpha-synuclein pathology (Kordower et al., 2008a, b; Li et al., 2008b, 2010). This suggests that the grafts might ultimately undergo the fate of the patients’ own dopaminergic neurons and succumb to the disease process of PD. The relevance of these recent post-mortem studies is not confined to the debate of VM grafting for patients with PD, but it throws up a whole series of questions related to what causes PD and how does the pathological spread of the disease occur (Brundin et al., 2008, 2010). However, that discussion lies beyond the scope of this review, and we will just briefly review the findings of post-mortem pathology in relation to the future of neural transplantation in PD. In two initial back-to-back papers, Li et al. (2008b) and Kordower et al. (2008a) reported on Lewy bodies in nigral transplants in patients who had been grafted over a decade before they died. Li and coworkers described two patients who under­ went the first of two sequential transplantation sur­ geries at age 48 (“patient 3” in the series) and 43 (patient 8), with 11- and 5-year disease history, respectively, of PD. Their second transplantation surgery took place 2–4 years later. They received dissociated fetal VM tissue from 6–8-week-old

aborted embryos into either the putamen only or both caudate and putamen. Patients 3 and 8 received immunosuppression with prednisolone and cyclosporine for 24 and 18 months and azathioprine for 20 and 6 months, respectively. Patient 3 was the first in the Lund series reported to show significant improvement (Lindvall et al., 1990) whereas patient 8 showed minimal clinical benefit from the graft surgery (Hagell et al., 1999). At post-mortem, 13–16 years after their respective first transplantation surgeries, both patients exhib­ ited numerous surviving and integrated grafted neu­ rons. Patient 3 had an estimated 12,100–29,500 grafted tyrosine hydroxylase (TH, the dopaminesynthesizing enzyme)-positive neurons in each of the multiple injection tracks (Li et al., 2008b), indi­ cating that he probably had over 100,000 surviving grafted TH neurons in total in each putamen. In both patients, the grafted neurons contained alpha-synuclein- and ubiquitin-positive Lewy bodies (Fig. 1) and Lewy neurites, morphologically

Fig. 1. Alpha-synuclein immunoreactive (arrow) Lewy body located in a neuron containing neuromelanin pigment (arrowheads) inside a VM graft in patient 3 from the Lund transplant series, as described in previously published papers (Li et al., 2008b, 2010). Photograph kindly provided by Dr J.-Y Li.

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indistinguishable from those seen in surviving neu­ rons in the host substantia nigra pars compacta. In young healthy individuals, cytoplasmic alpha-synu­ clein, even in non-aggregated form, is not detect­ able in the cell body. With normal aging it is upregulated and can be visualized by immunohisto­ chemisty in an increasing proportion of nigral TH neurons (Chu and Kordower, 2007). In patient 3, the proportion of TH neurons with detectable amounts of cytoplasmic alpha-synuclein in the cell body was 40% in the graft done 12 years ago, and 80% in the graft done 16 years ago, suggesting gradual changes in alpha-synuclein levels. Finally in this paper, it was shown that the Lewy bodies in the grafts also stained with an antibody to alpha­ synuclein phosphorylated at Ser129, indicating dis­ ease-related, post-translationally modified, and aggregated alpha-synuclein (Anderson et al., 2006). Whilst microglia accumulated around the graft in patient 3, they did not show significant activation (Li et al., 2008b). Kordower et al. reported on a 61-year-old patient with a 22-year history of PD who had received bilateral solid fetal VM transplants to the putamen from four, 6.5–9-week old, embryos 14 years earlier (Kordower et al., 2008a). The patient improved clinically for a period of �11 years after transplantation and then gradually deteriorated. Post-mortem analysis at death showed good graft survival with extensive TH innervation of the host striatum (Kordower et al., 1996). Kordower and coworkers also found cyto­ plasmic, aggregated and neuritic alpha-synuclein in grafted neurons. Moreover, the aggregates were ubiquitinated; some with the appearance of Lewy bodies were seen in grafted neurons. The transplants were also filled with activated micro­ glia to a level far greater than that seen in the host striatum. The investigators did not find any Lewy bodies in grafted neurons in two brains from patients who had died only 4 years after surgery (Kordower et al., 2008a). Simultaneously with the two reports describing Lewy bodies in grafted neurons, a third paper reported no neuropathological changes in

transplants in three patients operated 9–14 years before dying (Mendez et al., 2008). Later, how­ ever, the same team of investigators has reported that they now have found some Lewy bodies in at least one of the 9-year-old grafts (Isacson, perso­ nal communication and Kordower et al., 2008b). The initial reports on Lewy bodies in grafted neurons have been substantiated by more detailed descriptions and additional cases. Kordower and collaborators found Lewy bodies in one additional case 14 years after surgery (Kordower et al., 2008b). The Lewy bodies in grafted neurons have been found to be positive for thioflavin S, indicat­ ing that they contain the expected beta-pleated sheet structures (Kordower et al., 2008b; Li et al., 2010). Electron microscopic studies suggest that the Lewy bodies contain alpha-synuclein fibrils, further substantiating that they are indistinguishable from those seen in PD (Li et al., 2010). Interestingly, a detailed quantification of the proportion of pig­ mented (i.e., dopaminergic) neurons in the grafts that contained Lewy bodies in one of the patients, who received implants in two surgical sessions spaced by 4 years, revealed that they appear gra­ dually. Thus, in the 12-year-old graft 1.9% of the dopaminergic neurons had Lewy bodies, whereas the corresponding number was as high as 5.0% in the 16-year-old cells (Li et al., 2010). Thus the proportion of neurons with Lewy bodies in 12–16-year-old transplants is similar to the 3.6% reported for the substantia nigra pars compacta in PD patients (Greffard et al., 2010). The unexpected findings of Lewy bodies in implanted neurons in patients with PD suggest that the pathological process can affect young dopaminergic cells. Why this occurs is not known—it may relate to disease spread into the transplant via a prion-like mechanism (Brundin et al., 2008, 2010) or alternatively upregulation of alpha-synuclein in cells surviving in the inflamed environment of the grafted brain could eventually lead to aggregation of the protein (Brundin et al., 2008). Furthermore it remains unclear, though, how significant these findings are to the future use of cell therapies in PD. It is still only in a

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small proportion of cells and with an uncertain significance to the viability and efficacy of the graft. However, in addition to findings of increased cytoplasmic levels of non-aggregated alpha-synuclein that might indicate early aging, other observations suggest that even the trans­ planted dopamine neurons that do not display Lewy bodies eventually undergo some form of degenerative changes. First, the expression of the dopamine transporter (DAT) is downregulated in the grafted neurons several years after surgery (Chu and Kordower, 2010; Kordower et al., 2008b), in contrast to what is seen in younger grafts. Previous in vivo brain scan studies suggest that reductions in DAT in the striatum may be a pathological sign that precedes the development of PD (Sommer et al., 2004). Second, Kordower and coworkers reported that some of the neuro­ melanin-bearing neurons in the graft no longer express TH in the long-term surviving implants, which might also be a sign of neuronal dysfunction (Chu and Kordower, 2010). The same grafted neurons expressed apparently normal levels of vesicular monoamine transporter 2 (VMAT2) suggesting that there was not a general downregulation of protein expression, but that DAT and TH were particularly affected (Chu and Kordower, 2010). Taken together, the increase in cytoplasmic alpha-synuclein levels, as well as the decreased levels of DAT and TH, suggest that a significant proportion of the grafted dopamine neurons undergo some kind of degenerative change when they have been present in the PD brain environment for over a decade.

Cell transplantation in PD—where do we go from here? Evidently, cell-based therapies for PD using fetal VM have had a chequered history—showing in some cases significant long-lasting benefits, whilst in other patients troublesome side effects have arisen. As described above, there is now accumu­ lating data to suggest that some of the critical

factors in the success (or failure) of these fetal VM transplants for patients with PD have been identified and that the field is ready to move for­ ward again (although not all would agree—see Olanow et al., 2009). This will initially be with a further round of fetal VM transplantation trials before the next generation of stem cell-based treatments for PD are considered. Before per­ forming the next series of fetal VM transplant studies, there is a need to address several practical issues related to tissue procurement, dissection, and storage, which we describe in the following sections and this programme of work, including the new clinical trials, has now been funded by the European Commission (TRANSEURO). Ethics and tissue procurement of fetal neural tissue for grafting The use of fetal brain tissue for clinical transplan­ tation purposes is coupled to two major issues. First, whereas some countries have adopted the view that donor tissues obtained from fetuses should be treated the same way as tissues or organs from dead adults (i.e., governed by trans­ plantation law and including informed consent from the woman undergoing abortion), others prohibit the use of fetal tissue for transplantation on ethical or religious grounds (Boer, 1994). Sec­ ond, even when the use of fetal tissue is accepted by society, practical issues make the coordination of clinical trials difficult. The majority of clinical trials have used VM tissue from 2–6 embryos per surgical session. In most major hospitals, induced abortions are common practice, but only dopami­ nergic neurons from a minority of embryos/fetuses are suitable for transplantation. The age of the donor embryo is crucial. Thus, VM tissue from embryos smaller than 14 mm crown–rump length has proven difficult to dissect with contamination of non-neural tissue. On the other hand, if the tissue is taken from an embryo/fetus larger than around 28 mm crown–rump length, the survival of dopaminergic neurons is poor when the cell

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suspension transplantation technique is used (Freeman and Brundin, 2006). In accordance with ethical guidelines, when and where the abor­ tion is performed must not be affected by the desire to use fetal tissue for transplantation (Boer, 1994), which contributes to the fact that several potential donors fall outside the optimal developmental time window. Moreover, in a sig­ nificant proportion of cases the embryo/fetus is destroyed during the abortion, making it impossi­ ble to identify and dissect the VM. Taken together, the above factors make it difficult to predict if sufficient amounts of donor tissue will be available for transplantation to take place. Most clinical grafting programs that were active up until the publication of the NIH-sponsored trials struggled with this problem, and on several occasions planned surgeries have had to be post­ poned at the very last minute. During recent years the practicalities of using fetal tissue derived from induced abortions has been complicated further by yet one more factor. The clinical trials performed so far have utilized VM tissue obtained from routine induced surgical abortions performed using vacuum aspiration, quite often under ultrasound guidance (Gustavii, 1989). During the past decade, the use of medical abor­ tions has increased dramatically, with a concomitant decline in the use of surgical abortions. Mifepris­ tone was introduced in Europe in 1988 as an antiglucocorticoid and anti-progesterone drug that can induce terminations of early pregnancies (Schaff, 2010). Today it is often used to induce abortions during the first 9 weeks of gestation, i.e., the time window suitable for harvesting immature dopamine neurons for transplantation. Depending on which country in Europe one looks at, in extreme cases mifepristone accounts for over 50% of all induced abortions (Schaff, 2010), and the proportion is higher during the first 9 weeks. As a result the number of surgical abortions of embryos/fetuses that are suitable as donors in clinical transplantation trials has decreased dramatically. Whereas the sup­ ply of potentially useful neural donor tissue from surgical abortions was previously not a major

hurdle to clinical grafting, today it is very difficult or impossible to run a clinical program that relies only on surgical abortions. As a result it will be necessary to examine the use of VM donor tissue from medical abortions in the future. The potential drawback with this is that the viability of the brain donor tissue might be compromised during the medical abortion. The time taken from the start of the medical induction of the abortion to when the fetus is expelled is several hours (Schaff, 2010), and this might mean that the immature neurons have been anoxic for excessively long periods before they can be harvested and prepared for transplantation. Whether tissue from medical abortions can be used as donor material, when grafting dopamine neu­ rons, is currently being explored. Research teams are transplanting the tissue to immunosuppressed rodents and comparing the average yield of dopa­ mine neurons, and variability between donors, with that seen using tissue from surgical abortions. The outcome of these studies will be vital in determining whether transplantation using donor tissue from abortions has a future or not. Tissue dissection The dissection of fetal midbrain tissue is typically based on morphological landmarks where the ros­ tral limit is just caudal to the mesencephalic–dien­ cephalic border, the caudal limit is at the tuberculum interpedunculare of Hochstetter. The lateral border is set in the basal plate between the floor plate and sulcus limitans, such that the dis­ sected piece includes the ventral third of the neural tube (Freeman and Brundin, 2006). In recent years, many key regulators of dopamine neurogenesis, such as Lmx1a, Ngn2, FoxA2, and Pitx3 have been identified (Andersson et al., 2006, 2007; Kele et al., 2006; Maxwell et al., 2005) and spatiotemporal analysis of these transcription fac­ tors in the human fetal midbrain (Hebsgaard et al., 2009; Nelander et al., 2009) has aided in refining the dissection technique. A typical dissection of a human embryo at a stage equivalent to

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Fig. 2. Photograph illustrating a standard dissection. (a)VM from a human embryo that includes the entire mesencephalic dopamine domain. (b) A cross-section at the level of midway through the midbrain of similar stage embryo showing the dopamine domain as marked by TH (c), LMX1A (d), NGN2 (e), and FOXA2 (f). Cytospin of the resulting cell preparation showing a high proportion of DA progenitors as identified with the co-expression of LMX1a and FOXA2 (g–i). In the latter panels the cell nuclei are visualized using DAPI staining (blue). Photographs kindly provided by Jenny Nelander, Lund University. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this book.)

mid-dopaminergic neurogenesis is shown in Fig. 2a. This dissection is performed with the aim of including the maximum number of dopamine neurons and their progenitors (Fig. 2b) whilst excluding as many non-dopaminergic cells as pos­ sible. The resulting cell suspension from such dis­ sections performed on CRL 18 embryos yields approximately 5% post-mitotic dopamine neurons as determined by the number of cells immunopo­ sitive for TH, 40% FoxA2-positive cells, and 25% dopamine progenitors identified by co-expression of Lmx1a and FoxA2 (Fig. 2c). Thus, it is clear that better dissections of the developing human midbrain dopaminergic neurons is now possible with resultant higher yields of the important popu­ lation of substantia nigra dopamine neurons.

Storage of neural donor tissue for transplantation For all the reasons described above, it is likely that accessibility to suitable fetal donor tissue on a given planned day of surgery will be a limiting factor for future clinical transplantation programs. Even if the amount of suitable tissue available during one week is sufficient, coordinating the neurosurgical operation with tissue access will probably be very challenging. This means that development of effective tissue storage methods is now exceptionally important. This would allow pooling of brain tissue from abortions taking place on different days and thereby facilitate surgery. There are other advantages with tissue storage. For example, the delay period allows for more

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extensive bacteriological and virological examina­ tion of the donor tissue. If immunological match­ ing between donor and host proves to be important in the future, the storage period can also be used to have time for this. The time in storage can be used to expose the tissue to factors that promote its survival or, e.g., increase axonal growth. Finally, in some programs it has been deemed important that the woman undergoing abortion is not dependent upon the healthcare decision when she makes the decision to donate the tissue. Introducing prolonged tissue storage makes it possible for her to give her informed consent several days after the abortion. So far three different methods of storing embryonic VM tissue prior to grafting have been tested: explant or cell culture; freezing; and refrigeration. Regarding the use of cell culture as a means of storage, the two main options are dissociated and solid tissue cultures. Dissociated monolayer cell cultures are of limited usefulness as a storage method for fetal dopamine neurons, because when the neurons have matured in vitro they are very prone to die upon re-dissociation from the culture well (Brundin et al., 1988). Tissue explant cultures, on the other hand, are more versatile in this context. If the dopamine neurons mature within a small solid tissue piece they can be har­ vested and grafted without their integrity being disturbed. Freed and coworkers used this method to store donor tissue for 1–4 weeks in one of NIHsponsored clinical studies (Freed et al., 2001). One advantage is that the cells can be exposed to growth factors during the culture period (Clarkson et al., 2001), which might enhance their survival and func­ tion after grafting. A potential disadvantage, how­ ever, is that the culture period might significantly alter the relative proportions of different cell types in the VM graft tissue, thereby affecting the func­ tional efficacy of the graft. Precisely this phenom­ enon has been suggested (Bjorklund et al., 2003) to contribute to the GIDs observed in the first NIHsponsored trial (Freed et al., 2001), although there is still no experimental or clinical evidence that grafts of explant cultures of VM give rise to other

functional effects than those of freshly prepared VM tissue. Whereas freezing of fetal VM tissue down to –90–196�C allows for essentially unlimited storage periods, current protocols unfortunately result in significant additional loss of dopaminergic neurons when the tissue is grafted (Frodl et al., 1994). Despite this, freezing was used to store tissue in one of the early open-label clinical grafting trials in PD (Spencer et al., 1992) which resulted in very poor transplant survival as evidenced by post-mortem examination of one of the implanted patients (Red­ mond et al., 1990). Another more versatile method for tissue storage based on the same principle of slowing down the metabolism and maturation of the fetal neural cells is “hibernation”, which essen­ tially involves refrigeration of the tissue to 4�C in defined media. This method has proven very easy to use and is highly successful when it comes to storage of VM tissue. When rat VM tissue is hibernated at 4�C for 2–3 days, the survival of dopamine neurons upon subsequent intracerebral grafting is equivalent to what is obtained with fresh tissue (Nikkhah et al., 1995; Sauer and Brundin, 1991). The method has already been used to store tissue used in clinical trials (Freeman et al., 1995; Mendez et al., 2000; Olanow et al., 2003). When the VM tissue is stored for 5–12 days, a gradual drop-off in the survival rate of the grafted dopamine neurons is seen (Nikkhah et al., 1995; Sauer and Brundin, 1991). The reduction in survival of immature dopamine neurons can be counteracted by the addition of lipid peroxidation inhibitors called lazaroids (Grasbon-Frodl et al., 1996) and the immunophilin ligand FK506 (Castilho et al., 2000). It has also been reported that the addi­ tion of glial cell line-derived neurotrophic factor (GDNF) to the hibernation medium results in improved graft survival (Apostolides et al., 1998), but this claim has been challenged (Brundin et al., 1999; Petersen et al., 2000). Possibly, GDNF, which is known to be protective to dopamine neurons (Zigmond, 2006), cannot exert beneficial effects at low temperatures as it is not effective on dopamine neurons that are metabolically quiescent (Brundin et al., 1999). Notwithstanding the unclear effects of

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GDNF, it has been added to hibernation medium used in two series of clinical grafting trials (Mendez et al., 2000 and unpublished results). While hiberna­ tion of VM tissue is an effective storage method for a few days, and grafts obtained from such tissue clearly can exert functional effects in animal studies (Nik­ khah et al., 1995; Sauer and Brundin, 1991), it remains to be studied whether the hibernation per­ iod alters the relative cell composition (including non-dopaminergic neurons or glial precursors) in the grafts. This will be important to know before future clinical grafting studies employing long-term tissue hibernation are undertaken.

neurons are unlikely to constitute the main source of cells, even if all the practical problems discussed above are solved. There are simply too many vari­ ables concerning the tissue that cannot be finely controlled. Moreover, the protocols for procuring tissue are too labor-intense and are still not accepted for ethical reasons in many countries. One solution would be to develop methods that allows one to generate transplantable cells from renewable cell sources, such as stem cells that can generate large numbers of quality-controlled dopaminergic neurons (Fig. 3). This concept has been discussed widely for the past decade and has been the topic of numerous review articles. Indeed PD is often mentioned as one of the disorders where stem cell-derived cells might be applied clinically. There are several candidate stem cell types that can be subdivided into three main cate­ gories: lineage-specific stem cells, pluripotent stem

Stem cell-derived neurons for grafting in PD If cell transplantation is to become a widely used therapy for PD, embryo/fetus-derived dopamine Allogeneic cells ES cells

Syngeneic cells

Cell

replacement

therapy

Somatic cells (e.g. fibroblasts)

Dopaminergic neurons Patient (skin) biopsy

Blastocyst

Reprogramming

iPS cells

Stereotactic injection Direct reprogramming (iN cells)

Abortion

In vitro differentiation

Fetal ventral mesencephalon

Dopaminergic neurons

Dopaminergic neurons

Fig. 3. This schematic drawing describes the main sources of potential donor tissue that can be used for transplantation in PD. The left part of the figure depicts hESCs and fetal tissue sources, which both result in allografts. The right part of the figure illustrates the recently discovered iPS and iN cells that could be sources of syngeneic “personalized” donor tissue originating from the patient. The schematic drawing is modified from an original illustration made by Dr Laura E. Allan et al. (2010), and the brain drawings were kindly provided by Bengt Mattsson, both at Lund University.

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cells, and re-programmed somatic cells. In the following sections we describe the pros and cons of each stem cell type. During recent years, a set of criteria have been defined that stem cell-derived dopaminergic neurons need to fulfill before they can be considered for clinical application. These criteria are outlined in Table 1. Table 1. Ideal characteristics of a dopaminergic cell transplant derived from a stem cell source for possible use in patients with Parkinson’s disease Dopaminergic characteristics of cells in vitro: • Neuronal differentiation with morphology typical for midbrain dopaminergic neurons. • Differentiation that is efficient such that sufficient numbers (e.g., 100,000 dopaminergic cells per grafted side of brain) of cells could be derived from a reasonable starting number of cells. • The dopaminergic cells should express standard substantia nigra pars compacta markers such as TH; DAT; VMAT-2; and Girk-2. • The dopaminergic cells so derived should have an appropriate expression profile in keeping with normal dopaminergic neuronal development including Pitx3, Nurr 1, En1/2, etc. • The cells must show neurophysiological properties similar to that seen in mature nigral dopaminergic neurons and release dopamine. Other cells in the transplant: • The number of other neuronal cells derived from the stem cell source should be defined including Girk2-negative (non­ nigral) dopaminergic neurons and serotonin neurons. • The number of proliferating cells must be defined (e.g., using Ki-67) and the presence of such cells quantified and shown to be lost after short times to prevent graft overgrowth/tumor formation. • The absence of cells of ES/iPS cell origin, using markers such as Nanog, Sox 4, must be demonstrated. • The karyotype of the cells must be shown to be normal and stable. Cell behavior in experimental models of PD: • The dopaminergic cells so derived must continue to express the above nigral markers for long periods of times (i.e., months) after grafting into the adult brain. • The cells should extend processes and innervate the striatum with evidence of synapse formation. • The transplanted cells should have functional effects equivalent to that reported for dopaminergic cells derived from primary VM tissue (Grealish et al., 2010). • The cells grafted must not form tumors or express neither: (i) markers of early stem cells (e.g., Nanog; Oct 4, etc.) nor (ii) markers of cell proliferation beyond the immediate post-transplant period.

Briefly one can summarize the challenges facing stem cell therapy in PD into three areas. The first challenge is to develop protocols that efficiently promote the stem cells to differentiate in vitro into dopamine neurons of the midbrain phenotype. Second, it will be necessary to devise techniques to ensure that the differentiated dopamine neu­ rons survive intracerebral grafting and continue to function as dopamine neurons after the surgery. Experimental studies have shown that presence of a high proportion of dopamine neurons in cultures derived from stem cells or immortalized cell lines does not necessarily mean that the same cells will survive (Brederlau et al., 2006), or alternatively retain their dopaminergic phenotype (Paul et al., 2007), after grafting to the adult brain. Third, as we discuss further in later sections, it is essential that the stem cell-derived cells do not continue to proliferate excessively and generate tumors after transplantation to the brain (Li et al., 2008a). Lineage-specific stem cells as a source of dopamine neurons Neural stem cells exist at all rostro-caudal levels of the developing neural tube and in discrete regions of the adult brain (Gage, 2000; Kintner, 2002). Both fetal and adult neural stem cells are selfrenewing and give rise to the three major central nervous system (CNS) cell types: neurons, astro­ cytes, and oligodendrocytes in vivo as well as in vitro (Alvarez-Buylla et al., 2001; Kintner, 2002). In the early 1990s, it was found that it is possible to expand neural stem and progenitor cells from the fetal and adult CNS in vitro for long periods without the use of immortalization factors, com­ monly as free-floating aggregates of cells, termed neurospheres (Reynolds and Weiss, 1992; Reynolds et al., 1992) or other growth factorstimulated attached cultures (Buc-Caron, 1995; Laywell et al., 2000; Palmer et al., 1999). The neurosphere culture has long been the most com­ monly used expansion system and it offers some obvious advantages: neurospheres are simple to

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establish and maintain and they survive, integrate, migrate, and differentiate in the host brain after transplantation (Caldwell et al., 2001; Fricker et al., 1999; Rosser et al., 2000). However, neuro­ spheres are heterogeneous in their cell composi­ tion and tend to lose neuronal differentiation potential over time (Anderson et al., 2007; Parmar et al., 2002; Suslov et al., 2002). In 2005, Conti and colleagues introduced the neural stem (NS) cell culture system (Conti et al., 2005). Like the neuro­ spheres, NS cells are grown in defined serum-free conditions in the presence of the mitogens epider­ mal growth factor (EGF) and basic fibroblast growth factor (bFGF), but in adherent monolayered cultures as opposed to in free-floating aggregates. The NS cell cultures are highly homo­ genous with respect to morphology and neural progenitor marker expression; they show radial glia-like characteristics and retain their tripotent differentiation potential, including the capacity for neuronal differentiation even after prolonged expansion (Conti et al., 2005; Sun et al., 2008). Both NS and neurosphere cultures can readily be established from the developing VM of both rodents and humans (Caldwell and Svendsen, 1998; Caldwell et al., 2001; Hebsgaard et al., 2009; Ostenfeld et al., 2002). Whereas dopamine neurons can be formed in vitro from short-term expanded cells from the VM (Parish et al., 2008; Studer et al., 2000; Yan et al., 2001) or immature cortex (Lee et al., 2010), multi-passaged cells show a limited ability to generate dopamine neurons, even after genetic manipulation to induce a dopamine neuron fate (Andersson et al., 2007; Caldwell and Svend­ sen, 1998; Chung et al., 2006; Roybon et al., 2008). The relative difficulty with which dopamine neurons can be differentiated from tissue neural stem cells can be attributed to the fact that expan­ sion in EGF and bFGF tend to bias the cells to a GABA-ergic fate. An alternative explanation may be the special ontogeny of the midbrain dopamine neurons: a growing body of evidence shows that the dopamine neurons are uniquely derived from floor plate cells that become neurogenic only in the mid­ brain region (Bonilla et al., 2008; Hebsgaard et al.,

2009; Joksimovic et al., 2009; Ono et al., 2007). Floor plate cells may require different conditions for expansion and thus they are not maintained using standard culture conditions with EGF and bFGF, resulting in a diminished capacity to generate dopamine neurons. Taken together, therefore lineage-specific stem cells are currently not the most versatile or promising source for generation of dopamine neurons. Embryonic stem cells as a source of dopamine neurons Many of the limitations associated with expanded fetal and adult neural stem cells are overcome if one turns to an earlier stem cell type, the embryo­ nic stem cell (ESC). The ESCs are self-renewing and pluripotent cells that are derived from the inner cell mass of the pre-implantation blastocyst (Fig. 3). Like the cells of the epiblast, ESCs can give rise to all cell types of the embryo and adult organism (Evans and Kaufman, 1981; Martin, 1981; Nichols and Smith, 2009). In the mouse, ESCs have the capacity to re-aggregate with the epiblast and contribute to the formation of new embryos includ­ ing germline cells (Bradley et al., 1984). In 1998, the first successful derivation of human ESCs (hESCs) was reported (Thomson et al., 1998) and subsequently many hESC lines have been derived. Human ESCs maintain the developmental potential to contribute to cells of all three germ layers, even after clonal derivation (Amit et al., 2000), but whether hESCs correspond to pre-implantation epiblast cells of the mouse or the slightly more differ­ entiated post-implantation epiblast cells is debated (Nichols and Smith, 2009; Tesar et al., 2007). Never­ theless, it is clear that this cell type is of considerable interest for cell replacement therapies due to a vir­ tually unlimited self-renewal capacity and pluripotent differentiation spectrum. Over recent years, several protocols have been published describing the derivation of neural tissue from pluripotent human cells. Whereas the initial protocols utilized the ability of pluripotent cells to

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spontaneously give rise to neuroectoderm (Reubinoff et al., 2001), several factors have been identified which promote lineage-specific differen­ tiation including co-culture with stromal cells (Brederlau et al., 2006; Zeng et al., 2004), lowdensity culturing, and addition of fibroblast growth factors (FGFs) or inhibition of SMAD signaling (Chambers et al., 2009). Generation of several spe­ cific subtypes of neurons including midbrain dopa­ mine neurons from hESC have been reported (Fig. 4) (Cai et al., 2009; Correia et al., 2007; Hong et al., 2008; Iacovitti et al., 2007; Ko et al., 2007; Park and Lee, 2007; Park et al., 2005; Perrier et al., 2004), and more efficient protocols are con­ sistently being developed. From rodent studies, we know that correct mesencephalic identity of the transplanted dopaminergic neurons is important for proper survival, integration, reinnervation, and function of the cells after transplantation (Grealish et al., 2010). Since ESCs differentiate into neurons

Fig.4. Undifferentiated human ES cells grow in colonies. (a) Upon plating the cells under neural conversion conditions. (b) They differentiate via a characteristic rosette-like stage corresponding to neuroepithelial cells in vivo. (c) The cells in the rosettes are nestin positive (c, red). Neurons are starting to be formed at the rosette stage (c, green) and the neuronal differentiation proceeds. (d) After 28 days of differentiation, TH neurons can be detected (e). Photographs kindly provided by Dr Agnete Kirkeby. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this book.)

with characteristics of all levels of the CNS, it is therefore important to establish that the hESC­ derived dopamine neurons exhibit a correct VM identity to ensure functionality (Table 1). In ESC cultures, where positional identity is lost, VM char­ acter must be demonstrated with the combined expression of several midbrain markers such as Lmx1a/b, FoxA2, Nurr1, and Pitx3 in combination with TH (Fig. 4). The unlimited differentiation capacity and the pluripotent differentiation capacity of ESCs make them promising candidates as a potential source of transplantable neurons. On the other hand, these characteristics are also what make ESCs difficult to work with. Consequently, there are several crucial issues to be resolved when it comes to hESC differ­ entiation into functional dopamine neurons. For example, several studies have failed to demonstrate robustly that grafted hESC-derived neurons ame­ liorate behavioral deficits to the same degree as fetal VM-derived neurons (Christophersen and Brundin, 2007; Li et al., 2008). Part of this problem may be related to poor survival or loss of the dopaminergic neuron phenotype when the hESC­ derived neurons are grafted into the adult brain, despite the same cells strongly expressing TH in culture and other hESC-derived cells surviving the surgery (Li et al., 2008a) (Fig. 5). The immunogeni­ city of hESC-derived cells may also prove to be an issue in their clinical translation (Li et al., 2008a), but it is unlikely to represent more of a problem than that encountered to date with primary fetal neural transplants. Most importantly, the capacity of hESCs to form a multitude of somatic cell types means that one needs to take special care to ensure the purity of the differentiated cultures that are to be used for grafting. Particularly non-neural cells and neural cells of incorrect phenotype should be avoided, either by more efficient differentiation protocols or by positive or negative selection based on, for example, cell surface markers and fluorescence-activated cell sorting (FACS) (Hedlund et al., 2007, 2008; Nicholas et al., 2007; Placantonakis et al., 2009; Pruszak et al., 2009). Thus, a major obstacle to using ESC in a clinical

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A

B

Fig. 5. Survival of human ESC-derived dopamine neurons after grafting to the rat striatum. (a) Low-power image showing a section through one hemisphere of a rat brain immunostained for human nuclear antigen. This immunosuppressed rat received an instrastriatal graft of hESC that had been differentiated into around 20% dopamine neurons. In the striatum, a hESC-derived graft, with immunopositive (green nuclei) cells, is clearly visible. The white box marks the region where the cells in Panel B are located. (b) High magnification of a dual immunostained image from the same graft as in Panel A. The green fluorescent cells are immunopositive for human nuclear antigen, whereas the red cells are TH-positive, and most likely dopaminergic. Photographs kindly provided by Dr Asuka Morizane, when at Lund University. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this book.)

setting is their capacity to form teratomas and neural overgrowth in the host brain. During the coming years, we need to gain a better under­ standing of how to control and synchronize neural differentiation of hESCs and how to prevent pro­ liferation of the cells after transplantation. iPS cells represent a novel and interesting source of cells Several recent studies show that somatic cells can be re-programmed into pluripotent cells (reviewed in Nishikawa et al., 2008) or directly into mature cell types (Vierbuchen et al., 2010; Zhou et al., 2008). These exciting and groundbreaking findings open up new possibilities for cell replacement therapy, as it is now possible to generate patient-specific stem cells on demand. In 2006, Takahashi and Yamanaka showed in a pioneering study that fibroblasts from adult mice can be re-programmed into pluripotent cells by expressing only four factors (Oct4, Sox2, Klf4 and c-Myc). The resulting cells, termed induced pluripotent stem (iPS) cells, are phenotypically and morphologically very similar to ESCs, are germline-competent and contribute to chimeras at a reasonable frequency (Takahashi and Yamanaka,

2006; Takahashi et al., 2007). Human cells were sub­ sequently also found to be possible to re-program into iPS cells using the same four factors (Takahashi et al., 2007) or Nanog, Lin28, Oct4, and Sox2 (Yu et al., 2007) suggesting that patient-specific, genetically compatible cells for transplantation can be derived from skin biopsies from PD patients (Soldner et al., 2009). The initial iPS cells were derived using techniques not compatible with clinical applications as the reprogramming depended on oncogenes. The reprogramming genes were deliv­ ered using viral vectors and the genes were inserted in multiple sites of the genome. Several develop­ ments in iPS cell derivation technology have already been made. The number of genes required for repro­ gramming has been reduced, and iPS cells have now been generated using non-integrating viruses (Stadt­ feld et al., 2008; Yu et al., 2007), plasmid systems that result in single integration site and allow for subse­ quent excision of reprogramming factors (Kaji et al., 2009; Okita et al. 2010; Woltjen et al., 2009), and also by direct delivery of recombined reprogramming proteins (Lyssiotis et al., 2009). All these improve­ ments makes the iPS cells more clinically relevant, but further technological advances are needed in order to produce safe cells under good manufactur­ ing practice (GMP) conditions that can be used in a clinical setting.

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As was hoped for, iPS cells have been shown to respond to the same developmental patterning cues as ESCs. They use the same transcriptional network to generate neuroepithelium and func­ tionally appropriate neuronal types including dopamine neurons, albeit with reduced efficiency and increased variability (Cai et al., 2009; Chambers et al., 2009; Hu et al., 2010; Wernig et al., 2008). The iPS cells also have the same pro­ liferative capacity as ESC. Therefore, they share the risks for incomplete and unsynchronized differ­ entiation, coupled with risk of tumor formation, and neural overgrowth after grafting with ESC. An alternative approach may be direct repro­ gramming from one somatic cell type to another. Proof-of-concept of this comes from a study per­ formed by Melton and colleagues who reexpressed three key developmental regulators that reprogram differentiated pancreatic exocrine cells in adult mice into cells that closely resemble endocrine beta-cells (Zhou et al., 2008). Similarly, combinatorial expression of three neural-specific transcription factors was recently found to directly convert fibroblasts into functional neurons in vitro, and this new class of cells has been named induced neuronal (iN) cells (Vierbuchen et al., 2010) (Fig. 3). Although this is an exceptionally interesting finding, the field is in a very early era. For example, the iN cells generated so far have not been demonstrated to include dopamine neu­ rons. This initial study clearly provides evidence for the principle of cellular reprogramming of terminally differentiated cells without reversion to a pluripotent cell stage, which is a very attrac­ tive idea for generating patient-specific transplan­ table cells that differentiate into functional neurons after grafting. Importantly, the method can circumvent the issues with uncontrolled pro­ liferation and incomplete differentiation. How­ ever, the method introduces one new problem: since the reprogrammed cells do not proliferate, very large numbers of reprogrammed neurons must be generated for each patient in order to provide sufficient material for transplantation. Furthermore, it is not inconceivable that PD

patient-derived (thereby possible expressing PD susceptibility genes) iN cells or iPS cell-derived neurons might degenerate after grafting due to PD, in analogy to the patient’s own dopamine neurons

Safety and regulatory issues for clinical application of stem cells in the brain Transplantation of neurons derived from a highly proliferative population of stem cells into the brain involves potential safety risks, which we have reviewed extensively elsewhere (Li et al., 2008a), mainly associated with the possible inclu­ sion of proliferating cells that can form tumors. In any stem cell-based transplantation therapy, regardless of the target organ or tissue, tumor growth is a major safety concern. For stem cell therapy in the brain, it is a particularly grave con­ cern. The confined space of the intracranial cavity coupled to the vital functions coupled to many brain structures means that even modest or slow tumor growth in this locale can have neurologi­ cally disastrous and potentially lethal conse­ quences. The issue of graft-derived neural tumors was recently in the spotlight after the pub­ lication of a report of multifocal brain tumors in a boy suffering from ataxia teleangectasia who was treated with intrathecal and intracerebellar injec­ tions of neural stem cells (Amariglio et al., 2009). Furthermore, over the past few years the Califor­ nian biotech company Geron Corporation has worked hard to convince the Food and Drug Administration about the safety of their hESC­ derived oligodendrocytes (GRNOPC1) and their suitability for use in a transplantation therapy for spinal cord injury. In particular, fears of tumor growth and cysts forming in the grafts have been the source of great scrutiny (DeFrancesco, 2009). One possible cause for continued proliferation of stem cell-derived grafts, even after dedicated efforts to differentiate the cells into neurons, is the appearance of chromosomal aberrations in the stem cell cultures. While chromosomal

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aberrations occur commonly in rapidly dividing cells, most of them result in cell death. Only rarely are the cells viable, and in a few of these cases the aberration provides the cells with an advantage (e.g., upregulation of a growth factor receptor) that makes them proliferate more rapidly. It is well known that hESC cultures can accumulate chromosomal changes, particularly in chromo­ somes 12 and 17, and that the abnormal cells often outgrow their normal neighbors (Buzzard et al., 2004). A recent study showed that among different neural stem cell lines derived from human fetal brain, 24% developed trisomy in chromosome 7 and 5% exhibited trisomy in chro­ mosome 19 (Sareen et al., 2009). While these par­ ticular chromosomal changes did not result in tumor growth when the cells were grafted to the brain, they highlight the need to regularly monitor the cytogenetics of all cell lines considered for clinical use. Even epigenetic changes, e.g., altera­ tions in gene promoter methylation (Maitra et al., 2005), in stem cell cultures can lead to quantitative changes in gene expression and growth selection of a subpopulation of cells that is more prone to tumor formation. However, it is not sufficient to avoid changes at the chromosomal level in order to avoid tumor growth in stem cell neural grafts. Clearly, cells with normal chromosomes and epigenetic patterns also have the potential to proliferate. Thus, any remain­ ing pluripotent stem cells in “differentiated” hESC cultures must be avoided as they can continue to divide and form teratomas upon transplantation (Brederlau et al., 2006). When grafting hESC­ derived dopaminergic neurons, even residual neu­ roepithelial progenitors derived from stem cells, and present in the graft tissue, can continue to divide after intracerebral transplantation and give rise to large, and potentially harmful, tissue masses (Sareen et al., 2009). There exist several strategies that can be used to enrich the differentiated cell population in stem cell cultures or to deplete undifferentiated/ proliferating cells from the same cultures. Briefly, they include the use of pharmacological agents that kill proliferating cells: cell sorting using FACS or

magnetic activated cell sorting (MACS), and genetic manipulation of intracellular signaling pathways that govern cell proliferation. We recently reviewed these options in greater length elsewhere (Li et al., 2008a). The main challenge facing all these methods is that they must be 100% efficient. It is unaccepta­ ble to leave even one single pluripotent stem cell among the cells that are implanted into a brain. Whereas FACS and MACS are excellent techni­ ques to sort cells used in laboratory studies, they typically suffer from the shortcomings that the cell surface markers are not fully exclusive/inclusive of the undesired/desired cell populations and that the apparatus exhibits slight experimental errors. Furthermore, FACS is a relatively traumatic proce­ dure for neurons and as a result they can die during the procedure if they have long processes. None­ theless, a recent study showed that using FACS to select cells based on their expression levels of three cell surface cluster of differentiation molecules, it was possible to selectively enrich hESC-derived neuronal cells and drastically reduce risk of tumor formation after grafting (Pruszak et al., 2009). While all the safety issues we have just described are major scientific challenges, for them to be really relevant in a clinical setting they need to be addressed in the context of GMP (Unger et al., 2008). This adds yet another level of complexity because GMP requires that the issues are resolved using standardized protocols employing, e.g., ani­ mal-free (xeno-free) feeder cells and culture media in a setting with minimal risk of microbial infection. Although GMP-compatible protocols for hESC­ derived dopamine neurons are currently being developed (Swistowski et al., 2009), the application of GMP rules and generation of clinical-grade hESC-derived dopamine neurons is costly, time consuming, complex, and constitutes a significant practical hurdle to the clinical application of stem cell therapy in PD. For example, not just the differ­ entiation protocols, subsequent cell sorting and sto­ rage need to be performed according to GMP, but the hESC line itself (used to generate the dopamine neurons) has to be derived under GMP conditions in a certified laboratory.

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Future perspectives and concluding remarks Cell transplantation still remains a very promising therapeutic approach for PD. While the initial pro­ mise and excitement of the early open-label trials with fetal VM grafts in PD has been diminished and undermined by the subsequent placebo-con­ trolled, double-blind trials, we believe that the past 5–10 years has seen several developments that now make it possible once again to perform successful trials using fetal VM tissue in select groups of PD patients. By refining both the transplantation tech­ niques and patient selection, we think that this approach will give more reproducible and marked symptomatic relief in the patients, and that the prevalence and severity of the unwanted GIDs will be reduced. Notwithstanding our optimism regarding fetal tissue transplantation in PD, we still consider it necessary to develop new alterna­ tive sources of donor tissue in the future, since fetal tissue can never be the basis of a widely accessible cell therapy for PD. The past decade has seen dramatic developments in stem cell research, including substantially increased knowledge in hESC biology and the developmental processes underlying midbrain dopaminergic neurogenesis, as well as the discovery of the reprogramming of somatic cells into stem cells and neurons that opens up the door to patient-specific “personalized” cells (Fig. 3). Taken together, this progress is likely to lead to the creation of stem cell-based therapies for PD. Without dampening our enthusiasm for these future stem cell therapies, we recognize that sev­ eral safety issues and regulatory hurdles need to be negotiated during the coming 5–10 years before the PD patients will actually reap the benefits of this concerted research effort.

sponsored by the Swedish Parkinson Foundation and the Swedish Brain Foundation. PB and MP are part of the Linnéaus research environment BAGADILICO, sponsored by the Swedish Research Council. PB and MP are funded by the Swedish Research Council. RAB is supported by grants from the PDS (UK), the Cure-PD Trust, and an NIHR Biomedical Research Centre award to Addenbrooke’s University Hospital.

Abbreviations ADL bFGF DAT ESC EGF FACS F-dopa GID GMP hESC iPS MHC NIH NS TH UPDRS VM PD PET

Activities of Daily Living basic fibroblast growth factor dopamine transporter embryonic stem cell epidermal growth factor fluorescence activated cell sorting fluorodopa graft-induced dyskinesias good manufacturing practice human embryonic stem cell induced pluripotent major histocompatibility complex National Institutes of Health neural stem tyrosine hydroxylase Unified Parkinson’s Disease Rating Scale ventral mesencephalon Parkinson’s disease positron emission tomography

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

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

CHAPTER 15

Neural grafting in Parkinson’s disease: unraveling the mechanisms underlying graft-induced dyskinesia Emma L. Lane
,†, Anders Björklund‡, Stephen B. Dunnett§, and Christian Winkler{ † Welsh School of Pharmacy, Cardiff University, South Wales, UK

Wallenberg Neuroscience Center, Department of Experimental Medical Science, Lund University, Lund, Sweden

§ Brain Repair Group, School of Biosciences, Cardiff University, South Wales, UK

{ Department of Neurology, University Hospital Freiburg, Freiburg, Germany

‡

Abstract: The development of neural transplantation as a treatment for Parkinson’s disease has been compromised by a lack of functional efficacy and the appearance of transplant-induced motor side-effects in some patients. Since the first reports of these graft-induced dyskinesias (GID), and the realization of their impact on the progress of the field, a great deal of experimental work has been performed to determine the underlying cause(s) of this problematic side-effect. In this review we describe the clinical phenomenon of GID, explore the different representations of GID in rodent models, and examine the various hypotheses that have been postulated to be the cause. Based on the available clinical and preclinical data we outline strategies to avoid GID in future clinical trials using fetal cell transplants or cell preparations derived from stem cells. Keywords: Parkinson’s disease; Transplantation; Dopamine; Dyskinesia; Graft-induced dyskinesia; L-dopa-induced dyskinesia; Amphetamine-induced dyskinesia

of dopamine (DA) levels in the caudate–putamen. The cardinal motor symptoms of this disease— slowness of movement (bradykinesia), muscle rigidity, and resting tremor—can be effectively alleviated by pharmacotherapy using DA agonists or the DA-precursor L-dihydroxyphenylalanine (L-dopa). However, the efficacy of these drugs wanes as the disease progresses and long-term

treatment with L-dopa commonly leads to the

Introduction Parkinson’s disease (PD) is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra with concomitant reduction
Corresponding author.

Tel:. þ44-292-0874112; Fax: þ44-292-0876749; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84015-4

295

296 Table 1. Definitions of abnormal movements Dyskinesia

Chorea Dystonia Stereotypy LID GID AID

Disturbance of movement according to the original definition; very often used to describe an increase of movements (hyperkinesia) following pharmacotherapy. Dyskinesia induced by chronic L-dopa medication is of two types: (1) peak-dose dyskinesia, seen during the on-phase of each L-dopa dose; and (2) diphasic dyskinesia, seen at the beginning and end of the L-dopa dose when dopamine levels are rising or decreasing Derived from the Greek word for dance; movements are involuntary, usually short-lasting and quick, non-rhythmic and non-repetitive, primarily involving distal body parts Disturbance of muscle tone according to the original definition; usually associated with increased muscle tone due to involuntary, longer-lasting muscle contractions that put the head, body, or limbs into abnormal positions Coordinated repetitive movements; usually suppressible by will L-dopa-induced dyskinesia—abnormal movements following administration of L-dopa; changes of dyskinesia after transplantation usually mean changes in LID Graft-induced dyskinesia—abnormal movements following intracerebral transplantation and unrelated to drug stimulation Amphetamine-induced dyskinesia—abnormal movements in rodents with intracerebral dopaminergic transplants induced by administration of amphetamine; used as an animal model of GID

development of motor fluctuations and abnormal involuntary movements (AIMs) known as dyski­ nesia (see Table 1). These involuntary move­ ments, typically choreic or dystonic in nature, can in themselves be severely debilitating and affect the quality of life. With limited treatment options available in the later stages of PD, the need for alternative approaches was recognized several decades ago. After convincing results in animal models of PD, one of these approaches, neural transplantation (Bjorklund, 1992; Herman and Abrous, 1994; Winkler et al., 2000), moved into the clinic. To date, several hundred PD patients have received intracerebral transplants of fetal DAergic tissue obtained following elective surgical terminations of pregnancy. Scientific evaluation of grafted patients in several open-label trials demonstrated that fetal DAergic grafts survive and reinnervate the host striatum, release DA, and induce pro­ nounced and continuous improvement of motor deficits despite an ongoing disease process in the brain (Hagell and Brundin, 2001; Kordower et al., 2008a, b Li et al., 2008; Piccini et al., 1999; Winkler et al., 2005). To validate the findings of the openlabel trials and to guard against misinterpretation through placebo effects (de la Fuente-Fernandez and Stoessl, 2002; Goetz et al., 2008), two shamsurgery-controlled double-blind trials were funded by the NIH. However, in these trials improvements

of motor deficits were limited as compared to the open-label trials and improvements to motor func­ tion were mild and only observed in sub-populations of transplanted patients (Freed et al., 2001; Ma et al., 2010; Olanow et al., 2003). An additional development was the reports from both NIH trials of unanticipated side-effects, the appearance of a new type of dyskinesia unrelated to ongoing medi­ cation, now termed graft-induced dyskinesia (GID). During a temporary and self-imposed moratorium on transplantation for PD, there has been active discussion, alongside considerable new experi­ mentation, aimed at resolving the issues identified as key in maximizing the functional benefit derived from the graft and minimizing side-effects. In this review we focus specifically on the devel­ oping understanding of these side-effects.

The clinical problem of graft-induced dyskinesia (GID) The first reports of unanticipated side-effects of transplants came in the Denver–Columbia trial (Freed et al., 2001; Greene et al., 1999; Ma et al., 2002), in which severe involuntary, predominantly dystonic, movements were observed in 4 out of 33 grafted patients, 6–12 months after grafting (for details, see summary in Tables 2A and 2B). Unusually, these dyskinesias were unrelated to the

297

intake of L-dopa, persisting for several days after the drug had been withdrawn (Cho et al., 2005; Graff-Radford et al., 2006; Olanow et al., 2003). A video-based review of the patients transplanted in open-label studies in the Lund–London–Marburg cohort then revealed off-medication dyskinesia in 7 out of 14 grafted patients (Hagell and Cenci, 2005; Hagell et al., 2002). Severity peaked 24–48 months after grafting, reaching a significant clinical amplitude in two patients. In 2003, Olanow and colleagues reported off-medication dyskinesia

in 56% of their patients in a second NIH placebocontrolled trial (the Tampa–Mount Sinai trial, Olanow et al., 2003). In all studies, off-medication dyskinesia was clearly distinguishable from L-dopa­ induced dyskinesia, in being either more dystonic (Denver–Columbia trial), more stereotypic and rhythmic (Tampa–Mount Sinai trial), or both (Lund–London–Marburg cohort). In a recent video-based re-assessment of the patients in the Tampa–Mount Sinai trial the GIDs were described as repetitive, stereotypic movements in the lower

Table 2A. Explicit reports of graft-induced dyskinesia in grafted patients GID first described by…

No. patients No. of patients with GID Age of patients Time course of GID—onset– peak Phenomenology

Pre-graft LID Effect of transplant on PD symptoms (UPDRS)a Effect of transplant on LID Effect of dopaminergic drugs on GIDb Response to amantadine DBS, Type and no. of patients Response to DBS

Relationship to…..? Pre-graft FDOPA PET Post-graft FDOPA PET— graft area Post-graft FDOPA PET— ventral striatum (non-graft areas) Pre-graft UPDRS Post-graft UPDRS “on” Post-graft UPDRS “off” Pre-graft L-dopa response

Freed et al. (2001)

Hagell et al. (2002)

Olanow et al. (2003)

Double-blind 33 5 (15%) <60 6–12 months NR Severe cranial dystonia or persistent dyskinesia in arm or generalized dyskinesia Yes Improvement

Open-label 14 6 (43%)

Double-blind 23 13 (56%)

6–12 months 24–48 months Concurrent hyperkinesias and dystonias. Repetitive stereotypic ballistic movements Yes Improvement

6–12 months 12 months Repetitive stereotypic alternating contractions of flexor and extensor muscles at low frequency Yes Improvement

NR Worsen

Improvement Worsen

Improvement Worsen

Two responded well, one transiently GPi DBS (3)c/d NR

NR

NR

STN (1) or GPi (1) DBS GPi DBS reduced GID, STN DBS did note

GPi (1) STN (3) DBS GPi DBS 1 did well initially, then developed generalized GID, STN DBS improved allf

NR Yesci

Yesg No

Noh Noc

NR

Change in ventral striatum associated with poorer response, including GID j

NR

NR NR

NR Improvement

NR No, but predictive of positive graft effect

No NR

Noh Some improvement with GID. None without GID Noh Noh

(Continued)

298 Table 2A. Explicit reports of graft-induced dyskinesia in grafted patients (Continued ) GID first described by…

Freed et al. (2001)

Hagell et al. (2002)

Olanow et al. (2003)

Post-graft LID Immunosuppression

NR None used

Tissue storage

Yesl

Yesk Increased or development of GID on withdrawalj Yesm

Noh Used for first 6 months, onset coincided with withdrawal No

Summary of clinical trials reporting graft-induced dyskinesia, GID, following intracerebral transplantation of fetal ventral mesencephalon in patients with Parkinson’s disease. Details are provided with reference to the article that first reported the phenomena but additional details have also been obtained from subsequent analyses and are referenced separately. Abbreviations: DBS—deep brain stimulation; FDOPA PET—[18F] fluorodopa positron emission tomography; GID—graft-induced dyskinesia; GPi—internal segment of globus pallidus; LID—L-dopa-induced dyskinesia; NR—not reported; PD—Parkinson’s disease; STN—subthalamic nucleus; UPDRS—Unified Parkinson’s Disease Rating Scale a Data only available for first year (Freed et al., 2001), GID associated with residual PD symptoms (Olanow et al., 2003). b Some improvement in GID with dose reduction in L-dopa reported (Freed et al., 2001; Olanow et al., 2003). c Ma et al., 2002. d Freed et al., 2004. e Herzog et al., 2008. f Cho et al., 2009. g Reported as a trend, rs = -0.529, p = þ0.064 (Hagell et al., 2002) h Olanow et al 2009. i High FDOPA signal in “hotspot” areas of the graft was only present in patients with GID (Ma et al., 2002). j Piccini et al 2005. k p = 0.015 (Hagell et al., 2002). l All tissue was cultured for 4 weeks and transplanted as “noodles”, no relationship between this and GID has been reported. m Two of the patients most severely affected by GID had tissue stored in hibernation media at 4�C for 1–8 days (Hagell et al., 2002).

Table 2B. Implicit reports of graft-induced dyskinesia in grafted patients GID first described by…

No. patients in study No. with GID-like behaviors Age of patients Phenomenology

Pre-op LID Effect of transplant on PD symptoms (UPDRS) Effect of antiparkinsonian medication on dyskinesia Further treatment and response

Defer et al. (1996)

Jacques et al. (1999)

Hauser et al. (1999)

Open-label 5 3 (60%)

Open-label 60 1 (1.5%)

Open-label 6 1 (17%)

48–64 Worsened bilateral peak-dose “on” dyskinesia 2–6 months post-op, later development of asymmetric contralateral peak-dose dyskinesia Yes Improvement

55 Increased dyskinesia following transplantation (no other details) NR Improvement

50 Developed at 8 year posttransplant with severe “off” dyskinesia “groping movement” of right handa Yes Improvement

Appearance despite stable medication, variable regimes used

Not alleviated by change in medical regime Pallidotomy resolved

NR

NR

Improvement with GPi DBSa

Early reports by Defer et al. (1996) and Jaques et al. (1999) and the recent publication by Hauser et al. (1999) as well as subsequent publications on the

same patient cohorts mention changes in dyskinetic behaviors post-transplantation. There is not sufficient detail to conclusively determine whether

these are true GIDs; therefore, they are included but with the limited details available as Table 1B.

a Graff-Radford et al., 2006.

299

extremities with residual parkinsonism in other body regions, representing a prolonged form of diphasic dyskinesias (Olanow et al., 2009). Careful reading of transplantation studies published prior to the two controlled trials actually suggests that the alteration in the profile of dyskinesia after grafting is not a new phenomenon. Several early studies did report unusual dyskinetic behaviors both “on” and “off” L-dopa following transplanta­ tion, although details are limited (Defer et al., 1996; Hauser et al., 1999; Herzog et al., 2008; Jacques et al., 1999; Kopyov et al., 1997) (see Table 2B). While, for the most part, GIDs have been mild, some patients in each of the three studies described in Table 2A developed GIDs of a mag­ nitude and severity that has necessitated surgical intervention by deep brain stimulation (DBS) (Freed et al., 2001). In several of these cases the expression of GID has been effectively controlled by DBS of the subthalamic nucleus or the internal segment of the globus pallidus (Cho et al., 2005; Freed et al., 2004; Graff-Radford et al., 2006; Herzog et al., 2008). Clearly, this is not an accep­ table long-term solution to the problem of GID. A number of successful, individual transplantation cases have clearly demonstrated the potential ben­ efits afforded by cell transplantation in PD and unraveling the mechanism(s) underlying the slow and protracted development of GID is thus an urgent priority. This goes beyond the continuation of fetal tissue transplant trials, with implications for the clinical development of alternative sources of cells, such as cells derived from embryonic or adult stem cells, or from induced pluripotent cells.

Finding the cause of GID In an attempt to understand the root cause of GID, each clinical center has analyzed their patients as the basis for generating hypotheses for mechanisms (Hagell et al., 2002; Ma et al., 2002; Olanow et al., 2003). However, comparing PD transplantation studies, including those without any report of GID (e.g., Cochen et al., 2003; Mendez et al., 2000a, 2008),

has highlighted fundamental differences in the pro­ cedures used (for details, see Winkler et al., 2005). This has made it impossible to define a common cause of GID. Nevertheless, a number of technical and clinical parameters have been considered for their potential role in the development of GID. We categorize these as either related to the patients (e.g., the selection of patients, their striatal and extrastriatal DA loss before and after surgery, use of immunosuppression, etc.) or to the transplantation procedure itself (including the preparation of the tissue, storage, and surgical technique). An outline of the procedure and the leading hypotheses at each stage of the process are summarized in Fig. 1. However, if we are to clarify the mechanisms under­ lying these phenomena in patients, we need a valid animal model to allow more controlled experimental analysis.

Modeling GID in animals When GID was first described in patients, transplantinduced AIMs had been observed in rodent models of PD but only in response to L-dopa, i.e., “L-dopa­ induced dyskinesia” (LID). To quantify LIDs, clin­ ical rating scales were adapted to score AIMs of the head, limbs, and body that developed in rodents. First described by Cenci and colleagues (Cenci et al., 1998; Lee et al., 2000), this model has now been extensively characterized enabling exam­ ination of the molecular and behavioral impact of different PD therapies. Similarities to LID seen in PD patients, or in the more extensively used MPTPtreated primate model, have validated this approach (Dekundy et al., 2007; Lundblad et al., 2002; Maries et al., 2006; Winkler et al., 2002). Truly spontaneous GID, however, has only been described in two animal studies (Lane et al., 2006; Vinuela et al., 2008). 6-hydroxydopamine (6-OHDA)-lesioned rats with intrastriatal dopami­ nergic grafts displayed short bursts of mild AIMs at early post-grafting intervals but these were incon­ sistent and unreliable (Lane et al., 2006; Vinuela et al., 2008). Even non-pharmacological stimuli,

300 Patient selection

Dissect

DA A9 vs: A10 DA vs: 5-HT

Embryonic age

Surgical procedure

Collect

Age Severity of disease Preexisting LID Extrastriatal degeneration

Culture Fresh

No. of cells transplanted Location of cell deposits Tissue distribution, hotspots

Hibernate

Duration of culture

Culture process

Duration of hibernation Use of GDNF

Postoperative events Tissue preparation

Immunosupression Ongoing extrastriatal degeneration

Fig. 1. Schematic representation of factors hypothesized to contribute to GID.Many parameters vary between the different grafting studies, most of which have been or continue to be considered as factors that may contribute to graft-induced dyskinesia (GID), beginning with tissue-related components from as early as the dissection of the embryo. The dissected tissue may or may not include serotonergic (5-HT) neurons or different proportions of DAergic (DA) A9 vs. A10 neuronal subtypes, depending on the dissection landmarks and/or the age of the donor fetus. Tissue has variously been used fresh, stored in so-called hibernation medium or cultured for some time, and either dissociated into a crude cell suspension or cut into small tissue fragments. There has been some exploration of the use of glial cell line-derived neurotrophic factor (GDNF) in the hibernation procedure—its possible role in GID is unknown. Patient-related factors include pre-transplantation characteristics such as the severity of the disease and extent of DA denervation; the presence or absence of L-dopa-induced dyskinesia, LID; and age at the time of surgery. Surgical and post-surgical conditions that may play a role in the development of GID include surgical technique, the number of transplanted cells, and their location and distribution through the caudate putamen, the use of immunosuppression, and ongoing extrastriatal degeneration.

such as exposure to a novel environment or tail pinches, have been unable to consistently provoke spontaneous dyskinesia (Carlsson et al., 2006; Lane et al., 2006; Vinuela et al., 2008). With hindsight, observations in the 6-OHDA rat model of PD had already provided indications for aberrant graft-induced behavior, associated with adverse biochemical or cellular activity in the grafted striatum. In unilateral 6-OHDA-lesioned rats, the DA-releasing agent amphetamine evokes a profound rotational behavior contralateral to the lesioned and transplanted striatum (instead of restoring lesion-induced ipsilateral rotations back

to baseline). This aberrant contralateral graftmediated behavior reflects an overcompensation induced by DA released from the transplanted cells, does not correlate with the number of surviving DA neurons in a mature graft, and follows a different temporal profile to the turning behavior induced by DA released in the contralateral intact striatum seen in animals with lesions alone (Herman et al., 1993; Lane et al., 2006; Torres et al., 2007, 2008) (see Fig. 2). In an attempt to reproduce the clinical phe­ nomenon of GID, grafted rats were observed following injection of amphetamine and, using

301 L-dopa-induced

B

Amphetamine-induced rotations

Severity of AIMs

Magnitude of rotations Ipsilateral Contralateral

dyskinesia

Time (h)

Time (h) 6-OHDA-lesioned rats

C

Amphetamine-induced dyskinesia

Severity of AIMs

A

Time (h)

6-OHDA-lesioned rats with mesencephalic grafts

Fig. 2. The most common rodent model of PD is the unilateral 6-OHDA-lesioned rat.In response to chronic L-dopa administration, supersensitization of the DA receptors on the lesioned side causes the animal to perform circling movements, rotations, in a direction contralateral to the lesion for 3 h and develop severe abnormal involuntary movements (AIMs) known as L-dopa-induced dyskinesia, LID (Fig. 2a, grey line). Following transplantation of DA-rich ventral mesencephalic VM tissue the severity of these behaviors is significantly reduced (Fig. 2a, black line). In response to an injection of amphetamine, the DA imbalance causes a rat with a 6-OHDA lesion to turn in the direction ipsilateral to the lesion (Fig. 2b, grey line). With the addition of a DAergic graft into the lesioned striatum, the direction of the rotational response reversed to a contralateral bias with a biphasic time course (rapid onset and vigorous rotation at 15–30 min after injection, return to lower rotation rates during the next 2–3 h, and a further increase of rotations during the last 1–2 h (Herman et al., 1993; Lane et al., 2006), total duration 6 h, Fig. 2b, black line). Very low levels of motor and orolingual stereotypy are observed in lesion-only animals following amphetamine, which may contribute to the occasional low-level AID scores (Fig. 2c, grey line). Some grafted animals may also show this very mild stereotypic response. However, importantly, many of the grafted rats will develop amphetamine-induced dyskinesia (AID), a more severe behavioral phenotype reminiscent of the LID behaviors, over the 12–16 post-transplantation (Fig. 2c, black line). This behavior has a different time course to the rotational response, gradually increasing over the first 30–45 min and then remaining stable over the next 4–5 h before slowly returning to baseline (Cenci et al., 1998; Lane et al., 2006). Behaviorally they tend to be less severe than LIDs and have a greater hyperkinetic component with extensive orolingual and limb movements (Carlsson et al., 2006). The profile of behavior induced by amphetamine may relate to the location of the graft and the distribution of its DAergic innervation within the striatum. Different striatal regions are known to contribute to different behavioral patterns with amphetamine-induced orolingual stereotypies originating in the ventro­ lateral striatum (Kelley et al., 1988), overlapping with the caudal-lateral striatal areas known to contribute to LIDs (Cenci et al., 1998) (see Table 3 for other parameters that influence the expression of AIDs).

the rat LID scale, AIMs were observed in a proportion of transplanted animals, behaviors that are not present following amphetamine administration to lesion-only animals (Carlsson et al., 2006; Lane et al., 2006). These amphetamineinduced dyskinesias (AID) have since been observed in several further studies (Tables 2A and 2B) (Carlsson et al., 2007; Lane et al., 2008, 2009a, b). As spontaneous GID in rodent PD models is too inconsistent and unpredictable, AID has come to serve as an experimental model for the study of GID. Similar to GID seen in grafted patients, AID is observed in the

absence of any anti-PD medication and develops progressively following transplantation in a proportion of animals. Importantly, these involuntary movements can be clearly distinguished from amphetamine-induced rotation (see Fig. 2), and despite showing some similarities to LID, AID is comprised to a larger extent by dystonic axial movements and hyperkinetic limb and orofacial dyskinesia (Carlsson et al., 2006), which are characteristic features of clinical GID. There are alternative reported approaches to studying GID in rodent models, the main feature of which is the characterization of AIMs in grafted

302 Table 3. Animal models of post-grafting dyskinesia in PD (adapted from Lane et al. 2009a) Post-transplantation behaviors

Spontaneous transient forepaw tapping and body twisting

Amphetamineinduced AIMs

L-dopa-induced

Term

GID

AID

LID

LID

Observation period

2 × 1 min (sessions at repeated intervals) Presentc,d

9 × 1 min (every 20 min for 3h)a–l Improvea–l

1 × 2 mins (30 mins after a,b L-dopa) d,i,l Present

Improve with more cellsa,c,i Improved with more caudal graft vs rostralb

No effecti Less with larger graftsd Greater with “hotspot” graft of same cell number as diffuse graftd,l –

Graft size/No. of dopamine cells Location of dopamine cells in striatum

Expressed in small and large graft groupsc,d Not observedb

A9 vs A10 dopamine neurons Role of dopamine transporter (DAT)

Not observedf

18 × 1 min (every 20 mins for 6h)c,d,e,f,g Presentc,d,e,f Not observedf No within-group correlationc Worsened with more caudal graft vs rostralb Not observedf

Some observed in both DAT KO and WT groupsi

Not observed in WT or DAT KOi

5-HT neurons Inflammation

Not observedb,c Not observedg

No effect c,e,j,k No effectg

Dopamine neuron grafts

AIMs

A9 correlates with LID improvementf Greater LID improvement with DAT KO graftsi Worsen e,j No effectg Increased tappingh

L-dopa-induced forelimbfacial stereotypies

– – No effecth

All these models were produced in rats with unilateral 6-OHDA lesions of the nigrostriatal pathway, receiving transplants of embryonic VM tissue in

the DA-denervated striatum. Effects are reported on post-grafting dyskinesia, defined here as either spontaneous, non-drug-induced behaviors (GID),

dyskinetic behaviors following amphetamine administration (AID) or L-dopa (LID). Full paper citations are given in the reference list. - = issue not

reported/studied in this model; “Not observed” = behavior reported as not being present in these studies; “No effect” = reported as no difference

between groups. WT = transplanted cells from wildtype mice; DAT KO = transplanted cells from DA transporter knockout mice; AIMs = Abnormal

involuntary movements; 5-HT = serotonergic.

a Lee et al. (2000)

b Carlsson et al. (2006)

c Lane et al. (2006)

d Maries et al. (2006)

e Carlsson et al. (2007)

f Kuan et al. (2007)

g Lane et al. (2008)

h Soderstrom et al. (2008)

i Vinuela et al. (2008)

j Carlsson et al. (2009)

k Lane et al. (2009)

l Steece-Collier et al. (2009).

animals shortly after administration of L-dopa (Maries et al., 2006; Soderstrom et al., 2008; Steece-Collier et al., 2009; Vinuela et al., 2008) (Table 3). In these studies, grafted animals display repetitive and stereotypic grabbing and gnawing, or a tapping behavior. The increase in stereotypy observed in these experimental studies provides an interesting feature for further study (Cenci and Hagell, 2006; Olanow et al., 2003). Nonetheless,

one characteristic feature of GID in patients is that it is unrelated to L-dopa intake and persistent, in some cases after prolonged withdrawal from L-dopa. This suggests that mechanisms underlying GID and LID may be related but still distinct. In this review we term behaviors produced solely by the graft GID, while AID is used as an experi­ mental model to study a type of GID that shares some characteristic features, particularly its

303

dystonic and stereotypic nature, with GID seen in patients. In contrast, the use of L-dopa to stimulate dyskinesia is a parallel to the so-called peak-dose dyskinesia seen in the clinical “ON” state. There­ fore, changes to LID as a result of transplantation are referred to as post-transplantation LID.

Selection of patients to prevent GID In studies of grafted patients, correlation analyses have variously suggested that potential functional benefits derived from the graft are limited, if— prior to transplantation—PD patients are either too old, too severely affected by the disease, show a bad preoperative L-dopa response, or when degeneration of the DA system is not restricted to the putamen but extends to more ventral regions (Freed et al., 2001; Olanow et al., 2003; Piccini et al., 2005; Winkler et al., 2005). The Lund open-label trial demonstrated a trend toward a negative correlation between severity of GID and preoperative putaminal 18F-DOPA uptake, suggesting that more severely affected PD patients may carry a higher risk for the develop­ ment of GID (Hagell et al., 2002; Piccini et al., 2005). In this same study, there was a further correlation between the severities of post-operative peak on-phase dyskinesia and off-phase dyskinesia (Hagell and Cenci, 2005). In the Olanow et al. (2009) study patients with or without GID dis­ played similar levels of LID, and also similar striatal 18F-DOPA uptake on PET, both pre-and post-operatively. Nevertheless, the mechanisms of LID and GID may still be related in the sense that patients who have developed severe LID prior to surgery run an increased risk of developing GID after transplantation. Indeed, in one rodent study we have reproduced the positive correlation between pre-operative LID and post-operative AID (Lane et al., 2006). In a further study of the role of pre-transplantation L-dopa in GID we found that 6-OHDA-lesioned rats had a greater tendency to develop AID when the animals had been exposed to chronic L-DOPA prior to

transplantation (Lane et al., 2009b) irrespective of graft size. Most transplantation patients will have received long-term L-dopa treatment, and a good pre-operative L-dopa response has been part of the inclusion criteria for many transplantation stu­ dies. Indeed, a good L-dopa response was found to be predictive of a positive outcome in the Denver–Columbia trial (Freed et al., 2001). Thus, further studies have examined the relation­ ship of AID to the severity of pre-transplantation LID. Animals with moderate-to-severe LID showed a greater risk of developing AID, while animals with no or only minor LID, despite chronic L-dopa treatment, developed no or only very subtle GID (Lane et al., unpublished obser­ vations; Winkler et al., 2009). The implication of these clinical and preclinical data is therefore that patients with severe LID may be less appropriate candidates for neural transplantation. In conclu­ sion, PD patients selected for neural transplanta­ tion should not be too severely affected by the disease and with DAergic degeneration restricted to the putamen in order to obtain maximum functional benefit. Prior to grafting the patients should have shown a good therapeutic L-dopa response, but little or no LID, in order to reduce the risk for the development of GID.

Is immunosuppression required? It is now well established that the immune privi­ lege of the brain is not complete. Thus, while intracerebral DAergic grafts may survive for long periods after transplantation, they may be infiltrated by immune cells as suggested by post­ mortem analyses from the two US controlled trials (Freed et al., 2001; Olanow et al., 2003). Beha­ vioral recovery after grafting may therefore have been affected by an ongoing immune response induced by either lack of immunosuppression, as in the Denver–Columbia trial (Freed et al., 2001), or by withdrawal of low-dose cyclosporine by 6 months after grafting and concomitant waning of the behavioral benefit, as was the case in the

304

Tampa–Mount Sinai trial (Freed et al., 2001; Olanow et al., 2003). In contrast, in the Lund– London–Marburg patients, triple immunosuppres­ sion was continued for at least 12 months after grafting and behavioral improvement continued after gradual reduction and eventual withdrawal of immunosuppressive drugs (Piccini et al., 2000; Wenning et al., 1997). GID has so far not been convincingly associated with an ongoing nonrejecting inflammatory process within and around the graft. Indeed, in a recent animals study we did not observe any AID following either rejection, or a non-rejecting inflammation, of an intracerebral DAergic graft (Lane et al., 2008). Nonetheless, while GID developed and peaked 6–12 months after grafting in the two NIH-sponsored trials (Freed et al., 2001; Olanow et al., 2003), the peak of GID was only achieved at 24–48 months after grafting in patients that had received longterm triple immunosuppression in the Lund cohort (Piccini et al., 2005). Interestingly, the severity of GID increased in these patients after withdrawal of immunosuppression, suggestive of a delayed immune reaction (Piccini et al., 2005). Post­ mortem studies have so far not been performed on any patients with GID, and while it is clear that patients receiving cell suspension grafts with very little infiltration of immune cells seen in post­ mortem analysis did not show any GID (Mendez et al., 2005, 2008), these behaviors have not been described in patients that do show some immune reaction (Kordower et al., 1997). Until more con­ clusive data is available, we consider it advisable that grafted patients should continue to receive immunosuppression for at least 1 year after trans­ plantation, in order to maximize potential benefit from the graft and reduce the risk of GID.

Tissue preparation, graft composition, and surgical protocols Collection, preparation, and storage of tissue used for transplantation has been diverse between centers (Winkler et al., 2005). Patients in the

Tampa–Mount Sinai trial received grafts of tissue that had been stored in so-called hibernation med­ ium for 2 days, and patients in the Denver–Colum­ bia trial received grafts after culture of the tissue for up to 4 weeks. Interestingly, the two patients with the highest GID scores in the Lund trials had received tissue that had been hibernated for 1–8 days, whereas the other patients with transplants of fresh tissue displayed lower GID scores (Hagell et al., 2002). Although none of the parameters for collection, preparation, and storage of tissue have so far been correlated with GID development, further standardization of these procedures will be required before transplantation may be safely used in larger numbers of patients. One of the first hypotheses of the cause of GID was the possibility of a DA “overload”, too many DAergic cells producing an excess of DA into the striatum (Freed et al., 2001). Subsequent 18 F-DOPA PET scans did not find excessive DA synthesis and storage in patients with GID (Hagell et al., 2002; Olanow et al., 2009). In one study (Ma et al., 2002) a detailed analysis of the PET scans revealed “hotspots” of 18F-DOPA uptake within the grafted area suggesting a non-homogeneous cell distribution may underlie the emergence of GID. However, examination of the PET scans in the Lund series of patients did not support these findings, with no evidence of excessive or focal DA release (Hagell et al., 2002; Piccini et al., 2005) (Tables 2A and 2B). Animal experiments have also suggested that the number of DA neurons in the graft, whilst important in the reduction of LID, is not directly related to the development of AID or GID (Table 3). Various studies have reported similar levels of AID, despite a range in the order of 2000–17,000 DAergic cells (Carlsson et al., 2006; Lane et al., 2006). Nevertheless, a grafting cannula and microsyringe has been developed which pro­ duces a more even distribution of cells throughout the graft area (Mendez et al., 2000b). This tool should ensure that cell distribution in future clinical studies is as homogeneous as possible. The inclusion of different DA cell types in the transplant composition, i.e., neurons of the A9 and

305

A10 lineage forming the developing substantia nigra and ventral tegmental area, respectively, has been under debate. These two populations have different distributions in the mature graft as observed in rodent transplants, with A9 neurons being located around the periphery with axonal projections into the striatum, whereas the smaller A10 neurons lie more centrally with limited axonal outgrowth beyond the perimeter of the graft (Kuan et al., 2007; Thompson et al., 2005). The number of grafted A9 neurons has been correlated with the degree of both functional recovery and reduction of LID in rodents (Kuan et al., 2007), but negative effects of either the A9 or the A10 cell population within a graft have not yet been observed. Selec­ tion of either cell population for grafting is unlikely to be a viable approach for the continuation of fetal cell transplantation. However, these observation do emphasize the importance of ensuring precise pre-differentiation of stem cell populations des­ tined for transplantation, not simply into generic DAergic neurons but into the precise regional phe­ notype required for appropriate patterns of rein­ nervation of their distinct forebrain targets (e.g., Girk2þ, calbindin, and CCK neurons characteris­ tic of A9 nigral neurons).

Can serotonin neurons play a role? One aspect of graft composition that has been studied in more detail with respect to the develop­ ment of GID is the inclusion of serotonin neurons into the transplant. Serotonin neurons develop in close proximity to the DA neurons of the ventral mesencephalon and some contamination of the graft tissue is unavoidable, the degree of which depends on the precise dissection parameters. Since grafts of serotonin neurons have been shown to worsen LID in a rat model of PD (Carlsson et al., 2007, 2008), and as serotonin neurons have been demonstrated in large num­ bers in post-mortem analysis of transplants in PD patients (Mendez et al., 2008), the role of seroto­ nin in GID has been examined by transplantation

of brainstem raphé neurons and by pharma­ cotherapy (Tables 2A and 2B). In one rodent transplant study, grafts contained large numbers of both DA and serotonin neurons, increasing striatal serotonin innervation by 300%, but this was not correlated with the extent of AID observed in these animals (Lane et al., 2006). Another study has shown that even when the number of serotonin neurons exceeds the DA neurons in the graft tenfold, there is no impact on the severity of AIDs development (Garcia et al., 2009). AIDs develop in rats grafted with DA neurons regardless of the presence of sero­ tonin neurons (Carlsson et al., 2007; Lane et al., 2009a), suggesting that the development of GID is dependent on the DA component, and not the serotonin component, of the graft. Nevertheless, there is an apparent modulation of AID through serotonergic mechanisms (worsened by 5-HT reuptake inhibition, improved by 5HT1A inhibi­ tion) (Lane et al., 2006, 2009a). Lesion of the serotonin-containing host raphé nucleus did not affect the severity of GID in the grafted animals, suggesting that this pharmacological effect is on the grafted tissue. Although the grafted serotonin neurons are unlikely to be involved in the development of AID in the rodent model, there are some recent observations that point to a possible role in the expression of GID in grafted patients. Politis and coworkers report two patients that had received VM transplants in putamen or in both putamen and caudate nucleus 13 and 16 years earlier under­ went PET scanning using a ligand for the seroto­ nin transporter (SERT) by 11C-DASB PET (Politis et al., 2010). In these patients UPDRS motor scores in “off” were gradually reduced by about 70%, and from the fourth and eighth year after surgery they no longer needed any dopami­ nergic medication and 18F-dopa and 11C-raclo­ pride PET showed dopaminergic restoration and DA release within normal levels in the grafted putamen. Both of them, however, developed mod­ erately disabling GID over time. 11C-DASB PET revealed excessive binding, 2–3-fold above

306

normal, in the grafted parts of the striatum (puta­ men in one case, and putamen and caudate nucleus in the other). Interestingly, the GIDs were almost completely abolished by systemic administration of a 5-HT1A partial agonist, buspir­ one, which dampens transmitter release from ser­ otonergic neurons, indicating that they were caused by the serotonergic hyperinnervation. These data, consistent with findings in the rat model of AID (Lane et al., 2006), suggest a role of excess serotonin neuron activity in the induc­ tion of GID, either by a direct amphetamine-like action of serotonin on the dopaminergic nerve terminals, or by dysregulated release of DA as a “false transmitter” from the serotonergic term­ inals. It is known that extracellular DA under certain conditions can be eliminated by re-uptake into serotonergic terminals where it competes with 5-HT for vesicular storage and release (Kannari et al., 2006; Saldana and Barker, 2004; Schmidt and Lovenberg, 1985). Due to lack of normal auto­ regulatory feedback the release of DA from sero­ tonergic terminals will be dysregulated, with abnormal swings, resulting in GID. Additionally, local injection of serotonin into the striatum has been shown to induce dose-dependent stereotypic behaviors that are mediated by release of endogen­ ous DA, probably via reversal of the DA transpor­ ter (Jacocks and Cox, 1992; Yeghiayan and Kelley, 1995; Yeghiayan et al., 1997). In the Politis et al. study, therefore, it is proposed that, in areas of serotonin hyper-innervation, these two mechanisms may co-operate to induce excess, dysregulated DA release causing GID, and that the effective block­ ade of GID by buspirone is explained by its ability to dampen serotonin neuron activity by activation of the inhibitory 5-HT1A autoreceptors.

Conclusions The search for the underlying cause of GID is complicated by the fact that we may not be dealing with a single phenomenon. Although differences in design of the clinical trials make inter-trial

comparisons difficult, it seems clear that the GIDs observed in the Denver–Columbia and Tampa–Mount Sinai trials differ with respect to their expression and clinical phenotype, although the time course is approximately similar. Further­ more, whilst patients from each trial have come to post-mortem analysis, none of those patients have had any significant level of GID, leaving open questions about graft distribution, cellular compo­ sition, and immune/inflammatory response in this subgroup. Experimental studies of GID are ham­ pered by the fact that spontaneous, graft-induced AIMs analogous to GIDs in patients are not observed in any of the animal PD models studied so far. Nevertheless, the study of AIDs in 6­ OHDA-lesioned rats has provided a useful tool for the study of mechanisms underlying a type of GID that resembles, in both its protracted devel­ opment and motoric expression, the dystonic– stereotypic type of GID seen in patients. Despite these shortcomings, we are making significant pro­ gress in determining the combination of factors that interact in the development of GID in patients. Today there is growing optimism that avoidance of these abnormal behaviors, and indeed improved functional efficacy, can be achieved by better standardization of the trans­ plantation protocol across centers, more wide­ spread, homogenous distribution of the graft cell suspension, and careful selection of the most sui­ table candidates for DA cell replacement, charac­ terized by a good therapeutic response to L-dopa in the absence of any significant or troublesome L-dopa-induced dyskinesia and DAergic denerva­ tion which is restricted to the caudate–putamen. A new joint European initiative chaired by Dr Roger Barker (Cambridge University) and funded by the Seventh Framework Program of the European Union, is now underway. This new multi-center clinical trial is designed to re-estab­ lish fetal cell transplantation as a safe and effective treatment for PD and thereby opening the way for future clinical trials of alternative stem-cell based therapies that will avoid the practical and ethical problems associated with the use of fetal tissue.

307

Acknowledgments We would like to thank Bengt Mattsson for his assistance in generating the figures for this manu­ script. Our own studies have been funded by the UK Medical Research Council, Parkinson’s Disease Society of Great Britain, Michael J Fox Foundation, Parkinsonfonden, Swedish Research Council, and German Parkinson’s disease Foundation. The authors declare no financial conflicts of interest. References Bjorklund, A. (1992). Dopaminergic transplants in experimen­ tal parkinsonism: Cellular mechanisms of graft-induced functional recovery. Current Opinion in Neurobiology, 2, 683–689. Carlsson, T., Carta, M., Munoz, A., Mattsson, B., Winkler, C., Kirik, D., et al. (2008). Impact of grafted serotonin and dopamine neurons on development of L-DOPA-induced dyskinesias in parkinsonian rats is determined by the extent of dopamine neuron degeneration. Brain, 132, 319–335. Carlsson, T., Carta, M., Winkler, C., Bjorklund, A., & Kirik, D. (2007). Serotonin neuron transplants exacerbate L-DOPA­ induced dyskinesias in a rat model of Parkinson’s disease. Journal of Neuroscience, 27, 8011–8022. Carlsson, T., Winkler, C., Lundblad, M., Cenci, M. A., Bjorklund, A., & Kirik, D. (2006). Graft placement and uneven pattern of reinnervation in the striatum is important for development of graft-induced dyskinesia. Neurobiology of Disease, 21, 657–668. Cenci, M. A., & Hagell, P. (2006). Dyskinesia and neural graft­ ing in Parkinson’s disease. In P. Brundin & W. Olanow (Eds.), Restorative Therapies in Parkinson’s Disease, 184–224. Cenci, M. A., Lee, C. S., & Bjorklund, A. (1998). L-DOPA­ induced dyskinesia in the rat is associated with striatal overexpression of prodynorphin- and glutamic acid decar­ boxylase mRNA. European Journal of Neuroscience, 10, 2694–2706. Cho, C., Alterman, R., Miravite, J., Shils, J., & Taglati, M. (2005). Subthalamic DBS for the treatment of “runaway” dyskinesias after embryonic or fetal tissue transplant. Move­ ment Disorder, 20, 1237. Cochen, V., Ribeiro, M. J., Nguyen, J. P., Gurruchaga, J. M., Villafane, G., Loc’h, C., et al. (2003). Transplantation in Parkinson’s disease: PET changes correlate with the amount of grafted tissue. Movement Disorder, 18, 928–932. de la Fuente-Fernandez, R., & Stoessl, A. J. (2002). The pla­ cebo effect in Parkinson’s disease. Trends in Neurosciences, 25, 302–306.

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Thompson, L., Barraud, P., Andersson, E., Kirik, D., & Bjork­ lund, A. (2005). Identification of dopaminergic neurons of nigral and ventral tegmental area subtypes in grafts of fetal ventral mesencephalon based on cell morphology, protein expression, and efferent projections. Journal of Neu­ roscience, 25, 6467–6477. Torres, E. M., Dowd, E., & Dunnett, S. B. (2008). Recovery of functional deficits following early donor age ventral mesencephalic grafts in a rat model of Parkinson’s disease. Neuroscience, 154, 631–640. Torres, E. M., Monville, C., Gates, M. A., Bagga, V., & Dunnett, S. B. (2007). Improved survival of young donor age dopamine grafts in a rat model of Parkinson’s disease. Neu­ roscience, 146, 1606–1617. Vinuela, A., Hallett, P. J., Reske-Nielsen, C., Patterson, M., Sotnikova, T. D., Caron, M. G., et al. (2008). Implanted reuptake-deficient or wild-type dopaminergic neurons improve ON L-dopa dyskinesias without OFF-dyskinesias in a rat model of Parkinson’s disease. Brain, 131, 3361–3379. Wenning, G. K., Odin, P., Morrish, P., Rehncrona, S., Widner, H., Brundin, P., et al. (1997). Short- and long-term survival and function of unilateral intrastriatal dopaminergic grafts in Parkinson’s disease. Annals of Neurology, 42, 95–107. Winkler, C., Garcia, J. A., Carlsson, T., & Nikkhah, G. (2009). Predictive value of severity of preoperative L-dopa-induced dyskinesia for the development of graft-induced dyskinesia in the rat Parkinson model. Society for Neuroscience Abstracts 430.19/I15 Winkler, C., Kirik, D., & Bjorklund, A. (2005). Cell transplan­ tation in Parkinson’s disease: How can we make it work? Trends in Neurosciences, 28, 86–92. Winkler, C., Kirik, D., Bjorklund, A., & Cenci, M. A. (2002). L-DOPA-induced dyskinesia in the intrastriatal 6-hydroxy­ dopamine model of Parkinson’s disease: Relation to motor and cellular parameters of nigrostriatal function. Neurobiol­ ogy of Disease, 10, 165–186. Winkler, C., Kirik, D., Bjorklund, A., & Dunnett, S. B. (2000). Transplantation in the rat model of Parkinson’s disease: Ectopic versus homotopic graft placement. Progress in Brain Research, 127, 233–265. Yeghiayan, S. K., & Kelley, A. E. (1995). Serotonergic stimulation of the ventrolateral striatum induces orofacial stereotypy. Pharmacology Biochemistry and Behavior, 52, 493–501. Yeghiayan, S. K., Kelley, A. E., Kula, N. S., Campbell, A., & Baldessarini, R. J. (1997). Role of dopamine in behavioral effects of serotonin microinjected into rat striatum. Pharma­ cology Biochemistry and Behavior, 56, 251–259.

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 16

Deep brain stimulation: state of the art and novel stimulation targets Francisco A. Ponce†,‡ and Andres M. Lozano
,† † Division of Neurosurgery, University of Toronto, Toronto Western Hospital,Toronto, ON, Canada Division of Neurological Surgery, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix, AZ,USA

‡

Abstract: Levodopa therapy represents a major breakthrough in the treatment of Parkinson’s disease (PD). As time and disease severity progresses, however, the shortcomings and adverse effects of this neurotransmitter replacement strategy become apparent and patients develop disabilities despite best medical therapy. The heightened awareness of these difficulties has given birth to a re-examination of functional neurosurgery for advanced PD. In the 20 years since the renewed interest in deep brain stimulation (DBS), approximately 60,000 patients with PD have undergone this surgery, with an annual accrual of 8000–10,000 new patients per year worldwide. Clinical studies have confirmed the beneficial effects of DBS surgery for the treatment of the cardinal motor features of PD. The likelihood of improvement, however, varies from symptom to symptom and from patient to patient. Surgery is very effective in reducing the motor fluctuations and dyskinesias—the primary reasons for patients’ intolerance to medical therapy. Other problems are less or non-responsive. Further, despite the widespread use of this technology, the mechanism through which DBS alleviates symptoms is not fully understood. This review will discuss the patient population most likely to benefit from surgery, what aspects of the disease are most responsive, the current limitations of DBS, and new therapeutic targets that are being examined to address these limitations. Keywords: deep brain stimulation; Parkinson’s disease; functional neurosurgery were ablative, producing lesions in the brain using open or stereotactic techniques in the subcortical structures, the basal ganglia, and the tha­ lamus. Since the 1950s, neurosurgeons have performed temporary and reversible focal inacti­ vations or modulations of the intrinsic neural ele­ ments prior to making lesions. These tests used a variety of strategies, including injections of local

Introduction Surgery for Parkinson’s disease (PD) initially focused on treating tremor. Early treatments
Corresponding author. Tel.: þ1-416-603-6200; Fax: þ1-416-603-5298; E-mail: [email protected]

DOI: 10.1016/S0079-6123(16)8401-6

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anesthetics, cooling, or electrical stimulation, to both predict efficacy and warn of potential adverse effects with permanent lesions. It is in the course of these tests that the therapeutic effects of electrical stimulation were characterized. Benabid expanded and pursued the observation that tremor was reliably arrested during highfrequency stimulation in the thalamus (1987, 1989). He applied the same principle to another structure, the subthalamic nucleus (STN), and showed improvement of tremor, rigidity, and bradykinesia, as well as a reduction in the required levadopa dose (Benabid et al., 1994). Others targeted additional structures, including sites in the basal ganglia, thalamus, and cortex, and their input and output pathways. The use of deep brain stimulation (DBS) became widespread in the late 1990s, and several large studies have subsequently confirmed the efficacy and safety profile of the procedure (Burchiel et al., 1999; Deuschl et al., 2006; Hariz et al., 2008; Krack et al., 2003; Rodriguez-Oroz et al., 2005). Recently, a multicenter randomized controlled trial of DBS versus medical therapy concluded that, at 6 months, the surgery was more effective in improving motor function, quality of life, and reducing dyskinesias (Weaver et al., 2009). DBS has become an established tool in the management of patients with advanced PD. This review highlights the benefits and limitations of DBS in various brain targets and discusses some of the understanding of the mechanism of action.

Benefits Unlike ablative procedures, adjustable settings for DBS allow for maximization of benefits and mini­ mization of side effects. This, combined with the low morbidity of the procedure, has contributed to the appeal of DBS. For such reasons, ablative procedures have now largely been replaced by DBS. The DBS targets most often used to treat PD are the STN and the globus pallidus internus

(GPi). The choice between GPi and STN continues to be evaluated, and the best target still needs to be defined through a large and well-designed controlled clinical trial. A current study comparing DBS to best medical therapy includes randomiza­ tion of the surgical arm to either GPi or STN, and the final results will include comparison of the two targets (Weaver et al., 2009). The STN and the GPi are both components of the cortico-basal ganglia-thalamo-cortical loop. The cardinal motor features of PD are thought to arise as a consequence of the pathological beha­ vior within this loop. With regards to tremor, bradykinesia, and rigidity, there is considerable overlap in the clinical effects from stimulation of these two structures, with both having a levadopa­ like effect on the motor symptoms (Ghika et al., 1998; Houeto et al., 2003; Krack et al., 2003; Kumar et al., 1998). Patients who are most likely to benefit from DBS have symptoms consistent with idiopathic PD, without evidence of multiple system involvement. The best candidates typically have a history of having responded well to levodopa, and the most accurate predictor of long-term improvements following DBS is the magnitude of the levodopa response, which is tested preopera­ tively (Charles et al., 2002; Kleiner-Fisman et al., 2003). In such patients, medication-related com­ plications render medical management no longer satisfactory (Lang et al., 2006b). Complications may include dose failures, delayed “on” periods, diphasic dyskinesias, sudden “on–off” effects, severe peak-dose dyskinesias, or deeper or more frequent “off” periods that cannot be readily con­ trolled with medical approaches (Fahn, 2008). When stimulation parameters are optimized and dopamine medication doses are adjusted, the effect of treatment is analogous to a physiologic “continuous dopamine stimulation”, such that the fluctuating benefits seen after drug intake before DBS are replaced by a stable improvement (Ahlskog, 2007). The improvement from DBS approximates the best levodopa response, wherein the optimal “on” response can be cap­ tured and sustained in the absence of medication.

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Rigidity and tremor have been found to improve by 70–75% and akinesia by 50% (Krack et al., 2003). In general, improvements occur in the off-medica­ tion state, and symptoms are either only moderately or not improved by DBS during the on-medication state (Kumar et al., 1998; Limousin et al., 1998; Marin et al., 2006; Rodriguez-Oroz, et al., 2005). Levodopa dose and dyskinesias DBS reduces medication-related motor symptoms by a variable amount. One large study of patients treated with either STN or GPi DBS demon­ strated increased percentage of time during the day with good mobility without involuntary move­ ments from 27% to 74% in the STN group and 28% to 64% in the GPi group (DBSPDSG, 2001). While STN DBS may produce direct release of striatal dopamine in experimental animals (Bruet et al., 2001), it does not appear to do so in patients with advanced PD (Hilker et al., 2003; Strafella et al., 2003), Instead, it seems to functionally sub­ stitute for the dopamine effect (Welter et al., 2002), which results in marked and sustained reductions in, or withdrawal of, antiparkinsonian medications in most patients, typically on the order of 30–69% (Deuschl et al., 2006; Kleiner-Fisman et al., 2006; Mihalko et al., 2003). In addition, the disability and duration associated with the dyski­ nesias are also reduced by two complementary mechanisms—reduction in drug intake, and a direct anti-dyskinetic effect (Herzog et al., 2003; Limousin et al., 1998; Ostergaard and Aa Sunde, 2006; Rodriguez-Oroz et al., 2004; Schupbach et al., 2005). The improvement of dyskinesias is greater and more sustained with GPi DBS (Anderson et al., 2005; Durif et al., 2002; Krack et al., 1998; Limousin and Martinez-Torres, 2008; Rodriguez-Oroz et al., 2005; Volkmann et al., 1998). However, there has been less concomitant reduction in levodopa dosage seen with GPi DBS, suggest­ ing that whereas STN DBS results in the reduction of dykinesias, at least in part, indirectly, by per­ mitting decreased levodopa doses, stimulation at

the GPi likely has a more pronounced direct antidyskinetic effect. Indeed, due to the improved medication tolerance provided by GPi DBS, levodopa dosage may actually increase following stimulation (Lozano and Mahant, 2004). Dramatic and early reductions of levodopa after STN DBS are not always desirable, and have been associated with complications such as dysarthia, apathy, or depression (Benabid et al., 2003; Krack et al., 2003; Lang et al., 2003; Limousin and Martinez-Torres, 2008). Given that PD results in widespread dopamine deficiency affecting systems beyond the basal ganglia, these disturbances may reflect an ongoing dopamine deficiency state in non-motor areas. The decreased need for dopami­ nergic therapy at the basal ganglia following DBS does not imply a decreased need for dopamine else­ where in the brain, and caution should be taken when lowering levodopa dosages after surgery. Quality of life After STN and GPi stimulation, patients can expect improvements in quality of life and activ­ ities of daily living (Lezcano et al., 2004; Lyons and Pahwa, 2005), though this effect is more pro­ nounced in the younger patients than in the elderly (Derost et al., 2007). One large rando­ mized study comparing DBS versus optimum drug therapy alone found that stimulation resulted in improvements between 24% and 38% in the PDQ-39 subscales for mobility, activities of daily living, emotional well-being, stigma, and bodily discomfort (Deuschl et al., 2006). Another study reported early and sustained improvements in HrQoL outcomes in patients with baseline disabil­ ity who were treated with either STN or GPi DBS (Volkmann, et al., 2009). These findings confirm results from previous uncontrolled studies which consistently reported greater improvements in these areas than in social support, cognition, and communication (Diamond and Jankovic, 2005; Lagrange et al., 2002). The reduction in dyskinesias is also independently associated with improvements

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that have a major effect on quality of life (Deuschl et al., 2006; Fraix et al., 2000), and the quality of life of caregivers has also been noted to improve (Lezcano et al., 2004). Other symptoms Axial symptoms, such as gait difficulty, freezing, imbalance, and posture, as well as non-motor symp­ toms, show a more limited, short-lived and variable response to STN DBS (Krack et al., 1999; Loher et al., 2002). Improvements occur in early-morning dystonia as well as in night-time akinesia, and these are believed to be indirectly responsible for improvements seen in sleep architecture and qual­ ity, with up to 47% increase in total sleep time being reported (Arnulf et al., 2000; Cicolin et al., 2004; Hjort et al., 2004). Non-motor symptoms such as constipation, sensory complaints, and bladder com­ plaints may also improve with surgery, and bladder control may be improved by decreasing detrusor hyperreflexia (Finazzi-Agro et al., 2003; Seif et al., 2004; Winge et al., 2007; Zibetti et al., 2007). How­ ever, when symptoms are refractory to levodopa, they tend to be refractory to DBS as well. Speech is generally less improved, and can actu­ ally worsen following DBS (Krack et al., 2003; Limousin et al., 1998; Rodriguez-Oroz et al., 2005). In one study, speech improved only during the first year and then progressively returned to baseline by 5 years (Kleiner-Fisman et al., 2006). Hypophonia might improve, but dysarthria can be aggravated due to current diffusion to corticobul­ bar fibers (Pinto et al., 2005). Reduction of dopa­ minergic medication may also be a contributing factor. Patient satisfaction, particularly with regard to hypophonia and ability to communicate with their family, can decline after surgery. Long-term outcomes and disease progression The course of patients undergoing STN DBS appears to closely resemble the natural history of

PD on medical treatment, and likely reflects the progression of the underlying disease rather than a side effect of the treatment (Mihalko et al., 2003; Ostergaard and Aa Sunde, 2006; Rodriguez-Oroz et al., 2005; Schupbach et al., 2005). A longitudinal PET study has shown continuous decline of dopa­ minergic function in patients with advanced PD after clinically effective bilateral DBS, with rates of progression within the range of previous studies of non-stimulated patients (Hilker et al., 2005). While the motor symptomatic improvement with DBS has been shown to be relatively stable, over the long term there is a slow decline in the overall benefit from DBS (Herzog et al., 2003; Ostergaard et al., 2002; Voon et al., 2006). The observation that the time course and incidence of symptoms, such as dementia, after surgery is about the same as that reported in medically treated patients with similar risk factors suggests that neither stimula­ tion of the STN nor the GPi alter the natural course of the disease (Aybek et al., 2007).

Contraindications Contraindications to functional neurosurgical inter­ vention include serious systemic medical co-morbi­ tidies such as unstable heart disease, active infection, disabling cerebrovascular disease, or malignancy associated with markedly reduced life expectancy (Lang et al., 2006a). Candidates for surgery should have symptoms consistent with idiopathic PD without evidence of extensive mul­ tiple system involvement, since DBS is generally not effective in patients with non-levodopa respon­ sive features. Cognitive and neurobehavioral disor­ ders may be aggravated by surgery and therefore these represent relative contraindications. For example, depression, while a common co-morbid condition in this patient population, may be a contraindication when despite optimal antide­ pressant therapy it markedly impacts on the over­ all functioning (Benabid et al., 2009). Patients with dementia have been shown to have less overall benefit from DBS surgery, and patients

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with cognitive impairment are at risk of cognitive worsening with surgery (Hariz et al., 2000; Limousin et al., 1998).

Limitations of therapy The recent pathological characterization of a-synuclein-positive inclusions has provided a neuroanatomical basis for the symptoms of PD that predate the motor phase of the illness, as well as the symptoms that appear later in the disease course. The availability of antibodies to a-synuclein has allowed the mapping out of inclu­ sion pathology throughout the neuraxis in patients with PD. There is a good correlation between the presence and distribution of such inclusions and the symptomatology in PD, and Braak has used this to categorize the disease into six stages (2003). This approach indicates early involvement of the brainstem and olfactory regions, which can occur several years before the appearance of motor symptoms. According to this staging model, invol­ vement of the substantia nigra pars compacta (SNc) would be a relatively late pathological development, not occurring until stage 3 of the disease, at which point motor manifestations begin to appear. The a-synuclein-positive inclu­ sions eventually involve large cortical fields during the fifth and sixth stages, and this may in turn explain some of the cognitive and higher mental disorders associated with PD. Early pre-motor symptoms include loss of smell, REM behavioral disorders (RBD), and constipa­ tion. These symptoms have been shown to be pre­ dictive of later emergence of the more classic motor features of the disease (Hantz et al., 1994), and con­ stipation by itself corresponds to an increased risk of developing PD by as much as 4.5-fold (Abbott et al., 2001). Patients with RBD have been found to fre­ quently have associated dopaminergic changes on PET imaging, even in the absence of parkinsonian features (Juri et al., 2005), and as many as 50% of patients with RBD go on to develop PD (Comella et al., 1998; Schenck et al., 1986).

In the evolution of the treatments for PD, as levodopa-sensitive motor symptoms are better controlled due to improvements in pharmacother­ apy and advances in surgical treatments, the bur­ den of disability in current PD patients is shifting towards the non-dopaminergic deficits that appear as the disease progresses. These include symptoms involving pathways outside of the cortico-basal ganglia-thalamo-cortical loop, such as freezing and falling, depression, cognitive disturbances, and autonomic dysfunction. The initial benefits seen in postural instability and gait after DBS are typically lost by 1 year, with clear-cut progres­ sion of such symptoms by 5 years (Mihalko et al., 2003). Major contributors to loss of quality of life late in the disease include disorders of cognition, dementia, mood, depression, disabilities of sleep and speech, as well as autonomic disturbances (Agid et al., 2003; Welter et al., 2002; Xie et al., 2001). Stimulation of the current targets does not appear to help these symptoms and, indeed, may aggravate such deficits. Thus, patients manifesting degeneration of additional systems may only receive brief benefit from DBS because quality of life will be greatly impaired by the progressing cognitive disorders.

Novel stimulation targets Dopaminergic motor symptoms represent only one facet of the disease, and as the chronological sequence of pathological events is becoming bet­ ter characterized, there is an increasing apprecia­ tion of both the so-called non-motor components of the illness as well as the non-dopaminergic components. These include disorders of sleep and cognition, depression, olfactory disturbances, autonomic disturbances, gait and postural distur­ bances. It has been challenging to delineate the neuroanatomical substrates of these aspects of PD (Table 1), but this indeed is the crucial step if effective therapies are to be developed. In the effort to treat the late-onset doparefractory symptoms, old targets, such as the

316 Table 1. Parkinsonian symptoms and putative candidate areas for intervention Symptoms Dopaminergic Motor, emotional, and behavioral disordersa,b Non-dopaminergic Motor (gait disorders, disequilibrium, slowness)c Dementia, cognitive dysfunctiona,f,g Emotional and behavioral disorders, depressionj,k Hallucinationsb,f Sleep disturbances/RBDj Autonomic dysfunctiond,e,k

Olfactory dysfunctionc Weight lossh Pain

Candidate areas for investigation and in theory intervention

STN, GPi, SNr, nigral projections to the striatum, intrinsic striatal dopaminergic neurons, thalamus (principal nuclei, reticular n.), mesocortical dopaminergic pathway, A8 DA projections PPNa, gigantocellular reticular nucleus, coeruleus–subcoeruleus complex , thalamic nuclei (e.g., CM/Pf), pre-SMA Anterior cingulate gyrus, superior frontal gyrus, temporal cortex, entorhinal cortex, amygdaloid complex, CA2 sector of the hippocampus Amygdala, cingulate cortex, raphe nuclei, locus coeruleus, mesolimbic, mesocortical, mesothalamic dopaminergic systems, nucleus accumbens, anterior limb of internal capsule, subcallosal cingulate gyrus Amygdala, limbic cortex, nucleus basalis of Meynert PPNa, coeruleus/subcoeruleus, thalamus, hypothalamus Hypothalamus, sympathetic system (intermediolateral nucleus of the thoracic cord, sympathetic ganglia), parasympathetic system (dorsal vagal and sacral parasympathetic nuclei), enteric nervous system (alimentary tract, cardiac plexus, pelvic plexus) Olfactory bulb, anterior olfactory nucleus, perirhinal cortex, amygdale Hypothalamus, nucleus accumbens Thalamus, dorsal root ganglia, spinothalamic tracts, medial lemniscus, mortor cortex, cingulate cortex, insula

a

Hirsch (1994),

Del Tredici et al. (2002),

c Harding et al. (2002b),

d Kalaitzakis, et al. (2009),

e Bosboom et al. (2004),

f Paulus and Jellinger (1991),

g Braak et al. (1995),

h Harding et al. (2002a),

i Arnulf et al. (2008),

j Wakabayashi and Takahashi (1997),

k Braak et al. (1994),

l Manfredsson et al. (2009).

b

centromedian-parafascicular (CM/Pf) complex of the thalamus, are being reassessed. The CM/Pf has been targeted for the treatment of pain (Weigel and Krauss, 2004), though the report of concomi­ tant improvement of tremor and dyskinesias in two patients (Krauss et al., 2002) has prompted further evaluation of this target in both animals (Kerkerian-Le Goff et al., 2009; Nanda et al.,2009) and humans (Benabid 2009; Stefani et al., 2009). In addition, new targets, such as the radiation prelemniscalis, the caudal zona incerta, and the pedunculopontine nucleus area (PPNa) are being explored.

One of the most disabling aspects within these non-dopaminergic disorders is the disturbance in postural functioning and gait disturbances that accompany PD. Ten years into the illness, 50% of PD patients are falling. Within 20 years, approxi­ mately 50% have suffered a fracture related to a fall (Latt et al., 2009). Loss of balance and the ability to walk is associated with loss of indepen­ dence and reduced quality of life (Schrag et al., 2000). Recent experimental evidence suggest that the PPNa, a region in the brainstem, may play an important role in such late-stage symptoms as gait and postural instability and may be a potential

317

surgical target for intervention (Mazzone et al., 2005; Plaha and Gill, 2005; Stefani et al., 2007; Weinberger et al., 2008). The PPNa has both cho­ linergic and glutaminergic components, and autopsy studies of patients with advanced PD have shown striking degeneration in this area that is in keeping with the degree of degeneration seen in the substantia nigra (Hirsch et al., 1987). Experiments in animals have shown that activat­ ing the neurons in this region either electrically or by using neuroactive substances can drive locomo­ tion (Pahapill and Lozano, 2000; Whelan, 1996). This area has been implicated in symptoms such as gait freezing, imbalance, multi-system atrophy, and progressive supranuclear palsy (Nandi et al., 2008). These findings have led to small pilot stu­ dies to assess the effects of chronic stimulation in this area in patients with PD. The results of these early studies suggest falls can be improved by stimulation (Ferraye et al., 2010; Pereira et al., 2008). In addition, an unexpected finding when stimulating this area is that there appears to be improvements in vigilance and in sleep (Ferraye et al., 2010). These changes are consistent with the PET data that show major activation of the reti­ cular activating system in the central median par­ afascicular nucleus (Kinomura et al., 1996).

Mechanism of action Despite two decades having passed since the introduction of DBS, the precise mechanism of action remains unclear (Gradinaru et al., 2009). The effects of stimulation of the STN and GPi are similar to the effects of ablative procedures, though the reversibility of stimulation indicates that the effect is not due to a permanent lesion. This has been supported by primate studies, show­ ing only 5% decrease in cell count after 7 months of high-frequency stimulation (Wallace et al., 2007), as well as in postmortem studies in humans, showing little tissue damage from stimulation (Haberler et al., 2000). The similarity to ablative procedures suggests to some that DBS produces a

functional inhibition-like effect at both targets. Increasing evidence suggest that this is not so, and that the effects of DBS are complex and vary across the precise neural elements targeted and the parameters of stimulation used. Biochemical, metabolic, and electrophysiologi­ cal data in experimental models and in patients, together with modeling studies, provide consistent evidence in favor of activation (Hammond et al., 2008). Stimulating through an electrode is known to result in activation of nearby neurons or axons in the internal capsule, resulting in tonic contrac­ tion rather than paresis (Gorgulho et al., 2009), and stimulation of the STN in parkinsonian rhesus monkeys has been shown to produce predomi­ nantly activation of downstream targets (Hashi­ moto et al., 2003). Consistent with this notion, STN DBS in rodents releases glutamate at its target nuclei (Bruet et al., 2001). Further, the clinical effects of PPNa DBS seem to reflect acti­ vation (Limousin and Martinez-Torres, 2008), which is accomplished only at lower frequencies (Munro-Davies et al., 1999; Nandi et al., 2002) and mimicked by direct injection of agents that acti­ vate neuronal cell bodies (Miyazato et al., 1999). Increased cortical coherence in the beta band (12–30 Hz) has been found to correlate with the severity of symptoms in humans, suggesting that PD symptoms may result from pathological syn­ chronized oscillations that produce functional alterations in the basal ganglia network (Gatev et al., 2006; Hammond et al., 2007; Uhlhaas and Singer, 2006). Rather than inhibiting the target nucleus, high-frequency stimulation may result in orthodromic activation of target neurons of the STN, replacing the pathological activity by a sti­ mulus-driven firing pattern (Hammond et al., 2008). Such an effect has been seen in the GPi and substantia nigra pars reticulata (SNr) neurons in response to STN DBS (Galati et al., 2006; Mal­ tete et al., 2007). By locking electrophysiological activity to the harmonics of the stimulation fre­ quency, DBS may replace spontaneous pathologi­ cal synchronized group discharges and oscillations with noise that does not interfere with normal

318

cortical activity (Chomiak and Hu, 2007; McIntyre et al., 2004). Recent experiments have shown that disrupting such oscillations through stimulation of spinal sensory pathways is possible, suggesting a less invasive strategy to alleviate parkinsonian symptoms (Fuentes et al., 2009). DBS may also act through activation or inhibi­ tion of axons that project to the target nuclei, rather than on the nuclei themselves. By using optogenetics and solid-state optics to selectively activate or inhibit various targets in parkinsonian rodents, one study demonstrated that the thera­ peutic effects within the STN could be accounted for by direct selective stimulation of afferent axons projecting to this region (Gradinaru et al., 2009). Other studies have shown antidromic acti­ vation of afferent neurons to the STN, retrogra­ dely affecting cortical circuits and nuclei that send axons to, or close to, the stimulated site (Florio et al., 2007; Hammond et al., 1983). With this in mind, the best site of implantation of the DBS electrode may be in a region where the stimulation-driven activity spreads to most of the identified, dysrhythmic, neuronal populations, while preserving transmission of cortical informa­ tion and without causing additional side effects. Given the strategic position occupied by the STN as the only glutamatergic center of the basal gang­ lia network, receiving afferents from motorrelated cortical areas, and projecting to all nuclei of the basal ganglia, the effects of STN stimulation may be widespread due to activation of diverse pathways (Hammond, et al., 2008). The observation that STN DBS in levodopa­ responsive PD results in large reductions or dis­ continuation of levodopa has prompted investiga­ tion into the mechanisms by which STN DBS apparently amplifies the effect of the drug. Microdialysis studies of extracellular dopamine striatal concentrations in rats with SNc lesions have shown that DBS results in a twofold increase in levodopa-induced dopamine, suggesting a syner­ gistic interaction with levodopa (Lacombe et al., 2007). This effect may result from direct modula­ tion of the firing rates of the remaining

dopaminergic neurons (Lee et al., 2004; Meissner et al., 2003). In addition, some groups have started examining a possible neuroprotective effect of DBS in rats and primate models (Temel et al., 2006; Wallace et al., 2007), and others have hypothesized that early surgical interventions could reduce nigral degeneration (Rodriguez et al., 1998), though, to date, neuroprotection has not been clinically demonstrated. Finally, future progress in this field will require increasing our understanding of the cellular and molecular consequences of DBS. For example, recent observations suggest that DBS can alter excitability and influence plasticity (Prescott et al., 2009) and also drive neurogenesis, at least in the adult rodent hippocampus (Toda et al., 2008).

Conclusions DBS offers relief of the cardinal symptoms similar to the best response to levodopa, without the adverse side effects. Good candidate patients for DBS surgery continue to respond to levodopa but are disabled by the motor complications of medi­ cation therapy. The clinical benefits of DBS are likely due to disruption of the pathological activity in the cortical-thalamic-basal ganglia-cortical motor loop, and symptoms unrelated to this circuit, such as mood, cognition, gait, and posture, are typically not improved with DBS. As patients live longer and the disease progresses, these non-dopaminergic symptoms are increasingly becoming the main drivers of disability. Such symptoms present a new therapeutic challenge, and the future char­ acterization of the underlying anatomical and physiological causes of these features may lead to the identification and exploration of new ther­ apeutic targets. Acknowledgments AML is a Canada Research Chair (tier 1) in Neuroscience.

319

Abbreviations DBS GPi HrQoL PD PDQ-39 PET RBD REM SNc SNr STN UPDRS

deep brain stimulation; globus pallidus interna; health-related quality of life survey; Parkinson’s disease; 39 question Parkinson’s Disease Questionnaire; positron emission tomography; REM behavioral disorder; rapid eye movement; substantia nigra pars compacta; substantia nigra pars reticulata; subthalamic nucleus; Unified Parkinson’s Disease Rating Scale

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 17

The challenge of non-motor symptoms in Parkinson’s disease K. Ray Chaudhuri† and Per Odin‡ †

National Parkinson Foundation Centre of Excellence, Kings College Hospital and University Hospital Lewisham;

and Kings College and Institute of Psychiatry, London, UK

‡ Department of Neurology, Skane University Hospital, Lund, Sweden and Department of Neurology,

Central Hospital Bremerhaven, Germany

Abstract: The non-motor symptoms (NMS) of Parkinson’s disease (PD) are often poorly recognized and inadequately treated in contrast to motor symptoms and a modern holistic approach to treatment of PD should, therefore, include recognition and assessment of NMS. Certain aspects of the NMS complex of PD can be improved with currently available treatments, both dopaminergic and non-dopaminergic, but other features may be more refractory illustrating the importance of research into more effective drug therapies for the future. The American Academy of Neurology has recently published the first task force guidelines in relation to treatment of NMS of PD. Keywords: Non-motor; Non-motor symptoms quest; Non-motor symptoms scale; Parkinson’s disease; Rating scales

two ‘holistic’ prevalence studies of NMS in PD, the NMSQuest study in 2007 and the PRIAMO study in 2009, both indicating the importance of NMS and its presence across all stages of common PD (Barone et al., 2009; Martinez-Martin et al., 2007). These NMS significantly impair quality of life and may precipitate hospitalization and strongly contri­ bute to the cost of care for PD. NMS create a significant burden for people with PD and care­ givers, and studies suggest that NMS are a greater determinant of quality of life than motor features.

Introduction Non-motor symptoms (NMS) in Parkinson’s disease (PD) are common across all stages of PD but often receive little attention as NMS-related issues are often overshadowed by the motor symptoms. In spite of their importance there have been only
Corresponding author. Tel.: þ44-207-346-8336; Fax: þ44-208-333-3093; E-mail: [email protected]

DOI: 10.1016/S0079-6123(10)84017-8

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(Martinez-Martin et al., 2009). Some NMS, such as depression, dementia, dysautonomia, and sleep problems, are well established but others such as dysphagia, dribbling of saliva, weight changes, sex­ ual problems, and diplopia are poorly recognized. In recent years, a self-completed NMS question­ naire and NMS scale has been validated specifically for use in PD and a recent international survey showed that up to 62% of NMS in PD might remain undeclared to healthcare professionals because patients may be unaware that the symp­ toms are linked to PD. (Chaudhuri et al., 2010). In addition to the disease itself, some NMS can be precipitated by dopaminergic treatment of PD. Finally, non-motor fluctuations frequently compli­ cate treatment, the symptoms occurring when there is low level of central dopaminergic stimula­ tion (Muzerengi et al., 2007).

Pathogenesis It is clear that several NMS have a relatively poor response to dopaminergic therapy, and other pathways including the serotonergic and noradre­ nergic ones are involved (Zgaljardic et al., 2004). Braak et al. have introduced the concept of a sixstage pathological process, beginning at induction sites with degeneration of the olfactory bulb and the anterior olfactory nucleus (clinically manifest as olfactory dysfunction) at stage 1, while stage 2 reflects progression of the pathological process to the lower brainstem (Braak et al., 2003; Del Tre­ dici and Braak, 2004). The latter involves brain­ stem nuclei, which are thought to be key areas mediating NMS such as olfaction, sleep homeos­ tasis, depression and cognition, pain, constipation, and central autonomic control. Several of these symptoms are now recognized as possible premotor features of PD. The typical clinical motor triad of PD emerges at Braak stages 3 and 4 with the involvement of the substantia nigra. In line with the Braak staging, several NMS have been identified before the motor syndrome of PD emerges (Chaudhuri et al., 2006a). However, the

Braak staging is not without controversy as the concept relies on Lewy body deposition and not neuronal degeneration and also does not fully explain why cognitive problems occur early, for instance, in dementia with Lewy bodies.

Non-motor symptoms can predict the emergence of motor symptoms of PD It is well established that NMS of PD can present at any stage of the disease including a “pre-motor stage”. Prospective data suggest that at least four distinct NMS of PD such as olfactory problems, rapid eye movement behavior disorder (RBD), constipation, and depression may predate the motor signs (Abbot et al 2001; Berendse, 2006; Boeve et al., 2001; Chaudhuri et al., 2006a). Other symptoms include visual disturbances such as color vision disturbances and impairment in contrast sensitivity, apathy, constipation, central pain, excessive daytime sleepiness (EDS), and erectile dysfunction (Chaudhuri et al., 2006a). Among six NMS of PD, Diederich et al. have reported that visual dysfunction may have the best predictive discriminative power in early PD in a case–control study (Diederich et al., 2010). Early longitudinal studies suggest that NMS may appear early in the course of PD and become more prominent as the disease progresses, often dominating the later stages of the disease like in the 20-year follow-up study of the Sydney group (Hely et al., 2008). It is investigated if these “pre­ motor NMS” such as olfactory dysfunction in com­ bination with other symptoms such as RBD or constipation may open a possibility to identify a “Parkinson at risk” population, an issue particu­ larly clinically relevant if neuroprotective thera­ pies would become available (Table 1). Olfaction Olfactory dysfunction may affect up to 90% of PD patients. In 1975, Ansari and Johnson suggested

327 Table 1. The non-motor symptom complex of Parkinson’s disease (taken from: Metta et al. (2010)) Spectrum of non-motor symptoms in PD. Symptoms in italics indicate poorly understood or lesser known symptoms. MCI = minimal cognitive impairment Neuropsychiatric symptoms Depression

Anxiety

Apathy

Hallucinations, delusions, illusions

Delirium (may be drug-induced)

Cognitive impairment (Dementia, MCI)

Dopamine dysregulation syndrome (drug induced)

Impulse control disorders (drug induced)

Panic attacks Sleep disorders and symptoms REM sleep behavior disorder (possible pre-motor)

Excessive daytime somnolence, narcolepsy-type “sleep attack”

Restless legs syndrome, periodic leg movements

Insomnia

Sleep disordered breathing Non-REM parasomnias Fatigue Central fatigue Peripheral fatigue Sensory symptoms Pain Olfactory disturbance Visual disturbance (blurred vision, diplopia), impaired contrast-sensitivity Autonomic dysfunction Bladder urgency, frequency, nocturia Sexual dysfunction (may be drug-induced) Sweating abnormalities (Hyperhidrosis) Orthostatic hypotension Gastrointestinal symptoms Dribbling of saliva Dysphagia Constipation Nausea Vomiting Reflux Fecal incontinence Drug-induced NMS Hallucinations, delusions Dopamine dysregulation syndrome Impulse control disorders (e.g., compulsive gambling, hypersexuality, binge eating) Non-motor fluctuations Dysautonomic Cognitive/psychiatric Sensory/pain Other symptoms Weight loss

Weight gain (may be drug-related)

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the association between olfactory dysfunction and development of PD and subsequently olfactory dysfunction was established as a preclinical marker for PD (Ansari and Johnson, 1975; Doty et al., 1992; Hawkes, 2003; Montgomery et al., 1999; Ponsen et al., 2004; Ross et al., 2005; Side­ rowf et al., 2007). Studies have also reported olfac­ tory deficits in asymptomatic relatives of patients with PD, some of whom later became sympto­ matic (Siderowf et al., 2007). A longitudinal study of 2263 elderly men between 1991 and 1996 by Ross and colleagues assessed olfaction using a 12-odor smell identification test and reported an association between impaired olfac­ tion and incident PD (Ross et al., 2005). Both olfactory dysfunction and RBD have been reported in other disorders with abnormal synu­ clein pathology such as diffuse Lewy body disease (LBD) and multiple system atrophy (MSA). Non­ synucleinopathies, such as vascular parkinsonism, corticobasal degeneration, progressive supranuc­ lear palsy, and parkin-associated PD, tend to have intact olfactory function (Khan et al., 2004). Hyposmia however, appears to be common in genetic PD associated with the LRRK 2 mutation (Clarimon et al., 2008) and also in communitydwelling elderly people with mild parkinsonian signs (Clarimon et al., 2008). Lerner and Bagic have proposed that the pathology of PD may involve a pathogen which causes an anterograde spread from the olfactory bulb and nucleus through the primary, secondary, and tertiary con­ nections of the olfactory structures (Lerner and Bagic, 2008). REM behavior disorder (RBD) RBD is affecting REM sleep characterized by loss of the normal skeletal muscle atonia during REM sleep, thus enabling patients to physically enact often vivid and unpleasant dreams (Olson et al., 2000; Schenck et al., 1996). Like constipation and olfactory disturbance, RBD may precede the development of the motor signs of PD and data

suggest that RBD may precede the onset of motor symptoms in over 40% of PD patients (Chaudhuri et al., 2006a; Schenck et al., 1996). Tonic REM sleep atonia may be more predictive of future development of parkinsonism than phasic REM sleep atonia (Postuma et al., 2010). Imaging stu­ dies in patients with isolated RBD have indicated a small but significant symmetrical reduction in striatal dopaminergic uptake, which may be sug­ gestive of preclinical PD, and more recently patients with RBD have been studied with a com­ bined approach using olfactory testing, transcra­ nial ultrasound, and FP-CIT imaging (Eisensehr et al., 2000; Stiasny-Kolster et al., 2005; Unger et al., 2008). Uchiyama and colleagues reported an autopsy-proven case with the presence of inci­ dental striatal Lewy bodies in a patient with RBD for 20 years but no clinical evidence of PD (Uchiyama et al., 1995). It is suggested that that RBD may arise due to the degeneration of lower brainstem nuclei including the pedunculopontine and subcoeruleal nucleus areas affected in Braak stages 1 and 2 (Wolters and Braak, 2006). The sublatero-dorsal nucleus is thought to be the key regulatory center for RBD generation. The co­ occurrence of sleep disturbances and olfactory deficit in PD can be explained by Braak’s hypoth­ esis and in a community-based study, Henderson et al. reported that more patients with olfactory deficits than controls had excessive daytime som­ nolence (45 vs. 6%), restless legs (50 vs. 19%), abnormal movements during sleep (34 vs. 0%), and these generally occurred 3–5 years after diag­ nosis and were independent of mood disorders and drug therapy (Henderson et al., 2003). Depression Depression is one of the most common psychiatric complication of PD and typically affects around 40% (10–70%) of PD patients (Aarsland et al., 1999; Burn, 2002; Lauterbach, 2004; Thanvi et al., 2003). Depression may arise as a result of damage to serotonergic as well as limbic

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noradrenergic and dopaminergic neurotransmis­ sion. Remy et al. showed using 11C-RTI-32 PET—an in vivo marker of dopamine and NA transporter binding—that depressed PD cases had reduced 11C-RTI-32 PET binding versus non-depressed cases (in anterior cingulate, amyg­ dala, and the ventral striatum) along with reduced fluorodopa binding (Remy et al., 2005). In a recent study it has been reported that, in response to citalopram treatment, patients with solely major depression exhibited an expected decrease in 5-HIAA and MHPG levels which was not found in PD patients with major depression (Pålhagen et al., 2010). There were also other results in this study indicating that the biochemical basis and the response to citalopram differ between PD patients with major depression and patients with solely major depression. A cross-sectional study has reported an association between elevated plasma homocysteine levels, depression, and cognitive impairment in PD (O’Suilleabhain et al., 2004). Other studies have suggested that depression, like RBD and hyposmia, may precede the devel­ opment of PD (Lauterbach et al., 2004; Montgom­ ery et al., 2000; Nilsson et al., 2001; Schurmann et al., 2002;). Nilsson et al. reported that depressed patients are more likely to develop PD than osteoarthritis or diabetes (Nilsson et al., 2001), while a retrospective cohort study by Schurmann et al. reported that at the time of diagnosis of idiopathic PD, 9.2% had a lifetime diagnosis of depression compared to 4.2% in the controls (Schurmann et al., 2002). A PD test battery devel­ oped by Montgomery et al. (2000) combined tests for depression (Beck Depression Inventory), olfactory testing, and a simple motor task of wrist flexion and extension showed significant impairment in first-degree relatives of PD from controls (Montgomery et al., 2000) (Table 2).

Table 2. A list of non-motor symptoms suggested as preclinical (motor) feature in PD Constipation Olfactory deficit: (discrimination) REM behavior disorder Depression Possible links Restless legs syndrome Apathy Fatigue Anxiety Pain Male erectile dysfunction Visual disturbances (color vision)

oesophagal dysmotility can occur in advanced PD. Constipation is a common NMS of PD and may occur early and even precede development of PD (Abbot et al., 2001). Abbott et al. reported a prospective study which followed the bowel habits of 7000 men for 24 years and reported that those with initial constipation (< 1 bowel movement/ day) had a threefold risk of developing PD after a mean interval of 10 years from initial constipa­ tion (Abbot et al., 2001). Involvement of the dor­ sal vagal nucleus, as would occur in Braak stage 1, may explain the pre-motor appearance of consti­ pation (Wolters and Braak, 2006). However, a study by Benarroch et al. showed that there appears to be no correlation between the degree of cell loss or Lewy body counts in the dorsal vagal nucleus and the severity of constipation in PD (Cersosimo and Benarroch, 2008). In PD, there is severe loss of both central and colonic dopami­ nergic neurons, although constipation in PD does often not respond well to dopaminergic treatment (Metta et al., 2010). Prevalence of non-motor symptoms of PD

Constipation Gastrointestinal symptoms are common in PD and many such as dysphagia, dribbling of saliva,

A recent international study validating a self-com­ pleted non-motor questionnaire (NMSQuest) by Chaudhuri and colleagues revealed that NMS are highly prevalent in PD patients compared with

330

age-matched controls, and a typical patient reported 10–12 NMS (Chaudhuri et al., 2006b). This study also showed that NMS can occur in all disease stages and the number of symptoms corre­ lates with disease duration and severity (Chaud­ huri et al., 2006b). The NMS complex of PD is frequently unrecognized by healthcare profes­ sionals. In a prospective study of 101 patients, Shulman and colleagues demonstrated that neu­ rologists failed to identify major NMS such as depression, sleep disturbances, anxiety, and fati­ gue in more than 50% of the patients (Shulman et al., 2002). NMS symptoms can easily be missed as there is a tendency to concentrate on the motor aspects of PD. Frequently, physicians are unaware that NMS are related to PD (Chaudhuri et al., 2006b). In some cases the symptoms are not declared to the healthcare professional (Chaudhuri et al., 2006b). This might be because the patient does not know that the symptom is connected to PD or that she/he feels embarrassed to bring it up (for example, regarding incontinentia and sexual problems). Recently the first comprehensive clinic-based NMS questionnaire (NMSQuest), which allows easy identification of NMS by the physician, has been validated (Chaudhuri et al., 2006b). The NMS scale, which rates the symptoms in terms of frequency and severity, has also been validated in two major international studies in over 600 patients (Chaudhuri and Martinez-Martin, 2008; Martinez-Martin et al., 2009). The PRIAMO study has addressed NMS and quality of life in over 1000 patients across Italy and report high prevalence of several NMS such as sleep pro­ blems, pain, apathy, fatigue in PD across all stages (Barone et al., 2009). The symptoms: cognition The cognitive/neuropsychiatric NMS of PD range from anxiety state, apathy, and depression to frank dementia (Aarsland et al., 1999; Lauterbach, 2004; Shiba et al., 2000; Weisskopf et al., 2003). Psychosis is the key factor requiring nursing-home

placement, and depression causes a significant negative impact on the quality of life in PD (Aars­ land et al., 2000; Findley et al., 2003; Schrag et al., 2000). Anxiety, apathy, and fatigue Anxiety disorders are common in PD and may also be a preclinical risk factor (Shiba et al., 2000; Weisskopf et al., 2003). Several types of panic disorders have been described in PD but the most common forms are panic disorders, social phobia, and generalized anxiety disorder (Richard, 2005). The disorder may also be related to drug-induced NMS of PD (Singh et al., 2005). Apathy has now been established as a distinctive symptom of PD and is more common in PD patients than equally disabled osteoarthritic patients, indicating a neurodegenerative contribu­ tion (Pluck and Brown, 2002; Starkstein et al., 1992) Apathy may respond to dopaminergic med­ ication, such as levodopa, but involvement of other neurotransmitter pathways is likely (Brown and Pluck, 2000). Fatigue is related to depression and EDS, but cannot be explained by this comor­ bidity alone. In more than half of the patients mental fatigue is persistent and seems to be an independent symptom. Fatigue – both physical and mental – seems to be more common in PD patients compared to control populations (Alves et al., 2004; Lou, 2009). Psychosis and visual hallucinations Up to 40% of patients experience visual hallucina­ tions (Diederich et al., 2005). While visual hallu­ cinations are commonly viewed as a side effect of anti-PD treatments, neuronal degeneration itself may be causative (Fenelon et al., 2000). Delirium may occur in advanced dementia or may be induced by concurrent infection or in association with Parkinsonism—hyperpyrexia or neuroleptic malignant syndrome (Kipps et al., 2005). Onofrj

331

and colleagues have cited RBD as a possible risk factor for hallucinations in a follow-up study of PD patients with RBD and other risk factors include cognitive impairment, age, and duration of PD (Onofrj et al., 2002). Dopaminergic drugs can induce psychiatric symptoms by disrupting sleep, leading to vivid dreams, hallucinations, and finally delirium (Moskovitz et al., 1978). Cognitive dysfunction Dementia complicates up to 40% of PD cases, a rate approximately six times higher than normal healthy subjects (Emre, 2003). Hely and collea­ gues reported cognitive decline with rates as high as 84% in their long-term (15–18 years) follow-up study of PD patients (Hely et al., 2005). Dementia in PD is similar to that caused by lesions of the prefrontal cortex, characterized by a dysexecutive syndrome with impairment of visuo-spatial abil­ ities and memory on a background of loss of response to dopaminergic drugs (Aarsland et al., 2004; Apaydin et al., 2002). Nigral cellular degen­ eration, loss of cholinergic cells in the nucleus basalis of Meynert, and the presence of cortical and subcortical Lewy bodies have been implicated (Mattila et al., 2000). Like Alzheimer’s disease, hippocampal volume is diminished in PD with dementia (Laakso et al., 1996). Nocturnal NMS Nearly all PD patients have sleep disturbances which usually starts early in the disease (Chaud­ huri, 2003; Garcia-Borreguero et al., 2003; Lees et al., 1988).The pathogenesis of sleep disruption is multifactorial but degeneration of central sleep regulation centers in the brainstem and thalamo­ cortical pathways is likely to be important. The pedunculopontine nucleus, locus coeruleus, and the retrorubral nucleus influence normal REM atonia and phasic generator circuitry and have been implicated in the pathogenesis of RBD (Lai

and Siegel, 1990; Rye and Jankovic, 2002;). Other factors that may contribute to sleep disruption include motor symptoms, anxiety and depression, and dopaminergic treatment. Some NMS cause abnormalities in the primary sleep architecture and have a secondary effect on the quality of sleep, such as nocturia. Obstructive sleep apnea, not necessarily associated with obesity, and a nar­ coleptic pattern of rapid onset of sleep are also important causes of sleep-related morbidity in PD (Arnulf et al., 2002; Lees et al., 1988). Excessive daytime sleepiness EDS and involuntary dozing affects up to 50% of PD patients and may be a preclinical marker (Arnulf et al., 2002; Dhawan et al., 2006). EDS is important to recognize as it may considerably impact quality of life in PD (MacMahon, 2005). EDS is associated with poor concentration and memory, which may result in road accidents and accidents at work (Saper et al., 2001). Recently a secondary narcoleptic phenotype (narcolepsy without cataplexy) has been suggested in PD and may be linked to degeneration of hypocretin­ containing neurons in the hypothalamus (Haq et al., 2010). Saper and colleagues proposed the concept of a flip-flop switch, which is responsible for the sleep–wake cycle in primates (Saper et al., 2001). Dopaminergic dysfunction and neuronal degen­ eration can destabilize the switch and its regula­ tors, promoting rapid transitions to sleep. Dysautonomia and PD Autonomic dysfunction is associated with various movement disorders, most commonly PD and MSA (Chaudhuri, 2001). The symptoms of dysautonomia in PD may include orthostatic hypotension, bladder dysfunction, gastrointestinal dysfunction (particu­ larly constipation), sexual dysfunction, and hyperhi­ drosis. The pathophysiology is complicated and is thought to include dysfunction or degeneration of

332

the nucleus ambiguus, the dorsal vagal nucleus, and various medullary centers that modulate the activity of the sympathetic preganglionic neurons (these include the rostral ventrolateral medulla, ventrome­ dial medulla, and the caudal raphe nuclei) (Benar­ roch, 1999). Modulation of the central autonomic network is thought to be disrupted through degen­ eration of cholinergic, monoaminergic, and seroto­ nergic nuclei (Benarroch, 1999). Magerkurth and colleagues reported a statistically significant increase of orthostatic dizziness, bladder dysfunc­ tion (mainly urge incontinence and frequency), hyperhidrosis, and erectile dysfunction in PD patients compared with the controls (Magerkurth et al., 2005). Reports of bowel dysfunction, includ­ ing constipation and feeling full, were also increased in PD patients, but this was not statistically signifi­ cant (Magerkurth et al., 2005). Approximately 50% of PD patients rated the impact of the autonomic symptoms on their daily lives as “a lot” or “very much” (Magerkurth et al., 2005). The NMSQuest study also confirmed that dysautonomic symptoms were significantly more prevalent in PD patients than in controls (Chaudhuri et al., 2006b). Gold­ stein and colleagues used beat-to-beat blood pres­ sure measurements during performance of the Valsalva manoeuvre to detect sympathetic neuro­ circulatory failure in PD patients, and 6-[18F] fluor­ odopamine to estimate sympathetic cardiac innervation. They found that all nine of the PD patients who had sympathetic neurocirculatory fail­ ure, and 11 of the 15 PD patients who did not have neurocirculatory failure, demonstrated sympathetic cardiac denervation, relative to controls. This con­ trasted dramatically with the results from MSA patients, who did not show evidence of sympathetic cardiac denervation. The results suggest that car­ diac denervation is not related to severe or latestage disease and catecholamine function in PD may be defective not only in the brain but also in the heart (Goldstein et al., 2000). Meta-[123I] iodo­ benzylguanidine (MIBG) is a noradrenaline analog, taken up by postganglionic sympathetic neurons, and has been used to analyze the sympathetic car­ diac activity in PD patients with orthostatic

hypotension. It has been found to be reduced in patients with PD, both with and without orthostatic hypotension (Braune et al., 1999). This often differs from MSA, in which cardiac MIBG uptake is usually normal. Nocturia frequency and urgency are common complaints in PD patients and func­ tional imaging studies show that dopaminergic mechanisms may be involved in bladder control (Winge and Fowler, 2006). Fatigue Fatigue is a common complaint in PD and is reported to have a negative impact on the quality of life (Herlofson and Larsen, 2003). Fatigue may be related to other non-motor features of PD such as depression, sleep disturbances, and dementia (Karlsen et al., 1999). However, studies have shown that fatigue does occur independently of other NMS (Alves et al., 2004). Recent data invol­ ving positron imaging tomography in PD suggest that abnormalities of serotonin binding protein and limbic dopaminergic dysfunction may be responsible (Pavese et al., 2010). Sexual dysfunction Both reduced and increased sex drive has been reported in PD (Brown et al., 1990), and it is thought that this may represent another dysautonomic symp­ tom of the disease. One of the most common pro­ blems is erectile dysfunction, occurring in up to twothirds of the male PD population. Hypersexuality and other forms of aberrant sexual behaviors and drive are part of the impulse control disorder, which occurs with dopaminergic drug treatment, especially dopamine agonists (Pezzella et al., 2005). Pain Pain is one of the major clinical symptoms in PD but its pathophysiology is unclear. Aspects of pain

333

in PD may be mediated through a medial and lateral pain pathway, which relay via the parabra­ chial nucleus and locus coeruleus projecting to the secondary somatosensory cortex and the anterior cingulated cortex (Scherder, 2006; Waseem and Gwinn-Hardy, 2001). In early PD these pathways may be involved and this pain is also recognized as a possible pre-motor sign of PD (Wolters and Braak, 2006). Various pain syndromes described may be related to motor fluctuations, early-morn­ ing dystonia, or secondary causes such as muscu­ loskeletal pain (Goetz et al., 1986; Quinn et al., 1996). Chaudhuri and Schapira have proposed a modern classification of pain in PD (Chaudhuri and Schapira, 2009) which include central, periph­ eral, orofacial, and fluctuation-related pain.

Non-motor fluctuations Traditionally fluctuation of response to therapy in PD is recognized as motor fluctuations. However, non-motor fluctuations are prevalent and common and are arbitrarily subclassified as cognitive (clouding of thoughts, depression, apathy, panic), autonomic (sweating, lightheadedness), and sen­ sory (pain, parasthesae) (Chaudhuri and Schapira, 2009). Therapies aimed at continuous drug deliv­ ery such as long-acting dopamine agonists, apo­ morphine infusion, intra-jejunal levodopa infusion may improve both motor and non-motor fluctua­ tions. Thobosis et al. (2010) have proposed that non-motor fluctuations are related to mesolimbic dopaminergic denervation and apathy, depres­ sion, and anxiety can occur after stereotactic sur­ gery as a delayed dopamine withdrawal syndrome.

Non-recognition of NMS In a prospective study of PD patients followed up for 20 years, Hely et al. reported that non-levo­ dopa-responsive NMS were the most disabling, ahead of levodopa-induced dyskinesias in the Syd­ ney multicenter study (Hely et al., 2008).

Subsequently, although both NMSQuest and PRIAMO study have indicated the importance of the overall burden of NMS in PD, the NMS of PD are not well recognized in clinical practice. A clinic-based US study showed that existing depres­ sion, anxiety, and fatigue are not identified by neurologists in over 50% of consultations, and existing sleep disturbance in over 40% (Shulman et al., 2002). Another study reported correlation of NMS in PD at presentation retrospectively after clinico-pathological confirmation of diagnosis; 21% had NMS at presentation, including pain, anxiety, urinary dysfunction, and depression and were more likely to be misdiagnosed initially and had inappropriate medical interventions (O’Sulli­ van et al., 2008). More recently an international study using the NMS Quest has reported that irrespective of clinic and hospital background, PD patients reported 9–12 different NMS in their clinic visit, many of which had not been discussed with the doctor before being flagged by the NMSQuest with detrimental effect on quality of care (Chaudhuri et al., 2010).

Self-declaration of NMS and empowering patients Stacy et al. reported that NMS were common even in patients within 5 years of (motor) disease onset, and the use of a patient-completed questionnaire facilitated detection of these problems rather than routine clinic appraisal which is heavily biased towards motor symptoms. Recent studies using the non-motor questionnaire for PD (NMSQuest) have highlighted the significant occurrence of a range of 30 different NMS in PD in comparison with an age-matched control group (Fig. 1). These occurred across a range of PD patients from early to advanced disease, correlating strongly with advancing disease. In particular many NMS, such as dribbling of saliva, dysphagia, sexual problems, and pain, had not been discussed with the doctor before being flagged up by the NMSQuest (Chaudhuri et al., 2010). The study also high­ lighted that, irrespective of country of study and

334 80 70 60 50 40 PD patients

30

Controls 20 10

Dr Ta ibb ste lin Sw /sm g all el ow l Vo ing bo c m we on s itin l In inc tipa g em com ont tio pt ple ine n yin te nc g bo e we pa l ur ins ge no ncy ctu re w ria m eig los em h s o be t ha f in ring llu ter co cina est nc tio en ns tra tio n

0

Fig. 1. Distribution of PD patients and controls with various NMS (%).

disease stage, most PD patients are likely to flag up 9–12 different NMS in the NMSQuest at clinic visit. Additionally, further studies validating the first dedicated scale for NMS of PD, the PD nonmotor scale (NMSS), also indicated a strong rela­ tionship between the burden of NMS in PD and health-related QoL (Chaudhuri et al., 2007). The higher the burden of NMS as a whole, the worse is the health-related QoL.

Management of NMS Robust controlled studies are virtually nonexistent for the treatment of NMS in PD. However, there is some evidence from a handful of controlled trials for the treatment of certain NMS in PD, in particular depression, cognitive decline, psychosis,

and EDS. Moreover, the effect of these treatments on quality of life in PD is lacking, and many trials include only small numbers of patients. Dopami­ nergic treatment has some effect on depressive symptoms. Pramipexole has been investigated for its potential beneficial effect in depression, and Corrigan and colleagues reported antidepressant activity similar to fluoxetine (Corrigan et al., 2000). Other dopaminergic agonists including ropinirole (not pergolide) may have the same effect (Corrigan et al., 2000; Rektorova et al., 2003). However, some studies have reported pre­ cipitation of mania with pramipexole and ropinir­ ole (Singh et al., 2005). Tricyclic antidepressants and selective serotonin uptake inhibitors (SSRIs) have remained the major classes of drugs used for treatment of depression in PD (Byrne and Chaud­ huri, 2006). However, SSRIs such as fluoxetine or

335

fluvoxamine should be avoided in patients receiv­ ing selegiline as it can induce the potentially fatal serotonin syndrome (Byrne and Chaudhuri, 2006).Treatment of psychosis in PD remains com­ plicated as withdrawing dopaminergic treatment or introducing antipsychotics may worsen the par­ kinsonian state. However, there is good evidence that newer atypical antipsychotics, e.g., clozapine, may be beneficial (Factor et al., 2001). The evi­ dence of efficacy of quetiapine is controversial as a recent trial has shown no beneficial effect of que­ tiapine for PD psychosis (Ondo et al., 2005). Sev­ eral studies have shown that clozapine improves the psychosis rating scales (Factor and Brown, 1992; Factor et al., 2001). Dopaminergic treatment has a limited effect on cognitive impairment in PD. Loss of cholinergic cells form the basis of treatment for dementia in PD. The EXelon in PaRkinson’s disEaSe dementia Study (EXPRESS) for efficacy of rivastigmine in demen­ tia associated with PD represents advances in treatment of aspects of NMS in PD using nondopaminergic treatment (Emre et al., 2004). The cholinesterase inhibitor rivastigmine was shown to have a significant effect on dementia in PD as rated by dementia scores (Emre et al., 2004). In another double-blind, placebo-controlled trial donepezil was also shown to improve dementia (Ravina et al., 2005). There is evidence from two small trials to support the use of modafinil for EDS in PD (Adler et al., 2003; Hogl et al., 2002). Although the sample size was small, Adler and colleagues demonstrated that modafinil was effec­ tive for the treatment of EDS (Adler et al., 2003). However, a recent larger double-blind, placebocontrolled trial in 40 patients did not show efficacy of modafinil for EDS in PD (Ondo et al., 2005). There are no controlled trials for treatment of RBD but there are claims that night-time dosing with levodopa and use of clonazepam or prami­ pexole may reduce involuntary nocturnal move­ ments during sleep (Olson et al., 2000). Most clinical experience is based on use of clonazepam but it is necessary to exercise caution as sleepdisordered breathing may coexist with RBD and

can be worsened by clonazepam. Controlled trial evidence regarding the treatment of autonomic dysfunction in PD is only available for drooling and erectile dysfunction. Both botulinum toxin A and B injected into the parotid and/or submandib­ ular glands can be an effective treatment for drooling in PD (Lagalla et al., 2006). Erectile dys­ function can be treated effectively in PD with the use of sildenafil (an inhibitor of cGMP-specific phosphodiesterase type 5, an enzyme that regu­ lates blood flow in the penis) without the occur­ rence of side effects, in particular postural blood pressure (Raffaele et al., 2002).There is little research available for treatment of constipation, which is a very common presenting symptom in PD; however, in one study, macrogol was shown to be effective (Eichhorn and Oertel, 2001). Although deep brain stimulation of the subthala­ mic nucleus is an effective treatment for motor symptoms of PD, its effect on NMS is unclear (Parsons et al., 2006). Kalteis and colleagues reported an improvement in psychiatric symptoms such as depression, anxiety, and psychological symptoms in a study of 33 patients after subthala­ mic nucleus deep brain stimulation (Kalteis et al., 2005). There have been reports of decreased verbal and executive functioning after subthalamic nucleus deep brain stimulation (Castelli et al., 2006). Many NMS of PD may have a non-dopaminergic basis and symptoms usually only partly respond to dopaminergic treatment (Postuma et al., 2006). Indeed, dopaminergic therapy may precipitate some non-motor problems in PD such as the dopa­ mine dysregulation syndrome and orthostatic hypotension (Reuter et al., 2005). Recently dopaminergic therapies utilizing the concept of continuous dopaminergic stimulation have been shown to be effective for some NMS such as sleep-related problems in PD. Examples include the improvement of several non-motor problems, including sleep and mood with apomor­ phine and duodopa infusions (Chaudhuri and Schapira, 2009; Honig et al., 2009; Metta et al. 2010). The AAN has issued guidelines related to evidence base of treatment of NMS of PD and

336

highlight the fact that currently very few NMS have good-quality level I trial data (Zesiewicz et al., 2010).

Conclusions Delayed detection of NMS may lead to disability and poor quality of life, increasing the cost of care of PD in the society. NMS such as visual halluci­ nations, dementia, and falls are a major source of hospitalization and institutionalization. Recogni­ tion of NMS is therefore essential for the holistic management of PD and the importance of a multidisciplinary approach, including support for carers, cannot be overemphasized (Global Parkinson’s disease Survey Steering Committee, 2002).

Abbreviations AAN CSF EDS LBD MIBG MSA NMS NMSQuest PD QoL RBD SSRI UPDRS

American Academy of Neurology corticospinal fluid excessive daytime sleepiness Lewy body disease meta-[123I] iodobenzylguanidine multi-system atrophy non-motor symptoms Non-Motor Symptom Questionnaire Parkinson’s disease Quality of Life REM-sleep behavior disorder selective serotonin reuptake inhibitors Unified Parkinson’s Disease Rating Scale

Future perspective In future, it is anticipated that there will be a focus on development of pre-symptomatic tests in asymptomatic individuals who are at increased risk of PD although this raises ethical issues if robust neuroprotective strategies are not avail­ able. The development of the NMS-orientated PD-specific tools such as the SCOPA instruments, the NMSQuest and NMS Scale, and the modified UPDRS suggest a growing interest in recognition and awareness of NMS complex of PD among clinicians and researchers and will lend to robust studies addressing natural history, effect of drug therapy, and, in particular, disease-modifying therapies in the future. A key issue will be good quality-controlled studies addressing the effects of dopaminergic and non-dopaminergic therapies on NMS in PD, and studies such as the RECOVER study and also preliminary reports of ‘real life’ studies using apomorphine and duodopa infusions in using NMS as the primary end point, is a step in the right direction. Development of animal mod­ els to test therapies related to NMS of PD will also be keenly anticipated.

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Subject Index

truncated a-syn, lead to increased DAergic dysfunction and pathology, 96

AAV2 serotype, 90

AAV2 vector, 102

Abnormal metabolic networks, in PD

disease progression, evolves network activity, 166

assessed hemispheric progression, 164

CSPTC loops, functional changes in, 164

PDCP elevations, hemispheres, 165

PDRP and PDCP metabolic networks activity

quantification, 165

PDRP expression, baseline elevations in, 165

PD-related cognitive pattern (PDCP)

Alzheimer’s disease (AD)-type changes, 163

behavioral abnormalities, 163

characterized by, 163

computing PDCP expression, 163

Lewy body pathology, 163

metabolic imaging, for investigation, 163

PD-related spatial covariance patterns, metabolic changes

abnormal PD-related metabolic pattern, 162

CSPTC loops, 162

Parkinsonian tremor association with, 163

PDRP network activity, abnormal elevations,

162–163 treatment effects assessment, with network activity

DBS effect, 168

dopaminergic treatment, 166–168

gene therapy, 168–169

microlesion effect, 168

See also Parkinson’s disease (PD)

AAV-a-syn-transduced DA neurons, 98

AAV-mediated a-syn overexpression, 90, 93, 95

AAV-a-syn vector delivery, aspects, 102

AAV-a-syn vectors, transduction, 103

AAV-mediated protein expression

assessment, 104–105 immune reactions, 103–104 midbrain DA neurons, transduction efficacy, 105–106

non-specific toxicity, 102–103

working titers, 102–103

damage caused, toxic intermediates, 95

of human wild-type a-syn, 92, 96

motor behavior in rats, changes in, 93

neuroprotection studies, in AAV-a-syn model,

99–100 PD-like neuropathological changes induction axonal pathology, 91–93 cell death, 91–92 immune and inflammatory changes, 93–95 a-syn post-translational modifications, toxicity enhancement, 95–96

phosphorylation role in a
syn toxicity, 96

S129D phosphorylated a syn and PD

pathology, 96

a-syn mediated DAergic cell death,

mechanism, 95

a-syn phosphorylation, S87, 96

a-syn toxicity mechanism, in DA neurons,

96–97

DA re-uptake, effects on, 98–99

DA storage and release, effects on, 98

DA synthesis and a
syn interaction, 97–98

dopamine-dependent toxicity, 99

343

344

Abnormal movements, defined, 296

Adeno-associated virus (AAV), 90

serotypes, 90

vectors, 89–90

Adenosine-5’-triphosphate (ATP) depletion, 21

Adipsia, 36

Age-related loss, of striatal dopamine, 135

Aging, 123

Akinesia, 39

Alzheimer’s disease (AD), 162, 206

characteristic pattern, 207

pathology, 206

Alzheimer’s-like pathology, 117

Amantadine, 146

Amphetamines, 36–37, 40, 44

Animal models, of PD

based on systemic or local administration of

neurotoxins, 89

chemical exposures, role in

neurodegeneration, 26

cognitive impairment, 42–43

DRL paradigms, 42

specificity issue, 42–43

dopamine activation, rewarding effects,

43–44

emerging models, 25

genetic models, familial disease mutation,

18, 39

in genes LRRK2, Parkin, DJ-1, and PINK1

(Park family of genes), 18, 38

in a-synuclein gene, 38–39

of human disease, importance, 17, 35

ideal model, expectations, 26

instrumental learning, striatal dissection,

46–47

instrumental conditioning, 45

Pavlovian conditioning, 45

striatum association with, 46

Thorndike’s law of effect, 45

limitations, 89

motor and sensorimotor tests, for rat

locomotor activity, 39

rotation, functional outcome measure,

39–40

motor learning and, 45

dopamine and reinforcement, 43–44

MPTP model, rodents, 38

of neurodegenerative disease, 17

neurotoxicant-based models, basis for, See

neurotoxicant-based models, of PD

neurotoxin models, 18, 37–38

new models, 90

non-motor symptoms, 41–42

6-OHDA neurotoxin model

bilateral lesions, 36–37

dopamine depletion, 36–37

unilateral lesions, rotation method, 37

See also neurotoxicant-based models, of PD

overexpression of a-syn by viral vector, 90

pharmacological models, 36

procedural learning, 44–45

sensorimotor tests and neglect, 40

skilled reaching, 40–41

nigrostriatal deficit, 41

staircase test, 41

striatal dissection, instrumental learning,

45–47

See also Parkinson’s disease (PD)

Anxiety, 4, 42, 70–71, 80, 124, 178, 211

Anxiety disorders, in PD, 330

Aphagia, 36

A30P mutation, 78

Apomorphine, 36–37, 39

A30P proteins, 68

Arginine rich, mutated in early-stage tumors gene

(ARMET), 253

L-Aromatic amino acid decarboxylase (AADC),

97, 178, 180, 194, 208, 214, 222

Artemin (ARTN), 242

A53T mutation, 148

A53T proteins, 68

A53T variant, under control of mouse PrP

promoter, 68–70

Autoimmune diseases, 123

Axonal depolarization, 178

Axonal pathology, 91–93

Basal ganglia dysfunction, 211

Bilateral 6-OHDA lesions, 36–37

Bilateral parkinsonism, 135

345

Bilateral STN AAV-GAD gene therapy, 169

Blood–brain barrier (BBB), 117, 123

Braak staging, 206

Bradykinesia, 39, 124, 144

Brain-derived neurotrophic factor (BDNF), role

in PD, 238–239

alter synaptic transmission, 240

critical role in neurogenesis, 240

effect on DA neurons, 240–241

effects, and NT-4/5 on neuronal

protection, 242

isolation, 239

neuroprotective effects, 240

supports survival and differentiation, neurons,

239–240

therapy with neural transplantation, 240–241

Brain imaging, after transplantation

dopamine release, indirect assessment, 197

123 I-IBZM SPECT studies, 197

studies with, 11C-raclopride PET and, 197

graft function, indirect assessment, 197

graft-induced dyskinesias (GIDs)

dopaminergic function and, 199

graft composition and, 199

inflammatory and immune responses

and, 199

relation to fiber outgrowth, 198–200

striatal 18F-dopa uptake role, 199

graft survival, and presynaptic dopaminergic

system monitoring, 193–197

aromatic amino acid decarboxylase (AADC)

activity, 194

bilateral intraputaminal transplantation, 196

11 C-dihydrotetrabenazine PET, 194

18 F-dopa PET, functional imaging,

194–195

18 F-dopa uptake processes, 194

open-label study, 196

PET radioligands, 194

PET studies, 194

SPECT radioligands, 194

trials assessing, safety and, 193

UPDRS motor scores, 195–197

striato-cortical circuitries, restoration

assessment, 197–198

Caenorhabditis elegans, 8

CaM-aSyn mice, 77–78

transgene expression, 77

CaM promoter, 77–78

Catalepsy, 36

Catecholamine neurotoxin, 36

Catecholaminergic neurons, in PD, 54

Catecholamines, 125

CBA-driven vector construct, 91

11 C-DASB binding potential values, 212

11 C-DASB PET, demography of PD patients, 212

11 C-DASB PET, marker serotonin transporter

(SERT), 209–210

CD68 expression, 93

11 C-dihydrotetrabenazine PET, 194

Cell death, 93, 134

Cell replacement therapies, in PD, 193

Cell sorting using FACS, 286

Cholinergic neurotransmission, 205

Chronic fatigue, in PD, 211–213

Chronic neuroinflammation, potential triggers, 116

Cocaine, 44

Cognitive impairment, 146–147

Cognitive/neuropsychiatric NMS, of PD, 330–331

Conserved dopamine neurotrophic factor

(CDNF), 252–253

Constipation, common NMS of PD, 329

Corticobasal ganglionic degeneration (CBGD), 172

Corticostriato-pallido-thalamocortical (CSPTC)

loops, 162

COS7 cells, 8

Cre-loxP-based transgene, 78

11 C-RTI 32 binding, 210–211

11 C-RTI 32 PET, noradrenergic/dopaminergic

functions marker, 210

C-terminal-truncated a-syn, 96

11 C-WAY100635 PET, marker of serotonin

5-HT1A receptor, 210

Cyclooxygenase-1 and-2, 114

DA agonists, 178, 186

DA cell death, 115–116, 118, 121–122

DAergic cell death, 95–96

DAergic innervation, in striatum, 92

DAergic neurodegeneration, 96

346

DA homeostasis, 197

DA-mediated oxidative stress, 99

DA neurons, 6, 8, 81, 90, 93, 97, 105, 115, 117,

122, 179

and inflammation, 118–119

loss, 7

DA receptor stimulation, 184

DA synthesis, 97–98

activity of TH enzyme, 97–98

a-syn interaction with, 97

DAT binding, 180, 182, 207, 209–210, 213–215

measurement, 213

significant changes in, 194

DA transporter (DAT), 71, 82, 98–99, 134, 148,

179, 181, 184, 207, 214–215

DA turnover, 179, 184

D2-dopamine receptors, 148

D2/D3 receptors, 186

Deep brain stimulation (DBS), of SNT, 168

Deep brain stimulation (DBS) sugery, PD

treatment, 311–312

action mechanism, 317–318

axial, non-motor, speech symptoms, 314

benefits, and targets used STN/globus pallidus

internus (GPi), 312–313

complex effects of, 317–318

contraindications

cognitive and neurobehavioral disorders,

aggravated, 314–315

including systemic medical co-morbitidies, 314

electrode, implantation site, 318

improvements in quality of life

reduction in dyskinesias, 313

STN and GPi stimulation and, 312–314

improving motor function, 312

levodopa dose and dyskinesias, 313

new targets, stimulation

caudal zona incerta, 316

non-motor components, 315–316

pedunculopontine nucleus area (PPNa),

316–317

radiation prelemniscalis, 316

old targets, stimulation

centromedian-parafascicular (CM/Pf)

complex, 315–316

Parkinsonian symptoms, and candidate areas

for intervention, 316

therapy limitations, 315

tremor treatment, 311–312

Delirium, 330

Dementia, 4, 146

imaging in PD, 206

amyloid load, 209

cholinergic function, 208

glucose metabolism, 206–207

Depression, 4, 37, 42, 72, 119

complication of PD, 328–329

Diffusion tensor imaging (DTI), 206

Diffusion-weighted imaging (DWI), 206

[11C]Dihydrotetrabenazine (DTBZ), 179

Diphasic dyskinesia, 145

Disease progression

assessment of, 181–182

clinical and in vivo measurement, disparity,

182–183

network activity changes with, 164–166

DJ-1 deficiency, 10

Dopamine (DA), 35–36, 43, 90

agonists, 145

cell loss, in SNC, 135

depletion, 19–20, 26, 36, 42, 136, 205

cognitive change following, 42–43

dysfunction, 36

neurons, 113

release, indirect assessment, 197

123 I-IBZM SPECT studies, 197

studies with, 11C-raclopride PET and,

197

replacement, 4

therapies, 178

Dopamine-dependent toxicity, 99

Dopaminergic function, 181, 194, 207–208, 213

Dopaminergic neuronal loss, 4

Dopaminergic (DA) neurons, 4

Dopaminergic therapy, 167, 178, 186, 197

Dopamine-rich nigral grafts, 41

Dopamine synthesis, enzymatic processes for,

221–222

Dorsolateral striatum (DLS), 46

Dorsomedial striatum (DMS), 46

347

Dose–effect relationship, 69

D2 receptors, 180, 185, 197, 199

Drosophila

advantages, pink1/parkin pathway in, 11–12

contributions and future prespective, 11–12

as model system for PD, 5

pink1 and parkin pathway, 4–6, 8, 99

catalyzes ubiquitination, 5–6

in COS7 cells, 8

DA neuronal degeneration, 11

DJ-1 overexpression in, 10

endogenous functions, 6

function in flies relevant to humans, 7

heterozygosity for drp1, 8

mitofusin accumulation and ubiquitination, 9

null mutants, phenotypes, 7

Omi/HtrA2 function as downstream target, 10

other components in, 10

overexpression of DJ-1 in, 10

Parkin recruitment, depends on Pink1, 9

pink1 mutants, 6

promote mitochondrial fission/fusion, and

defects, 7–8

promoting mitophagy, 8–10

rapamycin and activating 4E-BP, 11

regulate mitochondrial integrity, 5–7, 11

PARK2 locus encodes Parkin, 5

regulates mitochondrial dynamics and

mitophagy, 9

spermatid morphology defects, 8

unique attributes, 12

Drug development, for PD, 134

Dynamin-related protein 1 (Drp1), 7

Dysautonomia, and PD

movement disorders, 331–332 symptoms of, 331–332

Dyskinesias, 145, 183–184

Dystrophic neurites, 91

Effective dopamine turnover (EDT), 179

Electrolytic lesions, of midbrain, 134

Enteric nervous system (ENS), 79

Environmental toxicants, 116

Epinephrine, 125, 214

Epworth sleepiness scale (ESS), 213

E3 ubiquitin ligase, 5

Excessive daytime sleepiness (EDS), in PD, 331

Familial forms, of PD, 4–5

Fatigue, in PD, 332

18 F-deoxyglucose (FDG) PET marker, 207

18 F-dopamine, AADC activity, 194

18 F-dopa PET, 194–195

F-DOPA uptake, 181

Fetal predisposition, for idiopathic PD, 123

18 F-fluorodopamine, 214

[18F]fluoro-L-m-tyrosine (FMT), 226

Fluorodopamine (FDA), 178–179, 184

Freezing, 124

Gastrointestinal symptoms, in PD, 329

Gene–environment interaction, 26

Gene therapy, 168–169

Genetic models, PD, 38–39

Genetic mouse models, PD

alterations in TH-aSyn mice

effects of expression of doubly mutated

gene, 66

microglial activation in, 66

CaMKII-tTA, 77–78

hamster prion protein, 72–73

of LRRK2 mutations, causing PD, 81

mice expressing A53T a-syn, under prion

promoter control

alterations in catecholamine systems, 71

mice overexpressing A30P variant, alterations in

under control of mouse prion promoter, 71–72

mice overexpressing a-syn

dopaminergic deficits and neuropathology in,

TH promoter role, 54, 65

molecular alterations, under PDGF-b

promoter control, 67–68

neuropathology and behavioral deficits,

under PDGF-b promoter control, 66–67 paraquat exposure, 65–66 under tyrosine hydroxylase promoter control, 54

mice overexpressing A53T variant

neuropathological alterations, PrP promoter

(PrPSynA53T) control, 68–70

348

Genetic mouse models, PD (Continued)

mouse prion protein (msprp) promoter

highest level of transgene expression, 68

mimic neuropathology observed in PD, 68

other models, 81–82

other promoters, 78–79

of recessive mutations, causing PD, 80–81

a-syn expression, under control of PrP

promoter, 70–71

a-syn models of PD, summary, 79–80

a-syn overexpression, under CaM promoter

control, 77–78

a-syn overexpression, under Thy-1 promoter

(Thy1-aSyn) control, 73

motor phenotype, relation to pathology, 73–75

non-motor phenotype, relation to

neurochemical pathology, 75–76

a-syn transgenic mice, overview, 55–64

Thy1-aSyn mice

neuropathologies in, 76

time course of deficits, in various lines

of, 76–77

Glial cell-derived neurotrophic factor (GDNF)

family of ligands(GFL), 237

GDNF, member, 243

AAV-GDNF therapy evaluation, 248

adeno-associated viral vector (AAV) system,

advantages, 247–248

adenoviral (Ad) vectors used, and

advantages, 247

Ad-GDNF delivery, effects of, 247

animal models of PD, efficacy in, 243–244

astrocytes production, protect adult

nigrostriatal DA neurons, 246

delivering ways, 245, 248

direct effects on DA neurons, 243

efficacy in PD patients, 244–246

ex-vivo cell therapy, 246

gene delivery of GDNF, therapeutic value of,

243, 250

GFRa-1 and Ret subunits, 243

in-vivo gene therapy, 246

lentiviral vectors (LV-GDNF), assess efficacy

of, 249–250

LV-a-synuclein model, 250

nonhuman primate PD models, positive

effects, 244

potential therapeutic value of, 243

recombinant lentiviral vectors (rLV) use, 249

Ret expression, 243

viral vectors, delivering in CNS, 246–247

glial cell line-derived neurotrophic factor, 243

neurturin (NTN), member of, 250–252

animal models of PD, efficacy in, 251

efficacy in PD patients, 252

ex vivo cell methods, 251

in vivo gene therapy, 251–252

pivotal role in biological processes, 242

See also Parkinson’s disease (PD)

Glial cell-line-derived neurotrophic factor

(GDNF), 100, 148, 182

Glial fibrillary acidic protein (GFAP), 69

Glutamic acid decarboxylase (GAD) gene, 168

G protein-coupled receptor kinase 5 (GRK5), 68

Graft-derived serotonergic neurons, 199

Graft function, indirect assessment, 197

Graft-induced dyskinesias (GIDs), 198–200,

297–299, 306

cause of, 299

clinical problem of, 296–299

controlled by DBS, 298–299

in grafted patients, explicit/implicit reports,

297–298

off-medication dyskinesia, 297

side-effects of transplants, 296

Tampa–Mount Sinai trial, 297

unrelated to intake of L-dopa, 296–297

factors hypothesized to contribute to, 300

graft composition, and surgical protocols,

304–305

DA cell types, in transplant composition, 304

DA overload, 304

immunosuppression requirement, 303–304

behavioral recovery and immune

response, 303

Denver–Columbia trial, 303

Tampa–Mount Sinai trial, 304

modeling in animals, 299–303

amphetamine-induced dyskinesias (AID)

and, 301–302

349

approaches studying, in rodent models, 301

L-dopa-induced dyskinesia (LID), 300–301

MPTP-treated primate model, 299

post-grafting dyskinesia animal models, in

PD, 302

rodent model of PD, unilateral 6-OHDA­ lesioned rat, 299, 301

patients selection, preventing

Lund open-label trial, 303

severe LID prior to surgery and GID high

risk, 303

serotonin neurons role, 305–306

tissue preparation, 304–305

Graft survival, and presynaptic dopaminergic

system monitoring, 193–197

bilateral intraputaminal transplantation, 196

11 C-dihydrotetrabenazine PET, 194

18 F-dopa PET, 194–195

open-label study, 196

PET studies, 194

trials assessing, safety and, 193

UPDRS motor scores, 195–197

GTP cyclohydrolase 1 (GCH1), 222

Hallucinations, visual and PD, 330–331

Hallucinatory-like behavior, 145

Haloperidol, 36, 146

Hamster prion protein, 72–73

Hemiparkinsonism, 135, 165

Herbicide paraquat, 8, 22–24, 65–66, 74, 82,

116, 121

Heroin, 44

Hippocampal dopamine transporter (DAT)

availability, 215

Human fetal VM grafts, 195

Human fetal VM tissue

striatal transplantation of, 198

transplantation studies, 194–196

unilateral putaminal and caudate implants

of, 195

Human wild-type a-syn, overexpression

in mice, 54

in midbrain and striatum, 103

in neocortex and septum, 101

in rat midbrain, 92

6-Hydroxydopamine (6-OHDA), 19–21, 36–38,

40, 42, 115–116, 124, 134

as dopaminergic neurotoxin, 19–21

-lesioned NHP, 134

-lesioned rats, 21

Hypokinesia, 36, 134

Hyposmia, 162, 206, 214–215

Idiopathic PD (iPD), 66, 95

pathogenesis, 114

123 I-FP-CIT SPECT

DAT marker, 207

olfactory testing, 214

123 I-iodobenzovesamicol (123I-BVM) SPECT, 208

IL-6, 125

IL-10, 123

Imaging

as biomarker, 183

cardiac sympathetic denervation, 214

dopamine storage capacity, 208

functional, 193

PD depression, 209–211

11 C-DASB PET, 209–210

11 C-RTI 32 PET, 210–211

11 C-WAY100635 PET, 210

123 I-b-CIT SPECT, 210

sleep disorders, in PD, 213–214

See also brain imaging, after transplantation;

neuroimaging, of nigrostriatal system

123 I-metaiodobenzylguanidine (MIBG), 214

Impulse control disorders (ICDs), 178, 186

Industrial chemical exposures, 26

Inflammation, stimuli facilitating PD, 123

aging, 123

psychological stress, 124–125

systemic (chronic) disease, 123–124

Inflammatory models, of PD

to cause NCD, 122–123

CNS injections of LPS, 120

inflammatory features, examination, 121

mimic, progressive nature of PD, 121

neuroinflammation and, 120

pathology, 119

PD-related issues, 120

to study pathogenesis, 119–120

350

Inflammatory models, of PD (Continued)

using lipopolysaccharide (LPS), 26

in vivo and in vitro, 120–121

iNOS gene, 115

Instrumental learning, striatal dissection,

45–47

Interferon-g, 114

Interleukin-1 beta (IL-1b), 93, 114, 118,

121, 125

Internal globus pallidus (GPi), 182

Intracranial self-stimulation (ICSS), 43

in vitro models of PD, 18

in vivo neurotoxicant-based models, See

neurotoxicant-based models, of PD Japanese encephalitis virus, 123

L-3,4-dihydroxyphenylalanine (L-DOPA), 221

L-dopa, 39

L-dopa-induced dyskinesia (LID), 299

L-dopa therapy, 36

Lentivirus (LV) vectors, 89–90

Leucine-rich repeat kinase 2 (LRRK2), 38

Levodopa-induced dyskinesia (LID), 166, 178, 184

Levodopa-induced motor fluctuations, 144–145

Levodopa-treated MPTP-lesioned primate, 145

Lewy bodies (LB), 39, 66, 90, 96, 113, 164, 206

pathology, 35, 206

in PD, 96

Lipopolysaccharide (LPS), 26, 70–71, 94, 117, 119,

121–122

Locus coeruleus (LC), 113

LRRK2 mutation, 39

Magnetic activated cell sorting (MACS), 286

Magnetic resonance imaging (MRI), 206

Major histocompatibility complex (MHC) II

expression, 93

MAO-B inhibitors, 134

Mesencephalic astrocyte-derived neurotrophic

factor (MANF), 252–253

Methamphetamine, 19

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine

(MPTP), 6, 21–22, 38, 66, 89, 133–134

chronic delivery of, 135

elicited parkinsonism, 21–22 -lesioned NHP models, See MPTP-lesioned Non-human primate (NHP) models

model, 21

protoxin, 134

in rodents, 38

sensitivity, 135

toxicity, 134

a-Methyltyrosine, 36

Mitochondrial dysfunction, 39

Mitochondrial fission/fusion dynamics, 7

Mitochondrial inhibitor rotenone, 99

Mitofusin (mfn), 7, 9–10, 12

Monoamine oxidase B (MAO-B), 134

Monoamine oxidase type A (MAO-A), 222

Monoubiquitination, 6

Motor behavior changes, in rats, 93

Motor learning, and reinforcement, 45

Motor tests, for rats, 39

Mouse prion protein promoter, 68

MPTP-lesioned non-human primate (NHP)

models, 134

facts update, 134–135

measurements improvent, 147

MPTP ability, produce nigral and striatal

dopamine cell loss, 134

MPTP-induced parkinsonism, pathology, 135

alpha synuclein pathology, 136, 143

basal ganglia, changes in outside of, 143

monoamine cell loss, 135–136

non-dopaminergic neurotransmitters, in

MPTP-lesioned primates, 136

premotor symptoms, 143

SNC, dopamine cell loss in, 135–136

phenomenology, motor/non-motor features

cognitive problems, 146–147

motor fluctuations, levodopa-induced, 144–145

motor phenotype, 143–144

psychosis-like behaviors, 145–146

sleep disorders, 146

in translational medicine, 147

in developing drugs for neuroprotection, 148

to mimic clinical endpoints in trials, 147

use in developing drugs, 148

MPTP-lesioned primate animals, 145

351

MPTP-parkinsonism in NHP, pathology, 135

alpha synuclein pathology, 136, 143

dopamine cell loss in SNC, 135

5-HT levels, 136

levodopa-induced dyskinesia, 136

monoamine cell loss, 135–136

non-dopaminergic neurotransmitters, 136–142

in outside of basal ganglia, 143

MPTP-parkinsonism, motor phenotype, 143–144 MPTP-primate to evaluate potential neuroprotective agents, 147–148

extranigral pathology, 143

as model of cognitive deficits in PD, 147

Multiple system atrophy (MSA), and abnormal metabolic networks, 161

Musculoskeletal abnormalities, 4

Mutations in DJ-1, 10

NADPH oxidase, 118

Nerve growth factor (NGF), 238–239

antioxidant enzymes, effect on, 239

discovery, 239

protects neurons, 239

receptors, 239

role in axonal guidance, 239

supports sympathetic and sensory neurons, 239

Network activity, assessing treatment effects, 166

deep brain stimulation (DBS) and, 168

dopaminergic treatment, 166–168

gene therapy, 168–169

Neural grafting, in PD, 265–266, 295

accommodating outcome differences, in both

trials, 270–271

design of trials, dopaminergic drug therapies,

273–274

dopaminergic cell transplant, ideal

characteristics, 281

double-blind placebo-controlled transplantation

trials

fetal VM transplantation in, 267

fetal VM transplants, utility and safety

concerns, 270

longer follow-up and improvement, 269

motor improvements, 268

negative results with side effects, 269

problematic issues, 268–269

second NIH-sponsored study, 269–270

trials failing, in achieving main outcomes, 268

UPDRS motor scores, 268

embryonic stem cells, as DA neurons source, 265

derivation of human ESCs (hESCs), 282–283

derivation of neural tissue from human tissue,

protocols, 282–283

give rise to all cell types, 282

hESC-derived cells, immunogenicity of, 283

hESCs, neural differentiation, 284

protocols for derivation of neural

tissue, 282–283

self-renewing and pluripotent, 282–283

teratomas formation, 283–284

ethical issues and tissue procurement, fetal neural tissue, 276–277

future perspectives, 287

graft-induced dyskinesias (GIDs),

development, 265–266, 272–273 L-dopa-induced dyskinesias (LIDs) role, 273

graft tissue, preparation and placement, 271–272

immunosuppression and, 272

iPS cells usage, 284–285

benefits, 284

derived, reprogramming, 284

similar to ES cells, 284–285

lineage-specific stem cells, as DA neurons source

neurons, astrocytes and oligodendrocytes, 281

neurospheres, expansion system, 281–282

NS cell cultures, 282

main sources of donor tissue, used for

transplantation, 280

neural donor tissue storage, 278–280

advantages, 278

cell culture, freezing and refrigeration, 279

neuropathological changes in grafts, issue, 274–276 alpha-synuclein pathology, 274–276 dopamine transporter (DAT) downregulation, 276

post-mortem pathology, 274

ubiquitin-positive Lewy bodies, in grafted

neurons, 274–276

VM grafting, safety, 274

352

Neural grafting, in PD (Continued) open-label transplantation trials in, 274

clinical improvements and evidence, 266–267

fetal dopamine neurons, for implantation, 266

fetal ventral mesencephalic (VM) tissue

used, 266

patient selection, role, 271

re-programmed somatic cells, as DA neurons

source, 281

safety and regulatory issues, SCs application in

brain

chromosomal aberrations, 285–286

epigenetic changes, 286

GMP-compatible protocols, 286

GRNOPC1, use in transplantation therapy, 285

pharmacological agents, destroying cell

proliferation, 286

tumors formation, 285

stem cell-derived neurons for, 280–281

stem cell therapy, challenges, 281

tissue dissection, 277–278

aim for max. number of dopamine

neurons, 278

regulators of dopamine neurogenesis,

role in, 277

standard, 278

Neurodegenerative diseases, 17

Neurodegenerative disorder, 4

Neuroimaging, of nigrostriatal system, 178

dopamine function, biochemistry, 178

F-DOPA PET studies, 181–182

functional brain imaging, 180

imaging role as biomarker, 183

PD-related spatial covariance pattern to, 180

post-synaptic imaging, 180–181

presynaptic imaging, 178–180

of treatment-related, behavioral complications

depression and cognitive impairment, 186

impulse control disorders (ICDs), 186

RAC binding, 186

of treatment-related, motor complications, 183

applications, future, 185–186

assessing DA release kinetics, 184

cerebral blood flow studies, 185

DA receptor stimulation pattern, 184

DA turnover, abnormality in, 184 dyskinesias, following fetal mesencephalic transplantation, 184–185 fluctuations in motor function, with dyskinesias, 184

levodopa therapy, 184

post-synaptic mechanisms, 185

presynaptic mechanisms, 183–185

RAC binding, 184

Neuroinflammatory consequences, of stressor, 125

Neuroinflammatory pathogenesis model, of PD

animal PD models, 114

considerations regarding ideal model

implementation, factors regarding, 119–120

inflammatory features, to be examined, 121

mimicing pathology, 119

mimic progressive nature of PD, 121

similar results use in, in vivo and in vitro,

120–121

variability associated, with model, 120

DA neurons, vulnerability to inflammation microglia, role in ROS generation, 118–119 nigral cell death, by cytokines IL-1b and TNF, 118–119

inflammation related enzymes and

cytokines, 114

microglia, role in coordination of

neuroinflammation, 114

PD progression, inflammation leading, 121–123

rodent models, inflammatory contribution, 114

fungicide, maneb, 116–117 herbicide, paraquat, 116–117 lipopolysaccharide, activate inflammatory response, 117–118 MPTP, neurotoxin to mimic PD in mice, 114–115 6-OHDA, gold standard rat model, 115–116 pesticide rotenone, 116–117 stimuli facilitate PD, through inflammation

aging, 123

psychological stress, 124–125

systemic/chronic diseases, 123–124

triggers contributing to chronic

neuroinflammation

and role in nigral cell death (NCD), 116

353

Neurological syndrome, 40 Neuronal cell

death, 66

inflammation and progression of PD, 94

Neuroprotection, 148

Neuropsychiatric-like behavior rating scale, 146

Neuropsychiatric symptoms, 145–146

Neurotoxicant-based models, of PD, 18–19

6-hydroxydopamine (6-OHDA) model, 19–21

attractive features, 20

as dopaminergic neurotoxin, 21

expansion into additional animal

species, 20–21

non-motor functional deficits modeling, 21

recent advances, 20

structure similar to dopamine, 20

MPTP model, cross blood–brain barrier, 21–22

functional deficits, 22

recent advances, 21–22

paraquat/maneb models, 23

paraquat model

association between PD and, 23

dosing produce behavioral features

characteristic of PD, 22

mixture model, 23

for modeling “preclinical” stages PD, 23

ROS production, 22

use as pesticide, 22

as useful compound in modeling, 23–24

rationale for, 18–19

reserpine model, limitations, 18–19

rotenone model

problem with, 24

produce systemic complex I inhibition, 24

similarities to human PD, 25

in vivo models, 19

dopamine depletion, 19

L-dopa supplementation, 19

methamphetamine use, 19

Neurotoxins, 37–38

Neurotrophic factors (NTFs), 237

Neurotrophic factor therapy, for PD, 237–238

Neurotrophin, 238–239

Neurotrophin-3 (NT-3), 238, 241–242

Neurotrophin-4/5 (NT-4/5), 238, 242

Neurotrophin family, 237–239 Neurturin (NTN), 242

compared with GDNF, 243

efficacy in animal models of PD, 251

CERE-120, advantages, 252

ex vivo cell methods used, 251

in vivo gene therapy, 251

efficacy in PD patients, 252

CERE-120 trial, 252

Nigral cell death, 118

Nigral DA neuron death, 122

Nigral degeneration, in PD, 205

Nigra pars compacta (SNc), 205

nigrostriatal system neuroimaging, See neuroimaging, of nigrostriatal system

N-methyl-D-aspartate (NMDA) receptor, 242

Nocturnal REM sleep, in PD, 214

Non-DA neurons, 4

Non-dopaminergic neurotransmitters, in

MPTP-lesioned primates, 137–142 Non-dopaminergic systems AAV vectors for overexpression of a-syn, 100–102

AAV6-a-syn, 101

AAV1 vector, 102

AAV2 vector, 102, 104

AAV8 vector, 102

AAV5 vectors, 101, 103–104

Non-human primate (NHP) models, of PD, 133

earliest and long lasting models, 133–134

MPTP-lesioned, See MPTP-lesioned

Non-human primate (NHP) models

unilateral model, 134

use of synthetic neurotoxin, 134

Non-motor fluctuations, in PD, 333

Non-motor phenotypes

in developing NHP models, 145

of Thy1-aSyn

neurochemical and pathological alterations, 75–76 other neuropathologies, 76

Non-motor problems, in PD, 145

Non-motor symptoms (NMS), in PD, 4, 41–42,

178, 325–326

cognitive symptoms, 330–331

354

Non-motor symptoms (NMS), in PD, (Continued)

complex, 327, 330

excessive daytime sleepiness, 331

fatigue, 332

future prespectives, 336

management

clonazepam, 335

clozapine, 335

controlled trials, 334

dopaminergic therapies, 335

macrogol, 335

pramipexole, 334

rivastigmine, 335

sildenafil, 335

SSRIs, 334–335

tricyclic antidepressants, 334

nocturnal, 331

non-motor questionnaire (NMSQuest), 329

non-recognition

NMSQuest and PRIAMO study, 333

pain, 332–333

pathogenesis, 326

as preclinical (motor) feature, 329

predict emergence, of motor symptoms, 326–329

depression, 328–329

gastrointestinal symptoms, constipation, 329

olfactory dysfunction, 326–328

REM behavior disorder (RBD), 328

and premotor NMS, 326

prevalence, cognitive/neuropsychiatric, 329–331

anxiety disorders, 330

apathy, and fatigue, 330

autonomic dysfunction, 331–332

dementia, 331

visual hallucinations and psychosis, 330–331

rapid eye movement behavior disorder

(RBD), 326

self-admission

and role of non-motor questionnaire for PD

(NMSQuest), 333–334

sexual dysfunction, 332

Noradrenaline, 36–37, 75, 136, 210–211

Obsession, 4

Olfactory bulb (OB), 71, 77–78, 119

DG and, 78

proteinase-K-resistant a-syn, 75

THþ neurons in aged mice, 67

Olfactory function, in PD, 214–215

Omi/HtrA2 in sporadic PD patients, 10

Omi/HtrA2 mutations, 11

Optic atrophy 1 (Opa-1), 7

Oxidative stress, 6, 10, 21, 38, 81, 94, 97, 115, 181

OX42 positive microglia, 117

Pain, clinical symptoms in PD, 332–333

PARK4, 4

Park family of genes, 38

PARKIN, 4

Parkin-dependent ubiquitination, 10

parkin function, to regulate mitochondrial

integrity, 5–7

parkin knockout mice, 6

parkin overexpression, 7

Parkinsonian conditions, differential diagnosis, 169

abnormal metabolic networks, 171

early parkinsonian symptoms, 169

clinicopathologic studies, 169

SPECT and TCS, 169

metabolic images, pattern analysis of, 169–170, 172

image-based classification, 170

logistic regression models, 170

See also abnormal metabolic networks, in PD

Parkinsonian rating scale (PPRS), 224

Parkinsonian tremor, 163

Parkinson’s disease (PD), 3–4, 113, 178, 237, 253,

296, 311

abnormal involuntary movements (AIMs), 296

abnormal metabolic brain networks in, See

abnormal metabolic networks, in PD animal models, See animal models, of PD application of trophic factors, therapeutic effects, 238

assessment of nigrostriatal DA function in, 177

Braak categorization, into six stages, 315

cardiac sympathetic denervation imaging in, 214

cardinal motor symptoms, 295–296

cell replacement therapies in, 193

cell transplantation in, 276

characterized clinically, 3–4, 162, 205, 237, 295

355

chronic fatigue in, 211–213 DA replacement, 238 dementia imaging in, 206 amyloid load, 209 cholinergic function, 208 dopaminergic function, 18F-dopa uptake, 207–208 glucose metabolism, 206–207 dementia with Lewy bodies (DLB), 206 depression imaging, 209–211 disorder involving, dopaminergic neuronal loss, 4 DJ-1 and Omi/HtrA2 genes associated, 3 dopamine replacement therapy, 3, 178 dopamine synthesis machinery, 222–223 feedback inhibition, by TH enzyme, 222 GTP cyclohydrolase 1 (GCH1), role, 222 tyrosine hydroxylase (TH) enzyme role, 222 Drosophila, as model system to study, 3 pink1 and parkin pathway, See Drosophila familial forms, clinical features, 4, 53 18 F-dopa PET role, 199 functional imaging with PET, progression assessment, 177, 187, 199–200 gene therapy for, dopamine replacement, 221 aromatic L-amino acid decarboxylase (AADC) role, 222 continuous DOPA delivery approach, 226–231 enzyme replacement strategies, for dopamine replacement, 225 viral vector-mediated replacement, 223–226 genetic factors role in, 53–54 genetic link, 114 goal of cell replacement therapies, 193 graft-induced dyskinesia and, See graft-induced dyskinesias (GIDs) imaging serotonin HT1A sites in, 210 levodopa-induced dyskinesia (LID), 178 levodopa treatment, 238, 311 loss of dopamine (DA) and, 237 measuring serotonin transporter (SERT) availability, 11C-DASB PET, 211 metabolic imaging, 161 modeling difficult task, pathological features, 18

ideal model, factors for, 26

neuroinflammatory pathogenesis, See neuroin­ flammatory pathogenesis model, of PD using toxins, See neurotoxicant-based models, of PD in vivo and in vitro, 120–121 motor symptoms, 237 multigenic forms, 4 network modeling, spatial covariance analysis, 161 neural grafting in, problems and possibilities, 265–266, 295 accommodating outcome differences, in both trials, 270–271

design of trials, 273–274

double-blind placebo-controlled

transplantation trials, 267–270 embryonic stem cells, as DA neurons source, 282–284 ethics and tissue procurement, fetal neural tissue, 276–277 graft-induced dyskinesias (GIDs), 272–273 graft tissue, preparation and placement, 271–272 immunosuppression and, 272 iPS cells use, 284–285 lineage-specific stem cells, as DA neurons source, 281–282 neural donor tissue storage, 278–280 neuropathological changes in grafts, issue, 274–276 open-label transplantation trials in, 266–267 patient selection, 271 safety and regulatory issues, SCs application in brain, 285–286

stem cell-derived neurons for, 280–281

tissue dissection, 277–278

neurotrophic factor therapy for, 237–238 brain-derived neurotrophic factor (BDNF), 239–241 glial cell-derived neurotrophic factor (GDNF) ligand family role, 242–250 Nerve Growth Factor (NGF) treatment, 239 neurotrophin-3–4/5 (NT-3, NT-4/5), 241–242 neurotrophin family, 238–239 neurturin (NTN) role, 250–252 novel neurotrophic factors, 252–253 in rodent models, NGF treatment, 239

356

Parkinson’s disease (PD), (Continued)

non-motor aspects imaging, 205–206

non-motor fluctuations in, 333

non-motor symptoms (NMS), 4, 325–326

complex, 327

management of, 334–336

nocturnal, 331

non-recognition of, 333

pathogenesis, 326

as preclinical (motor) feature, 329

predict emergence, of motor symptoms, 326–329

prevalence, symptoms, 329–333

self-admission of, 333–334

olfactory function in, 214–215

parkin, PINK1, and DJ-1 mutations in, 54

pathological hallmark, 113, 178, 205

patients having 11C-DASB PET,

demography, 212

presynaptic dopaminergic imaging, 177

progression and treatment-induced

complications monitoring

autopsy studies, 181

b-CIT imaging, 181

disparity between clinical and in vivo

measures, 182–183

ELLDOPA study, 182

F-DOPA PET, 181–182

F-DOPA uptake, in early PD, 181

metabolic network activity, changes in, 182

REAL-PET studies, 182

using UPRDS, 182–183

See also neuroimaging, of nigrostriatal system

radiotracer imaging (RTI) techniques, 178

reduced levels of BDNF and link between, 240

and related neurodegenerative disorders, 162

relationship between hyposmia level, and

hippocampal DAT availability, 215

rodent models, inflammatory contribution, 114

lipopolysaccharide, 117–118

MPTP, 114–115

6-OHDA, 115–116

pesticides, 116–117

See also neuroinflammatory pathogenesis

model, of PD

side-effects of transplants, 296

single gene-mediated, Mendelian forms of, 4

sleep disorders imaging in, 213–214

slowness of movement (bradykinesia), 295

a-synuclein role in pathogenesis, 89

See also viral vector model, of a-syn

overexpression

transplantation studies, and GID, 299

trichloroethylene (TCE) exposure, link to, 26

in vitro models, 18

Parkinson’s disease dementia (PDD), 206, 208–209

Parkinson’s Disease Rating Scale (UPDRS), 182

Parkinson’s disease-related spatial covariance

pattern (PDRP), 180, 182

Parkin ubiquitination, 5

PARK2 locus, 5

P1 artificial chromosome (PAC), 78

PDCP modulation, 168

PDGF-b promoter, overexpressing a-syn, 66–67

molecular alterations, 67–68

PD-like neurodegeneration, 92

PD-like neuropathological changes, 90

PD-like pathology, 120

PD-like syndrome, 6

PD-related cognitive pattern (PDCP), 163–165

PD-related motor pattern (PDRP), 162–163

PD-related tremor pattern, 164

PDRP expression, 162, 165, 168

PD tremor-related metabolic pattern (PDTP), 163

Persephin, 242

PET radioligands, 194

PET radiotracers, image b-amyloid plaque, 209

PET scans, 164, 171–172, 211

PFS-16 fatigue scores, 213

Pharmacological depletion, of dopamine

transmission, 36

Phosphatidyl-inositol 3-kinase, 10

PINK1-associated disease, 6

Pink1-dependent phosphorylation, 9

pink1 function, to regulate mitochondrial

integrity, 5–7

pink1/parkin pathway, 7

in Drosophila, advantages of study, 11–12

Platelet-derived growth factor

(PDGF)-b, 66–68

p75NTR receptor, 239

357

Positron emission tomography (PET), 177–178,

194, 206

Pramipexole, 186

presynaptic dopaminergic system, monitoring,

See graft survival, and presynaptic

dopaminergic system monitoring

Presynaptic, dopamine transporter (DAT), 194

Procedural learning, 44–45

Pro-inflammatory cytokines, 93–94

Proteasome-dependent protein degradation, 6

Protein Fis1, 8

Protein phosphatase 2A (PP2A), 97

PrP promoter

catecholamine systems in mice under, 71

external insults and a-syn expression,

interaction, 70–71

mice overexpressing

A30P variant under, 71–72

A53T variant, 68–70

Psychological stress, 124–125

Psychosis, in PD, 330–331

Psychosis-like behaviors, 145

PTEN-induced kinase 1, 4

6-Pyruvoyl-tetrahydropterin synthase, 222

RAC binding, 184, 186

Radiotracer imaging (RTI), 177–178

of nigrostriatal dopaminergic system, 183

as in vivo biomarker to assess, 182

Rapid eye movement (REM) behavior

disorder, 162

Rat, AAV-a-syn model in

motor impairments, 91

nigral cell loss, 91

TH-positive neurons, loss of, 91

time course variable, cell loss, 91

Reactive nitrogen (RON), 114

Reactive oxygen species (ROS), 6, 114, 116, 118, 121

Recombinant GDNF protein, 100

Regional cerebral flow (rCBF), 197–198

Regional cerebral glucose metabolism

(rCMRGlc) in humans, 206

REM behavior disorder (RBD), and PD, 328

REM sleep behavior, 172

REM sleep behavior disorders (RBD), 146

Reserpine, 36

Restless legs syndrome, 186

Rhomboid-7 protease, 9

RING-finger motifs, 5

Rotenone, 24–25, 117

S129A mutant a-syn, 96

Schizophrenia, 37

Sensorimotor tests, for rats, 39

Sepiapterin reductase, 222

Sexual dysfunction, in PD, 332

Single photon emission computerized tomography

(SPECT), 163, 178, 194, 206–207, 213–214

Skin lesions, 4

Sleep disorders, 146, 213–214

Sleep disruption, 4

Sleep regulatory centers, 213

SNc neurons, 164

Striatal DA nerve terminal density, 184

Striato-cortical circuitries, assessment of

restoration, 197–198

Substantia nigra (SN), 90, 92, 103, 105, 115, 119, 125

Substantia nigra neurons, 35

Substantia nigra pars compacta (SNC), 92, 113,

134–136, 148

Subthalamic nucleus (STN), 168

Supplementary motor area (SMA), 162

Supranuclear palsy (PSP), and abnormal

metabolic networks, 161

Synapsin promoters, 90–91

a
Syn119 expression, 78

a
Synuclein, 4–5, 18, 25, 38–39, 54

-mediated DAergic cell death, 95

-mediated neurodegeneration, 96

models, of PD, 79–80

overexpression, viral vector-mediated, See

AAV-mediated a-syn overexpression;

viral vector model, of a-syn overexpression

a-Syn toxicity mechanism, in DA neurons, 96–97

DA re-uptake, 98–99

DA storage and release, 98

DA synthesis, 97–98

dopamine-dependent toxicity, 99

and posttranslational modifications, 95

role of phosphorylation, 96

358

Targeting midbrain DA neurons, factors, 105–106

Target of rapamycin (TOR), 11

TCE exposure in rats, 26

5,6,7,8-Tetrahydro-L-biopterin (BH4), 222

TH-aSyn mice, alterations in, 66

TH enzyme, induced by PP2A, 98

Therapeutic STN lesioning, 168

TH promoter, and dopaminergic deficits, 54, 65–66

Thy1-aSyn mice

neuropathologies in, 76

time course of deficits in, 76–77

Thy-1 promoters, 66, 73

motor phenotype, 73–75

overexpression of a-syn under, 73

Transforming growth factor (TGF) superfamily, 242

Trichloroethylene (TCE), 26

Trophic factors, 238

Tumor necrosis factor (TNF), 93, 114, 121–122

Tyrosine hydroxylase (TH), 116, 124

enzyme, and inhibiton forms, 222–223

-positive neurons, 90

promoter

mutated gene and UPS impairment, 66

in overexpression of a-syn, 54–66

Tyrosine kinase A (TrkA) receptor, 239

Ubiquitinated VDAC1, 9

Ubiquitination, 96

dependent recruitment, 11

K27-linked, 6

K48-linked, 6

potential target of Parkin, 5

and removal of Mitofusin, 9

Ubiquitin proteasome system (UPS), 5, 66

Unified Parkinson’s Disease Rating Scale (UPDRS),

144, 147, 162, 182, 196–197, 213, 244

Unilateral hypokinesia, 134

Unilateral 6-OHDA lesions, 37

UPDRS motor ratings, 165

UPDRS motor scores, 196–197

UPSIT scores, and TRODAT uptake, 214

UPS/lysosomal degradation system, 95

Valsalva maneuvre, 214

VDAC1 in mammalian cells, 9

Ventral tegmental area (VTA), 91

Vesicular monoamine transporter 2 (VMAT2),

78, 98, 134, 148, 194, 222

Viral vector, 90

Viral vector-mediated dopamine replacement

clinical enzyme replacement, approaches AAV–AADC delivery approach, and drawback, 226–227 AAV mediated continuous DOPA delivery, efficacy, 227–229 behavioral and functional assessment, AAV DOPA delivery, 230 continuous DOPA delivery approach, advantages, 226–231 dopamine replacement strategy and results of, 224

four-gene delivery, 224

HSV-1/EIAV vectors, and drawback of,

224, 227

Prosavin vector, 226

early developments

BH4 in vivo, requirement for, 224

ex vivo gene transfer approach, 223

TH gene role, 223

in vivo gene transfer, 223–224

enzyme replacement strategies, 225

See also Parkinson’s disease (PD)

Viral vector model, of a-syn overexpression, 89–90

AAV-mediated a-syn overexpression, See AAV-mediated a-syn overexpression AAV vectors

to overexpress S129D mutant, 96

for rodents and primates, 90

use for overexpression of a-syn, 89–90, 100–

102

lentivirus (LV) vectors

transgene expression, in midbrain DA

neurons, 89–90

Viral vectors, delivering GDNF to PD

brain, 246

Virus H5N1, 124

VTA neurons, in mice, 91

WT protein, 78