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Environmental toxins and Parkinson's disease: what have we learned from pesticide-induced animal models?
Review
Environmental toxins and Parkinson’s disease: what have we learned from pesticide-induced animal models? Francesca Cicchetti1,2, Janelle Drouin-Ouellet1 and Robert E. Gross3,4 1
Centre de Recherche du CHUL (CHUQ), Axe Neuroscience, RC-9800, 2705, Boulevard Laurier, Que´bec, QC, G1V 4G2, Canada De´partement de Psychiatrie/Neuroscience, Universite´ Laval, Que´bec, QC, G1K 7P4, Canada 3 Department of Neurosurgery and Neurology, Emory University School of Medicine, 136B Clifton Road, Atlanta, GA 30322, USA 4 Center for Neurodegenerative Diseases, Emory University School of Medicine, 136B Clifton Road, Atlanta, GA 30322, USA 2
Parkinson’s disease (PD) is a common neurodegenerative disorder largely of idiopathic nature with the exceptions of rare familial forms, and is characterized by both motor and non-motor disturbances. Pathologically, most motor features are the result of a dramatic loss of ventral tier mesencephalic dopaminergic neurons and thus dopamine content at their target sites. Although the exact etiology of the disease remains to be elucidated, it is thought to be multifactorial, with a critical role for environmental factors, such as pesticides, that may act on genetically predisposed individuals. Arising from consideration of the potential environmental triggers of PD, in vivo animal models of the disease utilizing these compounds are increasingly reported in the literature. Here, we review recent advances in the predominant models employing the insecticide Rotenone, the herbicide Paraquat and the fungicide Maneb, discuss their scientific merit and evaluate their relevance in the study of PD pathogenesis. Introduction Parkinson’s disease (PD) is a neurodegenerative disorder typified in part by motor disturbances, including tremor, rigidity, and bradykinesia [1], originating from loss of dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). PD pathology is not restricted to the DA system, however, progressively involving noradrenergic and serotonergic neurons within the locus ceruleus and raphe nucleus, for example. Degeneration in these and other structures induces non-motor symptomatology including autonomic, mood, arousal and cognitive disturbances [2]. Additionally, predominant peripheral abnormalities (olfactory deficit or constipation) in PD are present early in the course, even before any pathological signs are visible in the central nervous system (CNS). The symptomatic features of familial and genetic forms of PD as compared to sporadic PD are distinguishable only by the occurrence of onset at young age (typically <40 years) in the former. On the cellular level, the pathological hallmark of PD is the presence of Lewy bodies - nuclear inclusions composed predominantly of the protein a-synuclein - found in both idiopathic and genetic forms of PD. Corresponding author: Cicchetti, F. ([email protected]).
The etiology of PD has yet to be convincingly established. Prevalence increases exponentially from ages 65 to 90. While a fraction of PD occurrence is related to mutations in genes such as a-synuclein and parkin, over 90% of PD is likely linked to environmental causes, in part due to pesticide exposure [3]. Specifically, the herbicide Paraquat (PQ) [4] and the fungicide Maneb (MB: manganese ethylene-bis-dithiocarbamate) [5] have been associated with the incidence of PD. However, a causal role for pesticides in the etiology of PD has yet to be definitively established. Current benchmark criteria for an adequate animal model of PD Animal models can provide a critical link in establishing a causal role for environmental toxins such as pesticides in the etiology of PD by allowing studies that are impossible to perform in patients. Their usefulness critically resides in the translatability of biochemical findings to the human condition. Ultimately, however, animal models are limited by their failure to mimic perfectly the disease state, related to several potential shortcomings. (i) Inadequate characterization of the disease state being modeled. Early appreciation of prominent motor signs resulting from the nigral DA neuronal loss [2] dominated the criteria for an adequate animal model of PD (Box 1), although now appreciation of the widespread pathological features must drive further model development. Additionally, recognition of the pathognomonic relevance of nigral Lewy bodies in human disease has designated their presence as another criterion for an adequate animal model (Box 1), although their true pathogenetic significance is debated [6]. (ii) Inadequate understanding of the effect of route of exposure on pathogenesis. In human disease, exposure to toxins is probable via ingestion or inhalation. Although one possibility is that the final effects are mediated through the blood brain barrier (BBB), recent advances in staging PD strongly suggest that route of entry into the nervous system may in fact be directly through neurons in the gastrointestinal system or the olfactory region. Similar routes of exposure, which may be ‘part and parcel’ to the disease state may not be feasible in animal models, especially over the protracted period of time over which human exposure probably occurs. As a result, realistic animal
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Review Box 1. Criteria for Pesticide Animal Models of PD Current criteria Behavioral motor impairments responsive to DA therapy Progressive and significant neurodegeneration of DA nigrostriatal neurons Inclusion bodies (Lewy bodies) in surviving DA neurons Updated criteria Degeneration in non-DA structures of the central nervous system implicated in PD Degeneration of other neurotransmitter systems (noradrenergic, serotonergic and cholinergic) Degeneration of other structures (locus ceruleus, pedunculopontine nucleus, raphe nucleus, dorsal motor nucleus of the vagus, anterior olfactory nucleus) Behavioral impairments associated with degeneration of non-DA central nervous system regions Autonomic dysfunctions, mood and cognitive disturbance Peripheral abnormalities (e.g. olfactory deficit) Reproducibility Attention to factors underlying variability (animal diets, mouse strain, gender, age, housing conditions) Reports of detailed methodology
models may not mirror the true human condition. (iii) Biochemical and/or anatomical differences between animals and humans. Differences in the BBB, neuroanatomical pathways, or even in nasal filtration of environmental toxins may affect the response of animals to exposure routes and doses similar to those seen by humans. Despite these potential shortcomings, recent years have seen a proliferation of studies examining the pathogenesis of nigrostriatal degeneration in animal models using exposure to environmental toxins, with a specific emphasis on the utilization of pesticides. Two distinct pesticideinduced animal models of PD have dominated this field of research: the Rotenone model and the Paraquat (PQ) plus Maneb (MB) model. Both were initiated to take account of the possible environmental origin of PD, yet probably neither mimics actual human exposure in terms of particular chemicals or timing and route of exposure. Both were also developed to reproduce human parkinsonian features, but neither has taken account of non-motor symptoms that predominate later in the disease. Improving reproducibility, a key component of any legitimate animal model, has been the goal of much recent research, as well as specificity both within the nervous system and outside of it. The intent of this review is to examine the data that have emerged from the use of environmental neurotoxins in rodents and discuss the experimental value of these new in vivo models in the study of PD. We will exclusively review the status of current animal models produced by exposure to Rotenone (insecticide), PQ (herbicide), and MB (fungicide). Rotenone, PQ and MB: The compounds and their mechanisms of action Rotenone, a chemical that belongs to the family of isoflavones naturally found in the roots and stems of several plants, is used as a broad-spectrum pesticide. Surprisingly, Rotenone can be used in organic food farming, based on its 476
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label as a natural product. Highly lipophilic, it easily crosses the BBB, and for cellular entry [1], does not depend on the dopamine transporter (DAT) – a transmembrane protein, residing in DA neurons, whose purpose is to clear DA from the synaptic cleft and which is a potential route for specific entry of neurotoxic agents into DA neurons (Figure 1). Once in the cell, Rotenone accumulates at mitochondrial complex I where it inhibits the transfer of electrons from iron-sulfur (Fe-S) centers to ubiquinone. Increased reactive oxygen species (ROS) production has been associated with complex I dysfunction induced by Rotenone, which may produce oxidative damage to DNA and proteins of neural cells. Nitric oxide (NO) can interact with ROS, especially superoxide and hydroxyl radicals, leading to peroxinitrite formation, ultimately resulting in cellular defects and damage to DA neurons [7]. More recently, Rotenone was shown to inhibit proteosome activity [8]. Dysfunction in proteosomes - intracellular structures that catalyze the degradation of ubiquitintagged proteins - has been implicated in the pathogenesis of both genetic and sporadic forms of PD [9]. PQ is one of the most widely used herbicides in the world. It is considered to be quick acting, non-selective, and seems to specifically destroy green plant tissue on contact. In the United States, PQ is classified under restricted use, whereas in the European Union PQ usage has been forbidden since July 2007. PQ is suspected to enter the brain by neutral amino acid transporters and subsequently the cells in a sodium-dependent fashion [10]. Once within cells of the CNS, PQ acts as a redox cycling compound at the cytosolic level, which potentially leads to indirect mitochondrial toxicity [11], although low affinity to mitochondrial complex I at high doses has been reported (Figure 1). In addition to these more classic mechanisms of action, recent reports show that PQ-induced apoptosis may involve Bak protein, a pro-apoptosis Bcl-2 family member [12], although the complexity of these newly suggested mechanisms is beyond the scope of this review. MB, which is used as a fungicide, is an irritant to respiratory tracts and is capable of inducing sensitization by skin contact. Mechanistically, and based on evidence discussed below, MB seems to cross the BBB. Although knowledge of the mechanisms of this toxin is very limited, MB preferentially inhibits mitochondrial complex III [13] (Figure 1). Further, MB was recently shown to induce apoptosis through Bak activation, whereas combination of PQ/MB inhibits the Bak-dependent pathway while potentiating apoptosis via Bax protein [14]. Rotenone models of PD Although the effects of Rotenone on the brain were first tested over 20 years ago, the model received the most attention when reproduced with a chronic mode of intravenous (i.v.) delivery [15]. Rotenone infusion via osmotic mini-pumps produced motor deficits reminiscent of several clinical features of PD – including hypokinesia, rigidity, hunched posture, unsteady movements and even resting tremor, the severity of which was associated with the extent of lesions. These motor impairments correlated with irreversible degeneration of nigral DA neurons. In two animals, amelioration of motor deficits was noted in
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Figure 1. Mechanisms of actions of various toxins used to produce PD models. Schematic illustration of mechanisms of actions of the most utilized toxins to induce PD features in rodents, based on current evidence. The precise mechanisms by which these toxins cross the BBB are not known. Rotenone likely crosses because it is highly lipophilic, whereas PQ has been suggested to use neutral amino acid and sodiumdependent transporters. The ability of MB to enter the brain has not been specifically demonstrated but is derived from studies showing its effects on this organ after systemic injection. All compounds, with the probable exception of PQ, target the mitochondrial complex. Complex I is more specifically targeted by Rotenone and MB attacks complex III, whereas mitochondrial dysfunction induced by PQ could emerge indirectly from redox cycling in the cytosol. This interaction of the compound at the respiratory cell level is extremely detrimental and ultimately leads to cell death. For comparative purposes, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the toxin most frequently used to generate animal models of PD, has been illustrated. MPTP is transported across the BBB and is converted to its toxic form 1-methyl-4phenylpyridinium (MPP+) in astrocytes via the enzyme MAO and ultimately released into the extracellular milieu by the plasma membrane transporter Oct3. MPP+ subsequently enters dopamine neurons by specific dopamine transporters and oxidative stress is generated followed by cell death. Abbreviations: AA: amino acid; ATP: Adenosine triphosphate; BBB: Blood brain barrier; DAT: dopamine transporter; MAO: monoamine oxidase; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine; UPS: ubiquitin proteosome system.
response to apomorphine treatment – a mixed D1/D2 agonist, supporting the hypothesis that the motor deficits were due to DA depletion [15]. Notably, ubiquitin and asynuclein inclusions reminiscent of Lewy bodies were discerned in nigral neurons in animals in which DAergic degeneration was observed, although the DAergic identity of neurons with inclusions was not examined. At the striatal level, loss of tyrosine hydroxylase (TH)-immuno-
reactive terminals was both dose and duration dependent, whereas levels of glutamic acid decarboxylase (GAD; indicative of GABAergic medium spiny striatal projection neurons) and acetylcholinesterase (AChE; indicative of cholinergic striatal interneurons) were not affected by the Rotenone treatments, arguing for DA specificity of the Rotenone effects. Importantly, Sprague–Dawley rats submitted to the same Rotenone regimen were ultimately 477
Review excluded from the study because of inconsistency in lesion production. Higher doses originally tested (up to 12 mg/kg) for short periods of time induced cardiovascular systemic toxicity and non-specific lesions. There are several issues that have hampered acceptance of the chronic i.v. Rotenone model, the first of which is variability. To address this, other routes of administration have been pursued. Similar to the i.v. route, the subcutaneous (s.c.) route is beset by variability (and mortality; see summary Table 1). Recently, daily intraperitoneal (i.p.) delivery for up to 60 days of similar doses as previously reported, with dissolution of Rotenone in a novel fatty-acid based vehicle, has achieved high reproducibility of the lesions [16]. Similar to previous reports, this model seems to generate specific DA lesions, accompanied by the presence of a-synuclein and poly-ubiquitin aggregates in remaining nigral DA neurons, although the striatal lesion still displays typical focal denervation. More recently, other modes of administration have been explored – such as oral [17] and intranasal [18] delivery – that are arguably more realistic with regard to potential entry sites for toxin exposure in human PD. Unfortunately, these novel routes have only been reported in single studies with limited pathological analysis (Table 1). The second important issue facing the Rotenone model is evidence demonstrating marked systemic non-specificity [15,19–27], leading to high mortality related to systemic adverse effects including cardiac, stomach and liver problems. The relationship of behavioral motor changes specifically to nigrostriatal DAergic degeneration has not been convincingly established. The hunched back posture and hypokinesia originally reported conceivably could have been related to severe peripheral abnormalities, observed by other investigators [22]. In the more recent i.p. model, despite an important body of literature demonstrating significant peripheral problems generated by various routes of Rotenone delivery in rodents, the apparently parkinsonian behavioral deficits were only considered as being the result of specific lesions [16]. Although apomorphine responsiveness of at least some of the symptoms suggests DA specificity to the behavioral dysfunction, no autopsies were conducted to evaluate peripheral abnormalities (even though all animals were sacrificed due to severe behavioral dysfunction), which might account for some of these behavioral changes as previously reported [22]. The third critical issue hampering the Rotenone model is non-specificity within the CNS, as has been observed by some laboratories. The striatal degeneration described is not closely related to human PD: a recent report assessing the last five years of research on the Rotenone model concluded that Rotenone does not reproduce the pathology of PD but rather induces a pattern of pathological changes more akin to atypical parkinsonism, characterized, for example, by striatal degeneration in addition to nigral neuronal loss [28]. Although a similar pattern of neuropathological changes to their initial i.v. model was observed [15], it will be critical for other laboratories to evaluate the new i.p. delivery approach [16] before its specificity can be considered established. 478
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What have we learned from the Rotenone model of PD? As discussed, issues of variability, mortality, non-specificity both within and outside the CNS, and reproducibility across laboratories remain major challenges for the chronic Rotenone model of PD. In addition, there remain critical issues regarding the translatability of the model: does the Rotenone model truly recapitulate human PD? The first issue is route of exposure. Although it is possible that in human disease Rotenone, or other toxins, gains access to the CNS via the BBB, and that i.v. or i.p. administration mimics this route, it is perhaps more likely that introduction is via direct exposure of neurons in the gut and/or olfactory region [29]. Thus the Rotenone model might fail to replicate a critical aspect of human disease, which could in fact bear as well on the second critical issue. As discussed above, non-specificity with respect to the nigrostriatal system is a feature that is now appreciated as characteristic of human PD. Could the non-specificity observed in Rotenone treated rodents reflect this feature of human PD? An analysis of the presence of Lewy body formation or a-synuclein accumulation has not been reported in structures other than SNpc and striatum (e.g. the dorsal motor nucleus of the vagus), as seen in human PD. Other non-DA system related dysfunction (e.g. cognitive aspects), has not yet been assessed in this model. On the other hand, perhaps the systemic effects observed – while not at all reminiscent of observations in human PD – could be re-evaluated in light of the fact that PD may originate from the peripheral system [2]. Future studies should include histopathological analyses of other systems and perhaps attempt to evaluate the model from a different angle, shedding light onto the potential value of these ‘‘nonspecific’’ effects in reproducing other aspects of PD pathology. PQ/MB: Synergistic effects of compounds and developmental vulnerability An important aspect of human pesticide exposure is that it is usually to a number of agents, rather than a single one, and it occurs over time. In addition, there is the possibility that in utero exposure to toxins, such as lipopolysaccharide as part of bacterial vaginosis during pregnancy, predisposes to specific nigral DA cell loss [30]. Based on these premises, several studies have been initiated to explore this concept in animals. Concurrent exposure to PQ and MB in adult mice led to significantly greater DA fiber loss, and greater 3,4-dihydroxyphenylacetic acid (DOPAC) and DA turnover, than exposure to either toxin alone. Locomotor activity was decreased after MB alone or after combined treatment [31,32]. A subsequent study, utilizing a twice a week for six weeks exposure scheme, showed DA damage, decrease in TH protein levels and DOPAC as well as nigral DA loss with combined PQ and MB only. The latter treatment alone also had adverse effects on behavior [33,34]. It was also recently shown that administration of PQ/ MB post-natally to mice led to a 38% loss of DA neurons in adulthood, whereas when post-natal exposure was followed by adult re-exposure there was a 70% loss of DA neurons in the nigra and a concomitant decrease in
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Table 1. Synopsis of Rotenone-induced animal models
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Review locomotor behavior [50]. This study suggests a period of ‘‘silent neurotoxicity’’ that predisposes adult animals to an exaggerated response to the same agents following readministration. A variation of this scheme has investigated the effects of prenatal s.c. MB injection in combination with adult i.p. PQ delivery. Nigral DA cell loss, and striatal decreases in DA, DA metabolites (DOPAC, homovanilic acid), DA turnover (reflecting decreased synthesis as well as breakdown, the latter mediated by DA neurons after DAT-mediated uptake), and serotonin were produced only after prenatal MB followed by adult i.p. PQ administration. This particular treatment also induced 95% decrease of locomotor activity in adult treated animals [35]. The findings reveal an interaction between age of exposure and the effects of neurotoxins. Older mice have been reported to be more susceptible to systemic PQ/MB treatment [34,36,37], perhaps due to changes in transport across the BBB [36,38]. We have found similar effects of PQ/MB exposure in older adult rats (six months), but especially with regard to the induction of lung pathology. In contrast to two month old rats, where only three out of 16 subjects became ill following four weeks of repeated exposures [39], 52% of older treated animals either died or had to be sacrificed as a result of severe weight loss and/or signs of respiratory distress following just two biweekly systemic injections of PQ/MB [40]. Others have also reported the presence of dichotomous populations in six week old rats in which one group suffers from systemic toxicity and another group recovers and displays no lung pathology [41]. We have made similar observations in mice [42], which have not been noted in previous studies. What have we learned from the PQ/MB-induced animal model of PD? The PQ/MB model has certainly been useful for its demonstration of potential synergistic effects of various environmental compounds in producing PD features in animals. The various experimental approaches utilized to develop this model have further provided support for a multi-hit hypothesis, which is interesting in that it replicates likely scenarios of intoxication in humans, although the routes of delivery remain, as for the Rotenone model, improbable occurrences and, contrary to the primary rationale, these compounds are not used simultaneously in agriculture [3]. Overall, the PQ/MB model meets several of the classic criteria, including behavioral motor impairments, significant DA-related degeneration and responsiveness to DA therapy. Importantly, it has met with more consistency among research groups. Conversely, to our knowledge, inclusion bodies have not been reported in the multiple variants that exist for this model (Table 2) and neither have non-motor impairments. Unfortunately, some nonspecific and undesirable peripheral effects [18,39–43], particularly to the lungs, provoking respiratory distress, have been reported for this model, limiting its utilization, especially since these effects are not related to human forms of PD. Future studies should be directed at examining for pathology in non-motor and gastrointestinal systems involved in early PD. 480
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Concluding remarks Are pesticide models useful in the investigation of the etiopathogenesis of human PD? First, the specificity of the pathological lesions induced raises doubts about the adequacy of these models, perhaps especially so for Rotenone. Lack of specificity may indicate disparate biochemical mechanisms underlying the degenerative process, or may reflect a failure to replicate the correct exposure route or time-course. Specificity may derive from the route of spread of the pathological process within the CNS, or indeed the peripheral nervous system, which is not replicated by more convenient routes and time-courses in rodents where it is produced by i.v., s.c. or i.p. administration. Non-specificity extends to the systemic toxicity observed with both Rotenone and PQ/MB exposure. At the least, this systemic toxicity adds a substantial experimental burden to utilizing these models. However, it too may reflect failure to replicate the appropriate exposure routes and time-course in human disease. Conversely, current models are in another sense too specific, not ostensibly incorporating the increasingly appreciated non-motor features of human PD. The validity of animal models heretofore has been predicated simply on modeling the motor features of PD. Indeed, until we have a full understanding of the extent of PD pathology, conceptual animal models will be difficult to generate and evaluate, and by the same token current animal models will provide a limited understanding of what precisely takes place in PD. Again, it is conceivable that route of exposure to toxins is a critical and integral part of the pathogenetic mechanism in generating the nature and range of pathological abnormalities that define bona fide PD. In that regard, a recent provocative hypothesis posits that all of the motor and non-motor systems observed to be involved pathologically can be accounted for by initial exposure to the olfactory system followed by extrasynaptic cell-to-cell transmission of aberrantly folded a-synuclein [29]. Perhaps more realistic routes of delivery would constitute a good starting point for producing models that mimic the disease’s pathological aspects and continued efforts should unquestionably put forward protocols that address this issue. Moreover, it may be that present efforts have failed to probe for these alterations, constituting another avenue for further exploration. In the end, however, it may be that the disease is so complex and develops over such a protracted period of time that it may be overly optimistic to expect to reproduce all these aspects in feasible small animal models, and defined models that recapitulate select aspects of the disease – as has been done with the motor aspects – may be the best that can be expected of animal models of PD. Finally, reproducibility is paramount to the value of any animal model. Variability in pesticide models is likely multifactorial. One factor, for example, may relate to animal diets, in that rodent chow composition varies depending on the provider and seasonal foods available. Several studies have reported the neuroprotective role of various diet components, like phytoestrogens, to the DA system. It is feasible that diets, which vary between laboratories, are responsible, at least in part, for the inconsistencies in the level of toxin-induced DA neurodegeneration reported. Mouse strain, gender, age, and housing con-
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Table 2. Synopsis of PQ/MB-induced animal models
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Review ditions also may contribute to these disparities. In order to move the field forward and to save resources, it is imperative to systematically report positive as well as negative data. The elaboration of new animal models of any disease is a challenging task and should not be undermined by limited sharing of experimental details. Reproducibility, which is a key factor in the development of valuable animal models of PD, will only find support on well-founded methodologies. Disclosure None of the authors has conflict of interests to disclose. Acknowledgments The authors wish to acknowledge the support of the Fondation Canadienne pour l’Innovation and the Canadian Institute of Health Research to Francesca Cicchetti. Janelle Drouin-Ouellet was initially supported by a master’s scholarship from Fonds de Recherche en Sante´ du Que´bec and subsequently by a Canada Frederick Banting and Charles Best doctoral scholarship. Robert Gross was supported in part by National Institutes of Health grants from the Emory Collaborative Center for Parkinson’s Disease Environmental Research from NIEHS and by a K08 Career Development Award (NS46322) from NINDS.
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Review 44 Alam, M. et al. (2004) The neurobehavioral changes induced by bilateral rotenone lesion in medial forebrain bundle of rats are reversed by L-DOPA. Behav Brain Res 151, 117–124 45 Antkiewicz-Michaluk, L. et al. (2004) Protective effect of 1-methyl-1,2,3,4-tetrahydroisoquinoline against dopaminergic neurodegeneration in the extrapyramidal structures produced by intracerebral injection of rotenone. Int J Neuropsychopharmacol 7, 155–163 46 Saravanan, K.S. et al. (2005) Acute intranigral infusion of rotenone in rats causes progressive biochemical lesions in the striatum similar to Parkinson’s disease. Brain Res 1049, 147–155 47 Sindhu, K.M. et al. (2005) Behavioral differences in a rotenone-induced hemiparkinsonian rat model developed following intranigral or median forebrain bundle infusion. Brain Res 1051, 25–34 48 Sindhu, K.M. et al. (2006) Rats with unilateral median forebrain bundle, but not striatal or nigral, lesions by the neurotoxins MPP+ or rotenone display differential sensitivity to amphetamine and apomorphine. Pharmacol Biochem Behav 84, 321–329 49 Saravanan, K.S. et al. (2006) L-deprenyl protects against rotenoneinduced, oxidative stress-mediated dopaminergic neurodegeneration in rats. Neurochem Int 49, 28–40 50 Garcia-Garcia, F. et al. (2005) Sleep disturbances in the rotenone animal model of Parkinson disease. Brain Res 1042, 160–168 51 Milusheva, E. et al. (2005) Increased sensitivity of striatal dopamine release to H2O2 upon chronic rotenone treatment. Free Radic Biol Med 39, 133–142 52 Sherer, T.B. et al. (2003) Subcutaneous rotenone exposure causes highly selective dopaminergic degeneration and alpha–synuclein aggregation. Exp Neurol 179, 9–16 53 Ling, Z. et al. (2004) Rotenone potentiates dopamine neuron loss in animals exposed to lipopolysaccharide prenatally. Exp Neurol 190, 373–383 54 Yang, Y. et al. (2005) Systematic administration of iptakalim, an ATPsensitive potassium channel opener, prevents rotenone-induced motor and neurochemical alterations in rats. J Neurosci Res 80, 442–449
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55 Huang, J. et al. (2006) A delivery strategy for rotenone microspheres in an animal model of Parkinson’s disease. Biomaterials 27, 937–946 56 Betarbet, R. et al. (2006) Intersecting pathways to neurodegeneration in Parkinson’s disease: effects of the pesticide rotenone on DJ-1, alphasynuclein, and the ubiquitin- proteasome system. Neurobiol Dis 22, 404–420 57 Phinney, A.L. et al. (2006) Enhanced sensitivity of dopaminergic neurons to rotenone- induced toxicity with aging. Parkinsonism Relat Disord 12, 228–238 58 He, Y. et al. (2003) Role of nitric oxide in rotenone-induced nigrostriatal injury. J Neurochem 86, 1338–1345 59 Manning-Bog, A.B. et al. (2002) The herbicide paraquat causes upregulation and aggregation of alpha-synuclein in mice: paraquat and alpha-synuclein. J Biol Chem 277, 1641–1644 60 Peng, J. et al. (2004) The herbicide paraquat induces dopaminergic nigral apoptosis through sustained activation of the JNK pathway. J Biol Chem 279, 32626–32632 61 Ossowska, K. et al. (2005) A slowly developing dysfunction of dopaminergic nigrostriatal neurons induced by long-term paraquat administration in rats: an animal model of preclinical stages of Parkinson’s disease? Eur J Neurosci 22, 1294–1304 62 Ossowska, K. et al. (2005) Influence of paraquat on dopaminergic transporter in the rat brain. Pharmacol Rep 57, 330–335 63 Li, X. et al. (2005) Paraquat induces selective dopaminergic nigrostriatal degeneration in aging C57BL/6 mice. Chin Med J (Engl) 118, 1357–1361 64 Shepherd, K.R. et al. (2006) The potentiating effects of 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) on paraquat-induced neurochemical and behavioral changes in mice. Pharmacol Biochem Behav 83, 349–359 65 Shimizu, K. et al. (2003) Paraquat induces long-lasting dopamine overflow through the excitotoxic pathway in the striatum of freely moving rats. Brain Res 976, 243–252 66 Kuter, K. et al. (2007) Toxic influence of subchronic paraquat administration on dopaminergic neurons in rats. Brain Res 1155, 196–207
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Environmental toxins and Parkinson’s disease: what have we learned from pesticide-induced animal models? Francesca Cicchetti1,2, Janelle Drouin-Ouellet1 and Robert E. Gross3,4 1
Centre de Recherche du CHUL (CHUQ), Axe Neuroscience, RC-9800, 2705, Boulevard Laurier, Que´bec, QC, G1V 4G2, Canada De´partement de Psychiatrie/Neuroscience, Universite´ Laval, Que´bec, QC, G1K 7P4, Canada 3 Department of Neurosurgery and Neurology, Emory University School of Medicine, 136B Clifton Road, Atlanta, GA 30322, USA 4 Center for Neurodegenerative Diseases, Emory University School of Medicine, 136B Clifton Road, Atlanta, GA 30322, USA 2
Parkinson’s disease (PD) is a common neurodegenerative disorder largely of idiopathic nature with the exceptions of rare familial forms, and is characterized by both motor and non-motor disturbances. Pathologically, most motor features are the result of a dramatic loss of ventral tier mesencephalic dopaminergic neurons and thus dopamine content at their target sites. Although the exact etiology of the disease remains to be elucidated, it is thought to be multifactorial, with a critical role for environmental factors, such as pesticides, that may act on genetically predisposed individuals. Arising from consideration of the potential environmental triggers of PD, in vivo animal models of the disease utilizing these compounds are increasingly reported in the literature. Here, we review recent advances in the predominant models employing the insecticide Rotenone, the herbicide Paraquat and the fungicide Maneb, discuss their scientific merit and evaluate their relevance in the study of PD pathogenesis. Introduction Parkinson’s disease (PD) is a neurodegenerative disorder typified in part by motor disturbances, including tremor, rigidity, and bradykinesia [1], originating from loss of dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). PD pathology is not restricted to the DA system, however, progressively involving noradrenergic and serotonergic neurons within the locus ceruleus and raphe nucleus, for example. Degeneration in these and other structures induces non-motor symptomatology including autonomic, mood, arousal and cognitive disturbances [2]. Additionally, predominant peripheral abnormalities (olfactory deficit or constipation) in PD are present early in the course, even before any pathological signs are visible in the central nervous system (CNS). The symptomatic features of familial and genetic forms of PD as compared to sporadic PD are distinguishable only by the occurrence of onset at young age (typically <40 years) in the former. On the cellular level, the pathological hallmark of PD is the presence of Lewy bodies - nuclear inclusions composed predominantly of the protein a-synuclein - found in both idiopathic and genetic forms of PD. Corresponding author: Cicchetti, F. ([email protected]).
The etiology of PD has yet to be convincingly established. Prevalence increases exponentially from ages 65 to 90. While a fraction of PD occurrence is related to mutations in genes such as a-synuclein and parkin, over 90% of PD is likely linked to environmental causes, in part due to pesticide exposure [3]. Specifically, the herbicide Paraquat (PQ) [4] and the fungicide Maneb (MB: manganese ethylene-bis-dithiocarbamate) [5] have been associated with the incidence of PD. However, a causal role for pesticides in the etiology of PD has yet to be definitively established. Current benchmark criteria for an adequate animal model of PD Animal models can provide a critical link in establishing a causal role for environmental toxins such as pesticides in the etiology of PD by allowing studies that are impossible to perform in patients. Their usefulness critically resides in the translatability of biochemical findings to the human condition. Ultimately, however, animal models are limited by their failure to mimic perfectly the disease state, related to several potential shortcomings. (i) Inadequate characterization of the disease state being modeled. Early appreciation of prominent motor signs resulting from the nigral DA neuronal loss [2] dominated the criteria for an adequate animal model of PD (Box 1), although now appreciation of the widespread pathological features must drive further model development. Additionally, recognition of the pathognomonic relevance of nigral Lewy bodies in human disease has designated their presence as another criterion for an adequate animal model (Box 1), although their true pathogenetic significance is debated [6]. (ii) Inadequate understanding of the effect of route of exposure on pathogenesis. In human disease, exposure to toxins is probable via ingestion or inhalation. Although one possibility is that the final effects are mediated through the blood brain barrier (BBB), recent advances in staging PD strongly suggest that route of entry into the nervous system may in fact be directly through neurons in the gastrointestinal system or the olfactory region. Similar routes of exposure, which may be ‘part and parcel’ to the disease state may not be feasible in animal models, especially over the protracted period of time over which human exposure probably occurs. As a result, realistic animal
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Review Box 1. Criteria for Pesticide Animal Models of PD Current criteria Behavioral motor impairments responsive to DA therapy Progressive and significant neurodegeneration of DA nigrostriatal neurons Inclusion bodies (Lewy bodies) in surviving DA neurons Updated criteria Degeneration in non-DA structures of the central nervous system implicated in PD Degeneration of other neurotransmitter systems (noradrenergic, serotonergic and cholinergic) Degeneration of other structures (locus ceruleus, pedunculopontine nucleus, raphe nucleus, dorsal motor nucleus of the vagus, anterior olfactory nucleus) Behavioral impairments associated with degeneration of non-DA central nervous system regions Autonomic dysfunctions, mood and cognitive disturbance Peripheral abnormalities (e.g. olfactory deficit) Reproducibility Attention to factors underlying variability (animal diets, mouse strain, gender, age, housing conditions) Reports of detailed methodology
models may not mirror the true human condition. (iii) Biochemical and/or anatomical differences between animals and humans. Differences in the BBB, neuroanatomical pathways, or even in nasal filtration of environmental toxins may affect the response of animals to exposure routes and doses similar to those seen by humans. Despite these potential shortcomings, recent years have seen a proliferation of studies examining the pathogenesis of nigrostriatal degeneration in animal models using exposure to environmental toxins, with a specific emphasis on the utilization of pesticides. Two distinct pesticideinduced animal models of PD have dominated this field of research: the Rotenone model and the Paraquat (PQ) plus Maneb (MB) model. Both were initiated to take account of the possible environmental origin of PD, yet probably neither mimics actual human exposure in terms of particular chemicals or timing and route of exposure. Both were also developed to reproduce human parkinsonian features, but neither has taken account of non-motor symptoms that predominate later in the disease. Improving reproducibility, a key component of any legitimate animal model, has been the goal of much recent research, as well as specificity both within the nervous system and outside of it. The intent of this review is to examine the data that have emerged from the use of environmental neurotoxins in rodents and discuss the experimental value of these new in vivo models in the study of PD. We will exclusively review the status of current animal models produced by exposure to Rotenone (insecticide), PQ (herbicide), and MB (fungicide). Rotenone, PQ and MB: The compounds and their mechanisms of action Rotenone, a chemical that belongs to the family of isoflavones naturally found in the roots and stems of several plants, is used as a broad-spectrum pesticide. Surprisingly, Rotenone can be used in organic food farming, based on its 476
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label as a natural product. Highly lipophilic, it easily crosses the BBB, and for cellular entry [1], does not depend on the dopamine transporter (DAT) – a transmembrane protein, residing in DA neurons, whose purpose is to clear DA from the synaptic cleft and which is a potential route for specific entry of neurotoxic agents into DA neurons (Figure 1). Once in the cell, Rotenone accumulates at mitochondrial complex I where it inhibits the transfer of electrons from iron-sulfur (Fe-S) centers to ubiquinone. Increased reactive oxygen species (ROS) production has been associated with complex I dysfunction induced by Rotenone, which may produce oxidative damage to DNA and proteins of neural cells. Nitric oxide (NO) can interact with ROS, especially superoxide and hydroxyl radicals, leading to peroxinitrite formation, ultimately resulting in cellular defects and damage to DA neurons [7]. More recently, Rotenone was shown to inhibit proteosome activity [8]. Dysfunction in proteosomes - intracellular structures that catalyze the degradation of ubiquitintagged proteins - has been implicated in the pathogenesis of both genetic and sporadic forms of PD [9]. PQ is one of the most widely used herbicides in the world. It is considered to be quick acting, non-selective, and seems to specifically destroy green plant tissue on contact. In the United States, PQ is classified under restricted use, whereas in the European Union PQ usage has been forbidden since July 2007. PQ is suspected to enter the brain by neutral amino acid transporters and subsequently the cells in a sodium-dependent fashion [10]. Once within cells of the CNS, PQ acts as a redox cycling compound at the cytosolic level, which potentially leads to indirect mitochondrial toxicity [11], although low affinity to mitochondrial complex I at high doses has been reported (Figure 1). In addition to these more classic mechanisms of action, recent reports show that PQ-induced apoptosis may involve Bak protein, a pro-apoptosis Bcl-2 family member [12], although the complexity of these newly suggested mechanisms is beyond the scope of this review. MB, which is used as a fungicide, is an irritant to respiratory tracts and is capable of inducing sensitization by skin contact. Mechanistically, and based on evidence discussed below, MB seems to cross the BBB. Although knowledge of the mechanisms of this toxin is very limited, MB preferentially inhibits mitochondrial complex III [13] (Figure 1). Further, MB was recently shown to induce apoptosis through Bak activation, whereas combination of PQ/MB inhibits the Bak-dependent pathway while potentiating apoptosis via Bax protein [14]. Rotenone models of PD Although the effects of Rotenone on the brain were first tested over 20 years ago, the model received the most attention when reproduced with a chronic mode of intravenous (i.v.) delivery [15]. Rotenone infusion via osmotic mini-pumps produced motor deficits reminiscent of several clinical features of PD – including hypokinesia, rigidity, hunched posture, unsteady movements and even resting tremor, the severity of which was associated with the extent of lesions. These motor impairments correlated with irreversible degeneration of nigral DA neurons. In two animals, amelioration of motor deficits was noted in
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Figure 1. Mechanisms of actions of various toxins used to produce PD models. Schematic illustration of mechanisms of actions of the most utilized toxins to induce PD features in rodents, based on current evidence. The precise mechanisms by which these toxins cross the BBB are not known. Rotenone likely crosses because it is highly lipophilic, whereas PQ has been suggested to use neutral amino acid and sodiumdependent transporters. The ability of MB to enter the brain has not been specifically demonstrated but is derived from studies showing its effects on this organ after systemic injection. All compounds, with the probable exception of PQ, target the mitochondrial complex. Complex I is more specifically targeted by Rotenone and MB attacks complex III, whereas mitochondrial dysfunction induced by PQ could emerge indirectly from redox cycling in the cytosol. This interaction of the compound at the respiratory cell level is extremely detrimental and ultimately leads to cell death. For comparative purposes, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the toxin most frequently used to generate animal models of PD, has been illustrated. MPTP is transported across the BBB and is converted to its toxic form 1-methyl-4phenylpyridinium (MPP+) in astrocytes via the enzyme MAO and ultimately released into the extracellular milieu by the plasma membrane transporter Oct3. MPP+ subsequently enters dopamine neurons by specific dopamine transporters and oxidative stress is generated followed by cell death. Abbreviations: AA: amino acid; ATP: Adenosine triphosphate; BBB: Blood brain barrier; DAT: dopamine transporter; MAO: monoamine oxidase; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine; UPS: ubiquitin proteosome system.
response to apomorphine treatment – a mixed D1/D2 agonist, supporting the hypothesis that the motor deficits were due to DA depletion [15]. Notably, ubiquitin and asynuclein inclusions reminiscent of Lewy bodies were discerned in nigral neurons in animals in which DAergic degeneration was observed, although the DAergic identity of neurons with inclusions was not examined. At the striatal level, loss of tyrosine hydroxylase (TH)-immuno-
reactive terminals was both dose and duration dependent, whereas levels of glutamic acid decarboxylase (GAD; indicative of GABAergic medium spiny striatal projection neurons) and acetylcholinesterase (AChE; indicative of cholinergic striatal interneurons) were not affected by the Rotenone treatments, arguing for DA specificity of the Rotenone effects. Importantly, Sprague–Dawley rats submitted to the same Rotenone regimen were ultimately 477
Review excluded from the study because of inconsistency in lesion production. Higher doses originally tested (up to 12 mg/kg) for short periods of time induced cardiovascular systemic toxicity and non-specific lesions. There are several issues that have hampered acceptance of the chronic i.v. Rotenone model, the first of which is variability. To address this, other routes of administration have been pursued. Similar to the i.v. route, the subcutaneous (s.c.) route is beset by variability (and mortality; see summary Table 1). Recently, daily intraperitoneal (i.p.) delivery for up to 60 days of similar doses as previously reported, with dissolution of Rotenone in a novel fatty-acid based vehicle, has achieved high reproducibility of the lesions [16]. Similar to previous reports, this model seems to generate specific DA lesions, accompanied by the presence of a-synuclein and poly-ubiquitin aggregates in remaining nigral DA neurons, although the striatal lesion still displays typical focal denervation. More recently, other modes of administration have been explored – such as oral [17] and intranasal [18] delivery – that are arguably more realistic with regard to potential entry sites for toxin exposure in human PD. Unfortunately, these novel routes have only been reported in single studies with limited pathological analysis (Table 1). The second important issue facing the Rotenone model is evidence demonstrating marked systemic non-specificity [15,19–27], leading to high mortality related to systemic adverse effects including cardiac, stomach and liver problems. The relationship of behavioral motor changes specifically to nigrostriatal DAergic degeneration has not been convincingly established. The hunched back posture and hypokinesia originally reported conceivably could have been related to severe peripheral abnormalities, observed by other investigators [22]. In the more recent i.p. model, despite an important body of literature demonstrating significant peripheral problems generated by various routes of Rotenone delivery in rodents, the apparently parkinsonian behavioral deficits were only considered as being the result of specific lesions [16]. Although apomorphine responsiveness of at least some of the symptoms suggests DA specificity to the behavioral dysfunction, no autopsies were conducted to evaluate peripheral abnormalities (even though all animals were sacrificed due to severe behavioral dysfunction), which might account for some of these behavioral changes as previously reported [22]. The third critical issue hampering the Rotenone model is non-specificity within the CNS, as has been observed by some laboratories. The striatal degeneration described is not closely related to human PD: a recent report assessing the last five years of research on the Rotenone model concluded that Rotenone does not reproduce the pathology of PD but rather induces a pattern of pathological changes more akin to atypical parkinsonism, characterized, for example, by striatal degeneration in addition to nigral neuronal loss [28]. Although a similar pattern of neuropathological changes to their initial i.v. model was observed [15], it will be critical for other laboratories to evaluate the new i.p. delivery approach [16] before its specificity can be considered established. 478
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What have we learned from the Rotenone model of PD? As discussed, issues of variability, mortality, non-specificity both within and outside the CNS, and reproducibility across laboratories remain major challenges for the chronic Rotenone model of PD. In addition, there remain critical issues regarding the translatability of the model: does the Rotenone model truly recapitulate human PD? The first issue is route of exposure. Although it is possible that in human disease Rotenone, or other toxins, gains access to the CNS via the BBB, and that i.v. or i.p. administration mimics this route, it is perhaps more likely that introduction is via direct exposure of neurons in the gut and/or olfactory region [29]. Thus the Rotenone model might fail to replicate a critical aspect of human disease, which could in fact bear as well on the second critical issue. As discussed above, non-specificity with respect to the nigrostriatal system is a feature that is now appreciated as characteristic of human PD. Could the non-specificity observed in Rotenone treated rodents reflect this feature of human PD? An analysis of the presence of Lewy body formation or a-synuclein accumulation has not been reported in structures other than SNpc and striatum (e.g. the dorsal motor nucleus of the vagus), as seen in human PD. Other non-DA system related dysfunction (e.g. cognitive aspects), has not yet been assessed in this model. On the other hand, perhaps the systemic effects observed – while not at all reminiscent of observations in human PD – could be re-evaluated in light of the fact that PD may originate from the peripheral system [2]. Future studies should include histopathological analyses of other systems and perhaps attempt to evaluate the model from a different angle, shedding light onto the potential value of these ‘‘nonspecific’’ effects in reproducing other aspects of PD pathology. PQ/MB: Synergistic effects of compounds and developmental vulnerability An important aspect of human pesticide exposure is that it is usually to a number of agents, rather than a single one, and it occurs over time. In addition, there is the possibility that in utero exposure to toxins, such as lipopolysaccharide as part of bacterial vaginosis during pregnancy, predisposes to specific nigral DA cell loss [30]. Based on these premises, several studies have been initiated to explore this concept in animals. Concurrent exposure to PQ and MB in adult mice led to significantly greater DA fiber loss, and greater 3,4-dihydroxyphenylacetic acid (DOPAC) and DA turnover, than exposure to either toxin alone. Locomotor activity was decreased after MB alone or after combined treatment [31,32]. A subsequent study, utilizing a twice a week for six weeks exposure scheme, showed DA damage, decrease in TH protein levels and DOPAC as well as nigral DA loss with combined PQ and MB only. The latter treatment alone also had adverse effects on behavior [33,34]. It was also recently shown that administration of PQ/ MB post-natally to mice led to a 38% loss of DA neurons in adulthood, whereas when post-natal exposure was followed by adult re-exposure there was a 70% loss of DA neurons in the nigra and a concomitant decrease in
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Table 1. Synopsis of Rotenone-induced animal models
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Review locomotor behavior [50]. This study suggests a period of ‘‘silent neurotoxicity’’ that predisposes adult animals to an exaggerated response to the same agents following readministration. A variation of this scheme has investigated the effects of prenatal s.c. MB injection in combination with adult i.p. PQ delivery. Nigral DA cell loss, and striatal decreases in DA, DA metabolites (DOPAC, homovanilic acid), DA turnover (reflecting decreased synthesis as well as breakdown, the latter mediated by DA neurons after DAT-mediated uptake), and serotonin were produced only after prenatal MB followed by adult i.p. PQ administration. This particular treatment also induced 95% decrease of locomotor activity in adult treated animals [35]. The findings reveal an interaction between age of exposure and the effects of neurotoxins. Older mice have been reported to be more susceptible to systemic PQ/MB treatment [34,36,37], perhaps due to changes in transport across the BBB [36,38]. We have found similar effects of PQ/MB exposure in older adult rats (six months), but especially with regard to the induction of lung pathology. In contrast to two month old rats, where only three out of 16 subjects became ill following four weeks of repeated exposures [39], 52% of older treated animals either died or had to be sacrificed as a result of severe weight loss and/or signs of respiratory distress following just two biweekly systemic injections of PQ/MB [40]. Others have also reported the presence of dichotomous populations in six week old rats in which one group suffers from systemic toxicity and another group recovers and displays no lung pathology [41]. We have made similar observations in mice [42], which have not been noted in previous studies. What have we learned from the PQ/MB-induced animal model of PD? The PQ/MB model has certainly been useful for its demonstration of potential synergistic effects of various environmental compounds in producing PD features in animals. The various experimental approaches utilized to develop this model have further provided support for a multi-hit hypothesis, which is interesting in that it replicates likely scenarios of intoxication in humans, although the routes of delivery remain, as for the Rotenone model, improbable occurrences and, contrary to the primary rationale, these compounds are not used simultaneously in agriculture [3]. Overall, the PQ/MB model meets several of the classic criteria, including behavioral motor impairments, significant DA-related degeneration and responsiveness to DA therapy. Importantly, it has met with more consistency among research groups. Conversely, to our knowledge, inclusion bodies have not been reported in the multiple variants that exist for this model (Table 2) and neither have non-motor impairments. Unfortunately, some nonspecific and undesirable peripheral effects [18,39–43], particularly to the lungs, provoking respiratory distress, have been reported for this model, limiting its utilization, especially since these effects are not related to human forms of PD. Future studies should be directed at examining for pathology in non-motor and gastrointestinal systems involved in early PD. 480
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Concluding remarks Are pesticide models useful in the investigation of the etiopathogenesis of human PD? First, the specificity of the pathological lesions induced raises doubts about the adequacy of these models, perhaps especially so for Rotenone. Lack of specificity may indicate disparate biochemical mechanisms underlying the degenerative process, or may reflect a failure to replicate the correct exposure route or time-course. Specificity may derive from the route of spread of the pathological process within the CNS, or indeed the peripheral nervous system, which is not replicated by more convenient routes and time-courses in rodents where it is produced by i.v., s.c. or i.p. administration. Non-specificity extends to the systemic toxicity observed with both Rotenone and PQ/MB exposure. At the least, this systemic toxicity adds a substantial experimental burden to utilizing these models. However, it too may reflect failure to replicate the appropriate exposure routes and time-course in human disease. Conversely, current models are in another sense too specific, not ostensibly incorporating the increasingly appreciated non-motor features of human PD. The validity of animal models heretofore has been predicated simply on modeling the motor features of PD. Indeed, until we have a full understanding of the extent of PD pathology, conceptual animal models will be difficult to generate and evaluate, and by the same token current animal models will provide a limited understanding of what precisely takes place in PD. Again, it is conceivable that route of exposure to toxins is a critical and integral part of the pathogenetic mechanism in generating the nature and range of pathological abnormalities that define bona fide PD. In that regard, a recent provocative hypothesis posits that all of the motor and non-motor systems observed to be involved pathologically can be accounted for by initial exposure to the olfactory system followed by extrasynaptic cell-to-cell transmission of aberrantly folded a-synuclein [29]. Perhaps more realistic routes of delivery would constitute a good starting point for producing models that mimic the disease’s pathological aspects and continued efforts should unquestionably put forward protocols that address this issue. Moreover, it may be that present efforts have failed to probe for these alterations, constituting another avenue for further exploration. In the end, however, it may be that the disease is so complex and develops over such a protracted period of time that it may be overly optimistic to expect to reproduce all these aspects in feasible small animal models, and defined models that recapitulate select aspects of the disease – as has been done with the motor aspects – may be the best that can be expected of animal models of PD. Finally, reproducibility is paramount to the value of any animal model. Variability in pesticide models is likely multifactorial. One factor, for example, may relate to animal diets, in that rodent chow composition varies depending on the provider and seasonal foods available. Several studies have reported the neuroprotective role of various diet components, like phytoestrogens, to the DA system. It is feasible that diets, which vary between laboratories, are responsible, at least in part, for the inconsistencies in the level of toxin-induced DA neurodegeneration reported. Mouse strain, gender, age, and housing con-
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Table 2. Synopsis of PQ/MB-induced animal models
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Review ditions also may contribute to these disparities. In order to move the field forward and to save resources, it is imperative to systematically report positive as well as negative data. The elaboration of new animal models of any disease is a challenging task and should not be undermined by limited sharing of experimental details. Reproducibility, which is a key factor in the development of valuable animal models of PD, will only find support on well-founded methodologies. Disclosure None of the authors has conflict of interests to disclose. Acknowledgments The authors wish to acknowledge the support of the Fondation Canadienne pour l’Innovation and the Canadian Institute of Health Research to Francesca Cicchetti. Janelle Drouin-Ouellet was initially supported by a master’s scholarship from Fonds de Recherche en Sante´ du Que´bec and subsequently by a Canada Frederick Banting and Charles Best doctoral scholarship. Robert Gross was supported in part by National Institutes of Health grants from the Emory Collaborative Center for Parkinson’s Disease Environmental Research from NIEHS and by a K08 Career Development Award (NS46322) from NINDS.
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