Neurotoxic in vivo models of Parkinson’s disease

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

Progress in Brain Research, Vol. 184

ISSN: 0079-6123

Copyright
2010 Elsevier B.V. All rights reserved.

CHAPTER 2

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

Pittsburgh, PA, USA

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

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

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

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

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

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

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

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increases, prediction of toxin selectivity improves. Much epidemiological data also supports the notion that environmental neurotoxin exposure contributes to some cases of sporadic PD (Di Monte, 2001; Fall et al., 1999; Gash et al., 2008; Gorell et al., 1997, 1998; Hageman et al., 1999; Kuhn et al., 1998; Liou et al., 1997; Pezzoli et al., 1996; Semchuk et al., 1993; Tanner et al., 2009). Therefore, the choice to use neurotoxins to model PD is rooted in both the etiological data and the capacity to induce relevant pathogenic features.

Classic in vivo neurotoxicant-based models Animal models of PD have now existed for some 50 years. While there have been many attempts and derivations, a few models have persisted through the decades to provide an immense amount of invaluable data that has significantly contributed to our understanding of PD.

Early modeling of behavior and dopamine depletion Perhaps the earliest pharmacologic attempt at a PD model resulted from work by Carlsson and Hilarp in the 1950s, in which they depleted brain catecholamines using reserpine (Carlsson et al., 1957). This depletion resulted in akinesia, and sub­ sequent work identified dopamine depletion as the cause of the behavioral phenotype. The phenotype was rescued by administration of L-DOPA, the biochemical precursor to dopamine (Bertler et al., 1958; Carlsson et al., 1958). Ultimately, the work led to the conclusions that (1) dopamine depletion in the basal ganglia was central to the cardinal behavioral features of PD and (2) alleviation of symptoms could be achieved through L-DOPA supplementation (Bertler et al., Carlsson, 1959). These were powerful early observations elucidat­ ing the proximal cause of motor-related PD symp­ toms and the discovery of a treatment regimen that remains the single most effective therapy for

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

6-Hydroxydopamine History and summary The use of 6-hydroxydopamine (6-OHDA) as an experimental dopaminergic neurotoxin is one of the oldest and most utilized models of PD (for

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

27

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

Abbreviations 6-OHDA GI LPS MPP+ MPTP PD ROS

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

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