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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mutant mice and is not prevented by Bcl-2. Experimental Neurology, 175(1), 87–97. Ross, O. A., Soto, A. I., Vilarino-Guell, C., Heckman, M. G., Diehl, N. N., Hulihan, M. M., et al. (2008). Genetic variation of Omi/HtrA2 and Parkinson’s disease. Parkinsonism and Related Disorders, 14, 539–543. Rubin, G. M., Yandell, M. D., Wortman, J. R., Gabor Miklos, G. L., Nelson, C. R., et al. (2000). Comparative genomics of the eukaryotes. Science, 287, 2204–2215 Sandebring, A., Thomas, K. J., Beilina, A., van der Brug, M., Cleland, M. M., Ahmad, R., et al. (2009). Mitochondrial alterations in PINK1 deficient cells are influenced by calci­ neurin-dependent dephosphorylation of dynamin-related protein 1. PLoS ONE, 4(5), e5701. Schneider, S.A., Paisan-Ruiz, C., Quinn, N.P., Lees, A.J., Houlden, H., Hardy, J., et al. (2010). ATP13A2 mutations (PARK9) cause neurodegeneration with brain iron accumu­ lation. Movement Disorders, 25, 979–984. Sherer, T. B., Kim, J. H., Betarbet, R., & Greenamyre, J. T. (2003). Subcutaneous rotenone exposure causes highly selec­ tive dopaminergic degeneration and alpha-synuclein aggre­ gation. Experimental Neurology, 179(1), 9–16. Simon-Sanchez, J., & Singleton, A. B. (2008). Sequencing ana­ lysis of OMI/HTRA2 shows previously reported pathogenic mutations in neurologically normal controls. Human Mole­ cular Genetics, 17, 1988–1993. Simuni, T., & Sethi, K. (2008). Nonmotor manifestations of Parkinson’s disease. Annals of Neurology, 64(S2), S65–S80. Singleton, A. B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J., et al. (2003). a-Synuclein locus triplication causes Parkinson’s disease. Science, 302(5646), 841. St Johnston, D. (2002). The art and design of genetic screens: Drosophila melanogaster. Nature Reviews Genetics, 3(3), 176–188. Strauss, K. M., Martins, L. M., Plun-Favreau, H., Marx, F. P., Kautzmann, S., Berg, D., et al. (2005). Loss of function mutations in the gene encoding Omi/HtrA2 in Parkinson’s disease. Human Molecular Genetics, 14(15), 2099–2111. Tain, L. S., Mortiboys, H., Tao, R. N., Ziviani, E., Bandmann, O., & Whitworth, A. J. (2009). Rapamycin activation of 4E­ BP prevents parkinsonian dopaminergic neuron loss. Natural Neuroscience, 12(9), 1129–1135. Tang, B., Xiong, H., Sun, P., Zhang, Y., Wang, D., Hu, Z., et al. (2006). Association of PINK1 and DJ-1 confers digenic inheritance of early-onset Parkinson’s disease. Human Mole­ cular Genetics, 15(11), 1816–1825. Trinh, K., Moore, K., Wes, P. D., Muchowski, P. J., Dey, J., Andrews, L., et al. (2008). Induction of the phase II detoxifica­ tion pathway suppresses neuron loss in Drosophila models of Parkinson’s disease. Journal of Neuroscience, 28 (2), 465–472. Twig, G., Elorza, A., Molina, A. J., Mohamed, H., Wikstrom, J. D., Walzer, G., et al. (2008). Fission and selective fusion govern mitochondrial segregation and elimination by autop­ hagy. EMBO Journal, 27(2), 433–446.

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

17

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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 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

105

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.

107

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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

134

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.

135

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

136

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 (90% specificity) even in the subgroups of early patients with very short symptom durations (i.e.,