Parkinson's disease: Insights from non-traditional model organisms

Progress in Neurobiology 92 (2010) 558–571

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Progress in Neurobiology journal homepage: www.elsevier.com/locate/pneurobio

Parkinson’s disease: Insights from non-traditional model organisms Ilse S. Pienaar a,*, Ju¨rgen Go¨tz b, Mel B. Feany c a

MRC Anatomical Neuropharmacology Unit, Department of Pharmacology, University of Oxford, South Parks Road, Oxford OX1 3TX, United Kingdom Brain & Mind Research Institute, Mallet Street Campus, Camperdown, University of Sydney, NSW, Sydney, Australia c Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 22 June 2010 Received in revised form 7 August 2010 Accepted 2 September 2010

Parkinson’s disease (PD) was one of the first neurological disorders to have aspects of the disease modeled faithfully in non-human animal species. A key feature of the disease is a diminished control over voluntary movement and progressive depletion of brain dopamine (DA) levels that stems from the large-scale loss of DA-producing neurons. Despite their inherent limitations, rodent and non-human primate models of PD have helped unravel several aspects of PD pathogenesis. Thus, we now have neurotransmitter replacement therapy for PD, and a number of neuroprotective compounds that can be assessed in clinical trials. However, no treatment is currently available that can halt or retard the progressive loss of DA neurons, which underlies PD pathology. Moreover, no therapies can permanently alleviate the clinical features of the disease. The lack of a cure or long-term effective treatment is paralled by our incomplete understanding of the underlying pathomechanisms of the disease. A range of robust, flexible, and complementary animal models will be an invaluable tool with which to unravel the pathogenesis of PD. Here we review the most important contributions made by non-mammalian model organisms. These include zebrafish (Danio rerio), flies (Drosophila melanogaster), anurans (frogs and toads) and nematodes (Caenorhabditis elegans). While it is not anticipated that they will replace rodent and primate-based ones, they offer convenient systems with which to explore the relative contribution made by genetic and environmental factors to PD pathology. In addition, they offer an economic and rapid alternative for testing compounds that target PD. Most importantly, the combined use of these models allow for ongoing research to uncover the basic mechanisms underlying PD pathogenesis. Crown Copyright ß 2010 Published by Elsevier Ltd. All rights reserved.

Keywords: Parkinson’s disease Animal models Zebrafish Danio rerio Fruit fly Drosophila melanogaster Nematode Caenorhabditis elegans Saccharomyces cerevisiae

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Parkinson’s disease: The defining pathology . . . . . . . . . . . . . . . . . . . . . . Criteria for animal models of PD and limitations of traditional models . Newly emerging models for PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Fish models of PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The Drosophila model of PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Anuran models of PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Nematode models of PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Yeast models of PD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: aSYN, Alpha-synuclein [protein]; SNCA, Alpha-synuclein [gene]; Catsup, Catecholamines-up; DBS, Deep-brain stimulation; DA, Dopamine; DAT, Dopamine transporter; ER, Endoplasmic reticulum; GstS1, Glutathione S-transferase S1; GFP, Green fluorescent protein; GTPCH, GTP cyclohydrolase I; hsp70, Heat-shock protein 70; KO, Knock-out; LRRK2, Leucine-rich repeat kinase 2; L-DOPA, Levo-dopa; LB, Lewy bodies; LNs, Lewy neurites; MPTP, 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MAO-B, Monoamine oxidase-B; PD, Parkinson’s disease; PPN, Pedunculopontine nucleus; PLD, Phospholipase D; Parl, Presenilin-associated rhomboid-like protease; p53, Protein 53; PINK1, PTEN induced putative kinase 1; Pu, Punch; RNAi, RNA interference; SIRT2, Sirtuin 2; 6-OHDA, 6-Hydroxydopamine; SNpc, Substantia Nigra pars compacta; STN, Subthalamic nucleus; TH, Tyrosine hydroxylase; VMAT2, Vesicular monoamine transporter. * Corresponding author. Tel.: +44 01865 272145; fax: +44 01865 285862. E-mail address: [email protected] (I.S. Pienaar). 0301-0082/$ – see front matter . Crown Copyright ß 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.pneurobio.2010.09.001

I.S. Pienaar et al. / Progress in Neurobiology 92 (2010) 558–571

1. Parkinson’s disease: The defining pathology Parkinson’s disease (PD) is the second most common neurodegenerative disorder after Alzheimer’s disease, affecting at least 2% of the population aged 65 and older (De Lau and Breteler, 2006; Go¨tz and Ittner, 2008). The defining symptoms of PD include muscle rigidity, postural instability, tremor and bradykinesia, while a range of cognitive and psychiatric dysfunctions often comanifest (Braak and Del Tredici, 2009). In recent years, it has become increasingly recognised that a range of other non-motor features, including olfactory dysfunction, dysautonomia, as well as mood and sleep alterations, may precede the classical motor features, possibly even by several decades (reviewed by Tolosa et al., 2009). The progressive loss of DA neurons in the Substantia Nigra pars compacta (SNpc) represents one of the most prominent neuropathological hallmarks of PD (Wooten, 1997). This loss is progressive and by the time PD symptoms appear, at least 50% of all nigral neurons have degenerated, accompanied by an 80% depletion of striatal DA levels (Marsden, 1990). Interestingly, neuronal loss in PD is not restricted to DA neurons, with cholinergic, serotonergic and noradrenergic neurons also being adversely affected (Jellinger, 2001). The relative contributions made by different neuronal subgroups towards the motor and non-motor clinical features of PD remain a highly active area of research interest. A key neurohistological signature of PD is intracytoplasmic inclusions termed Lewy bodies (LBs) and Lewy neurites (LNs) (Lewy, 1912; Forno, 1996). If present in patients with no clear neurological symptoms, they are considered representative of preclinical or presymptomatic PD, rather than non-specific, agerelated pathological changes (Mikolaenko et al., 2005; DelleDonne et al., 2008; Markesbery et al., 2009). In addition to PD, LBs are also detected in patients with Dementia with Lewy bodies (DLB) (Spillantini et al., 1998; McKeith et al., 2005). DLB as opposed to PD is diagnosed when dementia develops either prior to or within the first year of Parkinsonian symptoms appearing. LBs contain as a proteinaceous component the presynaptic protein a-synuclein (aSYN) in an aggregated, oligomeric or fibrillar form (Thomas and Beal, 2007), while, in its native state, aSYN is a highly insoluble and unfolded protein. Which species of aSYN contributes to neurodegeneration, and the mechanism of selective cell death in PD remains a topic of ongoing debate (Conforti et al., 2007; reviewed by Pan et al., 2009). Advanced age is a major risk factor for developing PD (Shaw and Ho¨glinger, 2008). Furthermore, epidemiological studies suggest that exposure to environmental agents, such as insecticides, herbicides, rodenticides, fungicides and fumigants may also increase the risk for developing PD (Migliore and Coppede`, 2009; Hatcher et al., 2008). Mitochondrial dysfunction, including oxidative damage to mitochondrial DNA and functional impairment of the oxidative phosphorylation system also biochemically characterizes PD (Malkus et al., 2009), in particular, due to inhibition of complex I of the mitochondrial electron transport chain. Support for this comes from animal models that use toxins to selectively target mitochondria, resulting in a relatively selective neuronal cell death (see Schapira, 2010, for a review). Although PD has traditionally been viewed as a sporadic disorder, modern genetic and genomic technologies, including genome-wide association studies (GWAS), suggest that there is a substantial genetic component to the disorder, with the sugestation that genetic causes may account for up to 10% of all known cases (Tan, 2007). Familial PD can be caused by mutations in several genes, including in SNCA (A30P, E46K and A53T singlenucleotide point-mutations, as well as gene duplications and triplications) (Polymeropoulos et al., 1997; Singleton et al., 2003; Zarranz et al., 2004; Chartier-Harlin et al., 2004). Linkage analysis

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has identified several other genes related to familial and also sporadic PD, which show either an autosomal dominant or an autosomal recessive inheritance pattern. These include PARK2, mutations of which cause autosomal recessive juvenile Parkinsonism (Hattori et al., 1998; Kitada et al., 1998), and which encodes parkin. Parkin is an ubiquitin E3 ligase that modifies a variety of proteins (Dawson and Dawson, 2010). Mutations in Leucine-rich repeat kinase 2 (LRRK2) also known as PARK8, comprise the most frequently diagnosed inherited cause of PD (see Dauer and Ho, 2010, for a review). Mutations in a second kinase, PINK1 (PARK6), is an additional cause of autosomal recessive early-onset PD (Valente et al., 2004). Likewise, mutations in DJ-1 (PARK7) associate with autosomal recessive juvenile PD (Bonifati et al., 2003a). Although the precise physiological function of DJ-1 remains to be determined, it may act as a neuroprotective sensor of oxidative stress (Yanagida et al., 2009). Mutations in ATP13A2 (PARK9) cause a recessive, juvenile-onset, atypical form of PD, characterized by pyramidal cell degeneration and cognitive dysfunction (Ramirez et al., 2006). Since the ATP13A2 protein is involved in the lysosomal degradation pathway, ATP13A2 mutations may contribute to PD due to abnormalities in aSYN clearance (Van de Warrenburg et al., 2001). Finally, a heterozygous missense mutation in the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) gene (PARK5) was identified in a German family. However, given the small number of affected individuals, UCHL1 may not be a genuine PD gene (Wintermeyer et al., 2000). The DA precursor Levo-dopa (L-DOPA) provides symptomatic relief, but fails to halt the progressive loss of DA neurons. Moreover, following its long-term use, severe dyskinesia is often seen despite optimal DA replacement therapy. Another treatment option, mainly for advanced PD, is deep-brain stimulation (DBS). This is undertaken when motor symptoms are inadequately controlled by medication alone or when patients suffer from medication-refractory tremor (Silberstein et al., 2009). Despite its efficacy (Sako et al., 2008; Fimm et al., 2009), DBS is complicated by the risk of intra-cranial haemorhage following the stereotactic implantation of the electrode for delivering DBS (Volkmann, 2007). The subthalamic nucleus (STN) is most frequently targeted for DBS delivery, however; gait abnormalities and freezing in advanced PD respond poorly to STN DBS (Krack et al., 2003; Moreau et al., 2008). Therefore, the pedunculopontine nucleus (PPN), located in the dorso-lateral segment of the ponto-mesencephalic tegmentum, which fulfills a role in maintaining postural stability, has become an attractive anatomical target for DBS delivery (Lee et al., 2000). Stefani et al. (2007) recently showed that combined targeting of the PPN and the STN improves the gait disturbance seen in advanced PD patients more than stimulation of either target alone. Nevertheless, the risk of fatal bleeding remains, and similar to the currently available pharmacological therapies, DBS is incapable of halting or delaying disease progression. 2. Criteria for animal models of PD and limitations of traditional models A suitable animal model of human PD is histopathologically characterized by the progressive and significant loss of DA neurons and the loss of some non-DA neurons, onset commencing during adulthood. Moreover, ideal animal models of PD are capable of mimicking the clinical manifestations of the human disease, including the motor phenotype that includes bradykinesia, rigidity, postural instability and resting tremor, with motor features being responsive to L-DOPA therapy. In light of the limitations of current PD therapies, rodent and primate models have been generated for dissecting the underlying disease mechanisms and improving therapeutic outcomes, either

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by administering neurotoxic substances or via genetic manipulation. A significant subset of the models make use of the stereotaxic or systemic delivery of neurotoxins such as 6-hydroxydopamine (6-OHDA), rotenone, paraquat or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which is converted to the potent neurotoxin 1-methyl-4-phenylpyridinium (MPP+). These toxins all selectively destroy nigrostriatal DA neurons, producing a syndrome that resembles idiopathic PD in animals (Burns et al., 1983). Some investigators rather use the rotenone rodent model for assessing neuroprotection, since its neurotoxic mechanism functions independent of the DA uptake transporter (DAT) (Pienaar et al., 2009; Tapias et al., 2010). Use of the rotenone rat model is further supported by recent studies showing a reduced glutathione and superoxide dismutase activities and increased SNCA expression, following stereotaxic rotenone infusion, similar to that seen in idiopathic PD (Xiong et al., 2009). The relative slow time-course of the rotenone model may more accurately model the pathogenesis of human PD, which follows a progressive course lasting several decades. Intense efforts have been made towards developing improved PD models to better understand the etiology and pathogenesis of PD, and to identify new drug targets. However, neither toxininduced nor transgenic animal models of PD, perfectly recapitulates all human symptoms. Toxin models hold merit for studying DA deficiency and the effects of DA replacement therapy. Transgenic approaches include the overexpression, knock-out, knock-in and knock-down of PD genes (Lewis et al., 2008). For instance, since Parkinsonism also characterizes individuals with fronto-temporal dementia, expression of a mutant form of the microtubule-associated protein tau, has been shown to cause severe early-onset Parkinsonism in mice (Ittner et al., 2008). However, in the context of the current emphasis on highthroughput screenings to identify genetic interactions or pharmacological therapies, murine or non-human primate models often prove costly and time-consuming (Faust et al., 2009), hence models using fish, worms, flies and anurans have become attractive alternatives. As discussed here, newly emerging PD models in zebrafish (Danio rerio), fruitflies (Drosophila melanogaster), nematodes (Caenorhabditis elegans) and anurans (frogs and toad) have made a significant contribution towards better understanding disease mechanisms and uncovering novel therapeutic strategies. 3. Newly emerging models for PD 3.1. Fish models of PD Zebrafish (D. rerio), a vertebrate species, have a number of characteristics that suggest potential utility as a model of PD. They have a rapid reproduction rate, producing hundreds of offspring every week. Also, embryos are transparent and develop outside the mother, allowing investigators to readily visualize the developing organs and tissues (Bretaud et al., 2004). Moreover, although a successful zebrafish breeding programme requires specialist facilities, such as controlled water temperature as well as adequate filtering and feeding devices, relative little skill is required to breed and maintain a school. Their small adult size (3–4 cm long) furthermore permits for easy and economic husbandry. Extensive information is available on the CNS patterning, pathfinding and connectivity of zebrafish, which show important similarities to the human CNS. Immunohistochemistry to detect for tyrosine hydroxylase (TH), the rate-limiting enzyme for synthesizing catecholamines (DA, norepinephrine and epinephrine), revealed many similarities between the zebrafish and mammalian catecholaminergic systems. For instance, large, pear-shaped TH-containing neurons located in the ventral diencephalon of zebrafish brains are believed to be homologous

to mammalian midbrain SN and ventral tegmental neurons (Son et al., 2003). In addition, a region has been identified in the zebrafish procencaphalon that displays a high degree of anatomical similarity to the mammalian striatum (Rink and Wullimann, 2001, 2002). There is extensive molecular and genomic information available on zebrafish (http://vega.sanger.ac.uk/Danio_rerio/index. html). PD-related genes, such as DJ-1 (Bai et al., 2006; Bretaud et al., 2006), UCH-L1 (Son et al., 2003), SNCA, PINK1, PARK2 and LRRK2 (Flinn et al., 2008) are evolutionarily conserved between humans and zebrafish, and their protein products are expressed in zebrafish ventral diencephalic DA neurons. Zebrafish display a much higher reproduction rate than rodents, making them highly suitable for large-scale chemical or retrovirusinduced mutagenesis screens. By microinjecting antisense morpholino oligonucleotides into single-cell embryos, McKinley et al. (2005) successfully disrupted DAT, which normally transports DA across the plasma membrane of DA neurons, thereby removing DA from the synaptic cleft following presynaptic release. Depletion of DAT protected neurons against MPTP-induced damage, supporting the notion that MPTP exerts its adverse effects through a DATmediated uptake mechanism. Prior to toxin exposure, morpholinomediated knock-down of DJ-1 resulted in increased protein 53 (p53) and Bax expression levels, but produced no detectable DA cell death, a finding consistent with studies done on mice (Chen et al., 2005; Goldberg et al., 2005). However, in the presence of both the superoxide radical H2O2 and the proteasome inhibitor MG132, DJ-1 reduction increased the tendency of DA neurons to undergo cell death (Bretaud et al., 2007). This suggests that pathological conditions associated with PD may evoke a sub-threshold activation of cell death pathways, when DJ-1 activity is altered (McKinley et al., 2005). Morpholino-mediated knock-down of PINK1 function in zebrafish did not result in DA neuron loss in the ventral diencephalon (Xi et al., 2010). Similarly, mice with targeted PINK1 disruption displayed no DA loss, although defects in DA release and impaired synaptic release were observed (Kitada et al., 2007). In zebrafish, investigators observed altered patterning of DA neurons in the ventral diencephalon, including altered axonal projections, accompanied by impaired locomotor behavior (Xi et al., 2010). Thus, in zebrafish at least, normal pink1 function is required for positioning DA neurons and for establishing appropriate neuronal circuits. Efforts are underway for improving the screening potential of zebrafish, by creating stable transgenic zebrafish lines that faithfully recapitulate relevant pathological mechanisms underlying human neurodegenerative disease. In this regard, Bai et al. (2007) isolated cis-acting regulatory elements capable of driving high-level transgene expression in differentiated neurons throughout the CNS of transgenic zebrafish. Meng et al. (2008) marked DA neurons in zebrafish by expressing the green fluorescent protein (GFP) under the control of tyrosine hydroxylase 1 (th1) promoter, thus reducing the need to identify DA neurons using laborious and expensive antibody-based techniques. Pink1 is a putative kinase, 581 amino acids in length, containing both mitochondrial targeting and serine/threonine kinase domains (Gandhi et al., 2006). PINK1 mutations in patients cause autosomal recessive PD (Li et al., 2005), while PINK1 KO zebrafish display a reduction in the number of DA neurons, impaired mitochondrial function, increased caspase-3 activity and elevated levels of reactive oxygen species (ROS). On the other hand, expression of wild-type human PINK1 in zebrafish and exposure to antioxidants reduced ROS levels and rescued the phenotype (Anichtchik et al., 2008). The effects of PINK1 knock-down may reflect a GSK3brelated mechanism, since morphant fish showed elevated GSK3b activity and the knock-down phenotypes could be partially abrogated by GSK3b inhibitors (Anichtchik et al., 2008).

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Owing to considerable sequence homology with humans at the amino acid level, zebrafish have been used for characterising the spatial and temporal expression patterns of the synuclein protein family, which includes alpha-, beta- and gamma-synuclein (Sun and Gitler, 2008). The superb genetic tractability in zebrafish provides a platform for exploring the functional consequences of disease-related mutations and the physiological functions of aSYN and its homologues that has remained enigmatic till now. Since hydrophilic substances can readily be added to the tank water and are then absorbed by zebrafish skin and gills (Lam et al., 2005) at any developmental stage (Crawford and Guarino, 1985), zebrafish are ideal for testing and screening drug effects (Peterson et al., 2000, 2004). For instance, the monoamine oxidase-B (MAOB) blocker L-deprenyl was shown to protect zebrafish embryos against oxidative stress-inducing toxins, a mechanism that has previously been implicated in PD pathogenesis (Parng et al., 2006). Prior to or simultaneous with MG132 exposure, a pharmacological inhibitor of p53, a protein implicated in neuronal cell death pathways, has been shown to prevent DA cell death in vivo. Interestingly, DJ-1 knock-down leads to increased p53 expression, similar to proteasome inhibition, and is accompanied by widespread neuronal death (Bretaud et al., 2007). These findings suggest that p53 inhibition is a potential therapeutic target for treating PD. Whereas humans, monkeys and some mouse strains are highly vulnerable to the neurotoxic effects of MPP+, the active metabolite of MPTP, rats are less sensitive to MPTP-induced toxicity (Burns et al., 1983; Mori et al., 1988). In larvae and adult zebrafish, a doserelated and area-specific neurodegeneration of DA neurons is induced by MPP+. In particular, exposure results in a significant reduction of TH-positive cells in the posterior tuberculum of the ventral diencephalon (Lam et al., 2005). Also, using an enhancer trap transgenic zebrafish line, in which the reporter gene of ETvmat2:GFP was inserted into the second intron of the vesicular monoamine transporter 2 (VMAT2), Wen et al. (2008) labeled monoaminergic neurons with GFP during embryogenesis. The results showed that MPTP treatment reduces the number of GFPpositive monoaminergic neurons in the pretectal cluster, while the

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telencephalon, pretectum, raphe nuclei and locus coeruleus was left unaffected. Since similar effects were reported in mammals, this indicates that MPP+’s actions affects similar neural pathways in the zebrafish, as in the human brain (McKinley et al., 2005). Neuromelanin is a dark polymer pigment, produced in DA and noradrenergic neurons (Saper and Petito, 1982). There has recently been increased interest in neuromelanin due to a possible link between the presence of neuromelanin and the vulnerability of DA neurons to undergo oxidative damage, which could eventuate in their apoptotically induced cell death (Fedorow et al., 2005). In fact, aberrant regulation of neuromelanogenesis and turnover has been proposed to play a key role in PD pathology (Zecca et al., 2006), supported by the loss of neuromelanin-containing DA neurons seen in PD patients (Hirsch et al., 1988). In screening and evaluating putative melanogenic regulatory compounds, the rapidity, cost-effectiveness and physiological relevance of zebrafish, provides an attractive alternative to mammalian model systems (Fig. 1 and Table 1). Similar to data obtained in frogs, peripheral effects have been observed following zebrafish exposure to MPTP, such as increased depigmentation and respiratory difficulties (Bretaud et al., 2004). These effects might be due to the degeneration of catecholaminergic nerve terminals that innervate pigmented cells, since the catecholaminergic neurotoxin 6-OHDA produces similar effects (Anichtchik et al., 2004; Ryan et al., 2002). Thus, skin pigment modifications may be a useful tool by which to screen zebrafish mutants for resistance to MPTP. DA serves as a key neuromodulator of locomotory circuits in both vertebrates and invertebrates (Crisp and Mesce, 2004; Kiehn and Kjaerulff, 1996). Thirumalai and Cline (2008) found that application of exogenous DA as well as exposure to the DA reuptake inhibitor bupropion, markedly reduced spontaneous swimming behavior in zebrafish larvae at 3 days post-fertilization (dpf). However, bupropion had no effect on episode frequency at 5 dpf, even though DA neurons and projections to the spinal cord were present at this developmental stage (McLean and Fetcho, 2004; Rink and Wullimann, 2002). However, interpretation of these findings may be limited, since bupropion is also capable of blocking noradrenaline reuptake; therefore, some of the effects

Fig. 1. The fraction of homologous overlap (given as a percentage) in five genes, which, in mutated form may underlie genetic forms of PD. Species reviewed here in this article (Xenopus laevis, Danio rerio, D. melanogaster and C. elegans) are compared to more traditional experimental species (Macaca mulatta, Mus musculus and Rattus norvegicus), and to the relative human gene (100%). See Table 1 for shared identity values and the accession identity numbers of the peptides presented here. Symbols used above: y annotated ortholog is fragmented, z no ortholog annotation identified.

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Table 1 A summary table of the species-related differences between the 5 human PD-related gene homologues (SNCA, Parkin, PINK1, DJ-1, LRRK2), present in the novel species used for modelling PD (Xenopus, D. melanogaster, Danio rerio, C. elegans, S. cerevisiae), as discussed here, compared to those seen in more traditional model species (M. Mulatta, M. Musculus, Rattus norvegicus). A summary list is provided of differences seen in the amino acid length of the gene products, identifiable via the peptide accession number (brackets contain information on the genomic browser used). The right-most column provides the degree of identity overlap shared with the human gene. PD-related gene

Species

Peptide length (amino acids)

Peptide accession #

SNCA (PARK1)

H. sapiens M. mulatta M. musculus Rattus norvegicus Xenopus laevis Danio rerio D. melanogaster C. elegans S. cerevisiae H. sapiens M. mulatta M. musculus Rattus norvegicus Xenopus tropicalis Danio rerio D. melanogaster C. elegans S. cerevisiae H. sapiens M. mulatta M. musculus Rattus norvegicus Xenopus Danio rerio D. melanogaster Xenopus C. elegans S. cerevisiae H. sapiens M. mulatta

140 140 140 140 141 – – – – 465 465 464 465 – 458 482 386 – 581 581 580 580 – 574 721 – 641 – 189 190

P37840 (UniProt) P61143 (UniProt) 055042 (UniProt) P37377 (UniProt) AAH54200 (NCBI) (X. laevis) – – – – 060260 (UniProt) ENSMMUP00000026859 (Ensembl) Q9WVS6 (UniProt) Q9JK66 (UniProt) – Q561U2 (UniProt) Q7KTX7 (UniProt) Q9XUS3 (UniProt) – Q9BXM7 (UniProt) ENSMMUP00000000169 (Ensembl) Q99MQ3 (UniProt) B5DFG1 (UniProt) – Q5PRB2 (UniProt) FBpp0070917 (Ensembl) – Q09298 (UniProt) – Q99497 (Ensembl) ENSMMUP00000025846 (Ensembl)

M. musculus Rattus norvegicus Xenopus. laevis Danio rerio D. melanogaster C. elegans S. cerevisiae H. sapiens M. mulatta M. musculus Rattus norvegicus Xenopus tropicalis Danio rerio D. melanogaster C. elegans S. cerevisiae

189 189 189 189 217 187 – 2527 2037 2527 2526 668 2533 2445 2393 –

Q99LX0 (UniProt) O88767 (UniProt) Q6NTU4 (UniProt) Q5XJ36 (UniProt) FBpp0086741 (Ensembl) P90994 (UniProt) – Q5S007 (UniProt) ENSMMUP00000020845 (Ensembl) Q5S006 (UniProt) ENSRNOP00000005438 (Ensembl) ENSXETP00000033900 (Ensembl) ENSDARP00000018692 (Ensembl) Q9VDJ9 (UniProt) Q9TZM3 (UniProt) –

Parkin (PARK2)

PINK1 (PARK6)

DJ-1 (PARK7)

LRRK2 (PARK8/Dardarin)

assigned to bupropion may have been mediated by noradrenaline instead of DA. In contrast, pharmacologically blocking the D2 DA receptors seemingly increase the frequency of swimming bouts, while activation of adenylyl cyclase, which acts as a downstream target that receives inhibition from D2 receptor signalling, blocked DA’s inhibitory effect (Thirumalai and Cline, 2008). Since the swimming bouts were much more frequent and of shorter duration at 5 dpf compared with those seen at 3 dpf, it remains possible that endogenous DA may be unable to suppress output of the swimming circuit at 5 dpf, due to other ontogenic events, such as the maturation of other modulatory inputs (Brustein et al., 2003) or increased excitatory synaptic drive within the notocord (Buss and Drapeau, 2001). Therefore, the initiation of swimming episodes is seemingly suppressed by endogenously released DA in zebrafish larvae at 3 dpf, while, at subsequent larval stages, endogenously released DA is unable to suppress the initiation of swimming, with larvae exhibiting more frequent swimming bouts.

Comments

98.6% identical to H. sapiens 95.0% identical to H. sapiens 95.0% identical to H. sapiens 85.5% identical to H. sapiens No clear ortholog detected No clear ortholog detected No clear ortholog detected No clear ortholog detected 97.2% identical to H. sapiens 84.4% identical to H. sapiens 85.6% identical to H. sapiens No clear ortholog detected 63.9% identical to H. sapiens 45.0% identical to H. sapiens 33.0% identical to H. sapiens No clear ortholog identified 97.2% identical to H. sapiens 81.6% identical to H. sapiens 82.1% identical to H. sapiens No clear ortholog identified 56.7% identical to H. sapiens 34.0% identical to H. sapiens No clear ortholog identified 30.4% identical to H. sapiens No clear ortholog identified Possible gene prediction error identified Fragmented gene is 100% identical to H. sapiens 91.5% identical to H. sapiens 91.5% identical to H. sapiens 77.8% identical to H. sapiens 83.1% identical to H. sapiens 53.2% identical to H. sapiens 53.8% identical to H. sapiens No clear ortholog identified 97.3% identical to H. sapiens 86.8% identical to H. sapiens 86.6% identical to H. sapiens 68.2% identical to H. sapiens 49.4% identical to H. sapiens 24.3% identical to H. sapiens 23.5% identical to H. sapiens No clear ortholog identified

Zebrafish larvae have recently become an attractive model for studying neurological disease, due to the possibility they offer for imaging whole neurotransmitter systems three-dimensionally (Panula et al., 2006). Application of this technique showed that zebrafish larvae catecholaminergic neurons are left unaffected by MPTP exposure (McKinley et al., 2005; Bretaud et al., 2004; Lam et al., 2005). However, others have reported that MPTP can affect the motor function of zebrafish larvae, with abnormalities becoming evident 1–2 days following toxin exposure (Sallinen et al., 2009). The change in swimming behavior was transient, with animals showing spontaneous recovery at 5–6 days post-exposure. In addition, a detailed cell count following behavioral analysis, revealed a transient decline of approximately 50% in the number of TH-expressing neurons. These effects, induced by MPTP, were sensitive to the MAO-B inhibitor L-deprenyl, a drug prescribed for alleviating motor symptoms in PD patients (Lam et al., 2005; McKinley et al., 2005). The drug’s protective effects have also been demonstrated in the MPTP primate (Cohen, 1984; Langston et al.,

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1984) and MPTP mouse model of PD (Heikkila et al., 1984; Tatton and Greenwood, 1991), confirming the model’s suitability for testing the clinical effectiveness of therapeutic compounds. The authors’ proposed explanation for the spontaneous recovery and lack of cell death seen in this study was due to zebrafish neurons being incapable of producing MPP+, MPTP’s toxic metabolite, in sufficient enough quantities to produce significant toxic effects (Sallinen et al., 2009). This finding was previously also reported by Anachtchik et al. (2006). The study by Sallinen et al. (2009) also introduced novel concepts for the developing catecholaminergic system, including the suggestion that the diencephalic cell population, consisting of groups 5, 6 and 11, may resemble the mammalian SN. The study further elucidated on the previously-held belief that adult catecholaminergic cells are only detectable at 4 dpf (McKinley et al., 2005), by showing that they are already distinguishable at 72 h post-fertilization (Sallinen et al., 2009). In contrast, adult zebrafish exposed to either MPTP or to 6-OHDA displayed significant and chronically decreased DA and noradrenaline levels, although concomitant changes in the number of DA neurons or TH and caspase-3 protein levels were not detected (Anichtchik et al., 2004). Locomotor perturbations were also seen; including a decrease in velocity and in the total distance moved. However, no significant morphological alterations in catecholaminergic cell clusters were seen. The authors proposed that the absence of DA cell death could be explained by the fact that the fish had received only a single, acute exposure to the toxin (Sallinen et al., 2009). Following exposure to the mitochondrial poisons rotenone and paraquat, larval and adult zebrafish display altered swimming behavior (Bretaud et al., 2004). However, in adult fish, effects were only seen at sublethal doses, while larvae were left unaffected. Why zebrafish embryos and larvae are protected from Parkinsonian neurotoxic compounds, compared to adults, remains to be clarified. Many genetic mutations affecting the zebrafish CNS manifest phenotypically as abnormal swimming behavior. Therefore, analysis of swimming behavior may provide insight into how complex neuronal networks produce motor behavior, and how neuronal pathologies affect locomotor circuits (Panula et al., 2006). However, the relevance of a swimming abnormality to human PD is limited by the fact that the neural mechanism responsible for producing intact swimming behavior by zebrafish is currently unknown. Moreover, since others (i.e. Bretaud et al., 2004) have shown locomotor deficits by zebrafish in the absence of an underlying DA neuron loss, the validity of the model for studying PD will require further investigation. 3.2. The Drosophila model of PD Over the last decade, the small fruit fly (D. melanogaster) has played a key role in increasing knowledge of the molecular underpinnings of PD and has been particularly useful for unraveling the function of genes associated with PD. Completion of the fly genome sequencing project (http://www.sanger.ac.uk/ Projects/D_melanogaster) has allowed for an analysis of the cellular pathways in which PD-related genes act, making Drosophila one of the most genetically metazoans. The core pathology seen in human PD can be reproduced in Drosophila to a remarkably accurate extent; including an areaspecific and age-dependent loss of DA neurons, as well as the formation of LBs and LNs (Feany and Bender, 2000). For instance, transgenic flies that overexpress wild-type or mutant forms (A30P or A53T) of human SNCA display adult-onset loss of DA neurons, accumulation of filamentous aSYN-containing LB-like inclusions and locomotor dysfunction (Feany and Bender, 2000). This loss was prevented by expression of the chaperone heat-shock protein 70 (hsp70) (Auluck et al., 2002), while a nucleotide exchange of serine

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129 with alanine, to create the S129A point-mutation, abrogated

aSYN toxicity (Chen and Feany, 2005). This highlights the important role played by phosphorylation in neurodegenerative disease (Ittner et al., 2008). Discovery of parkin’s native function as a ubiquitin ligase (Shimura et al., 1999, 2000), led to the proposal that disrupted parkin function results in accumulated ubiquitylated proteins, which aggregate to form inclusion-bodies in neurodegenerative disease. Moreover, the protein may play a role in promoting autophagy of dysfunctional mitochondria following loss of mitochondrial membrane potential (for a review, see Bu¨eler, 2010). Drosophila PARK2 null mutants exhibit a reduced lifespan, locomotor defects, and male sterility. Locomotor defects are secondary to muscle degeneration with muscle cell apoptosis, accompanied by mitochondrial structural abnormalities, including swelling and disintegrated cristae (Greene et al., 2003). PARK2 mutants also showed reduced body weight, while displaying reduced cell size compared to wild-type controls (Pesah et al., 2004). Furthermore, pan-neuronal expression of the parkin substrate Pael-R caused an age-dependent degeneration of DA neurons, while co-expression with parkin caused degradation of Pael-R and suppressed its toxicity (Yang et al., 2003). Toxicity of Pael-R was also suppressed by the antioxidant and chaperone, thioredoxin (UmedaKameyama et al., 2007). The neurodegenerative phenotype of Parkin mutants was enhanced by loss-of-function mutations of glutathione S-transferase S1 (GstS1) (Whitworth et al., 2005), previously identified as a modifier gene (Singh et al., 2001). A key question in PD genetics and pathogenesis is whether loci associated with PD are in the same cellular pathway, or if multiple independent insults cause DA neuron loss, resulting in PD. Here, Drosophila models have provided evidence for a genetic interaction between the PD-linked genes PARK2 and PINK1 (Clark et al., 2006; Park et al., 2006; Yang et al., 2006), while functional analyses revealed that overexpression of trA2, the Drosophila homologue of the mitochondrial protease OMI, genetically interacts with PINK1, resulting in a more severe eye pathology (Whitworth et al., 2008). This work also revealed that overexpression of Rhomboid-7, a homolog of the human mitochondrial protease presenilin-associated rhomboid-like protease (Parl), induces a rough eye phenotype. It is likely that rhomboid-7 is positioned upstream of either PINK1, PARK2, or omi, since this phenotype was suppressed when these genes were removed. Overexpression of PARK2 rescued male sterility and mitochondrial defects of PINK1 mutants, whereas double-mutants lacking both PINK1 and PARK2 show muscle wasting identical to that observed in either mutant alone (Clark et al., 2006). Thus, PINK1 and PARK2 appear to share the same functional pathway, with PINK1 located upstream of PARK2 (Clark et al., 2006). Similar findings were also reported by a second independent group (Yang et al., 2006). Overexpression of PINK1 produces disorganization of the Drosophila retina (Whitworth et al., 2008). The so-called ‘‘rough eye’’ phenotype, which is characterised by roughening of the eye surface of adult flies, is an easy read-out of gene function. The eye assay helped to identify two novel proteases that possibly interact with PINK1 and PARK2. These included HtrA2 also called OMI/ PARK13, a member of the family of Htr proteins, which localizes to the mitochondrial intermembrane space and/or the endoplasmic reticulum (ER) (Huttunen et al., 2007). OMI selections or pointmutations in the protease domain have been implicated in PD pathogenesis, with the encoded proteins having been detected in LBs in the post-mortem brains of PD patients (Strauss et al., 2005). Moreover, mice lacking this protease showed a Parkinsonian syndrome. Abnormalities included neurodegeneration of striatal neurons at approximately 3 weeks of age, accompanied by astrogliosis and microglial activation. Moreover, the mice rarely

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survived 40 days post-natally (Jones et al., 2003). However, recent findings revealed that Drosophila Omi/HtrA2 null mutants, unlike PINK1 or PARK2 null mutants, lack mitochondrial morphological defects (Yun et al., 2008). Furthermore, the Omi/HtrA2 mutant (G399S) previously reported in sporadic PD patients, retained near-complete function. Taken together, this report, in addition to recent clinical studies (Ross et al., 2008; Simon-Sanchez and Singleton, 2008), does not support the previous hypothesis that Omi/HtrA2 is essential for regulating mitochondrial integrity and dynamics. These conflicting findings highlight the importance of future experiments for resolving the endogenous function of Omi/ HtrA2 as it relates to PINK1, and whether such a genetic interaction indeed underlies aspects of Parkinsonism. Reducing function of drp1, which encodes a key mitochondrial fission-promoting protein, causes lethality in a PINK1 or PARK2 mutant background (Poole et al., 2008). Conversely, muscle and mitochondrial defects were suppressed by increased drp1 levels and by heterozygous loss-of-function mutations in genes encoding the mitochondrial fusion-promoting factors, OPA1 and Mfn2. An eye phenotype associated with increased PINK1/PARK2 pathway activity was suppressed by perturbations that reduce mitochondrial fission, while being enhanced by perturbations that reduce mitochondrial fusion. These observations support the view that the pink1/parkin pathway promotes mitochondrial fission and that the loss of mitochondrial and tissue integrity in PINK1 and PARK2 mutants derives from reduced mitochondrial fission. Several studies have attempted to explain the role played by DJ1 (PARK7) in PD pathogenesis. A role was deduced for DJ-1 for protecting against oxidative stress, with an acidic isoform of the protein that accumulates, following exposure to such toxic conditions (Meulener et al., 2005). Genetically-modified DJ-1 Drosophila provide an opportunity for understanding cellular pathways that protect against neuronal damage, while the study of DJ-1 potentially links a genetic cause with critical environmental risk factors. In one study, double KOs of the Drosophila DJ-1 alleles (a and b) showed no obvious external phenotype, being viable and fertile and having a normal lifespan. In addition, DA neurons in these fly brains revealed no change in cell numbers, when comparing young (1 day old) flies to aged (30 days old) flies. This finding is reminiscent of humans null for DJ-1 function, who are in general good health, apart from acquiring Parkinsonism in later life (Bonifati et al., 2003b). DJ-1a and DJ-1b flies displayed sensitivity to the oxidative stress-evoking agents paraquat and rotenone, which act through mitochondrial pathways implicated in inherited PD (Dawson and Dawson, 2003), as well as being environmental toxins considered to be risk factors for sporadic PD (Uversky, 2004). The increased sensitivity to paraquat resulted primarily from the loss of DJ-1b function, which, compared to DJ-1a is expressed significantly in the CNS (Meulener et al., 2005). DJ-1b displayed a biochemical modification when exposed to paraquat. This included a shift in its isoelectric point, reminiscent to that observed with human DJ-1 in cultured cells (Canet-Aviles et al., 2004). Moreover, SDS-PAGE analyses revealed a modification of DJ-1b, not previously reported, as slower migration upon exposure to paraquat (Meulener et al., 2005). Phosphatase treatment revealed that this particular modification was unlikely to be due to phosphorylation (Meulener et al., 2005). Overall, the findings from this study demonstrate that, at least in Drosophila, DJ-1 protects from environmentally-derived oxidative stress. The Drosophila DJ-1 model may therefore serve as a tool by which to elucidate on gene/environment integration and the role this might play in PD pathogenesis. Paraquat exposure initiates an oxidative cascade by generating superoxide radicals, including H2O2, which forms the substrate for catalase activity. Hence, elevated catalase levels in neurons can be

regarded as a reliable early indicator of a paraquat-induced oxidative stress response in cells (Rohrdanz et al., 2001). In a study by Chaudhuri et al. (2007), Drosophila treated with paraquat for 12 and 24 h resulted in significant elevations of catalase activity, detected in the heads of adult flies, compared to age-matched, untreated control flies. Moreover, catalase activity levels in paraquat-fed males showed greater elevation compared to female flies, at both time-points. In addition to motor difficulties, such as climbing deficits, wildtype flies exposed to paraquat developed a Parkinsonian syndrome, including resting tremor, bradykinesis, rotational behavior and postural instability (Chaudhuri et al., 2007). Again, male flies exhibited these symptoms earlier (by 12 h) than females did. Moreover, whereas male flies showed near-complete immobility after treatment with paraquat for 24 h, female flies retained a degree of motor ability. The study furthermore shows that in both genders the paraquat-induced mobility deficit could be rescued by treating wild-type adult flies with L-DOPA or DA, which illustrates the clinical validity of the paraquat Drosophila model of PD. The study also showed a linear relation between the dosage of paraquat and lifespan reduction. Again, a gender bias was noted, with females surviving, on average, 12–16 h longer than male flies. The study further revealed that paraquat ingestion reduces the number of surviving DA neurons (but with no effect on non-DA neurons), accompanied by changes in neuronal morphology, which correlated with the onset of movement deficits (Chaudhuri et al., 2007). A number of studies have suggested that generation of oxidative stress associated with the metabolism of endogenous DA may be an important contributor to neuronal damage in PD (Tsang and Chung, 2009). Recent human genetic data supports such a notion with significant association between polymorphisms detected in NAT2, MAO-B, GSTT1, tRNAGlu and PD (Tan et al., 2000). To further investigate the role of DA and oxidative stress in PD, Chaudhuri et al. (2007) made use of Drosophila mutant strains for three genes involved in DA homeostasis: pale, which encodes the TH enzyme, involved in DA biosynthesis (Neckameyer and White, 1993), Punch (Pu) which encodes for GTP cyclohydrolase I (GTPCH) (Mackay and O’Donnell, 1983), the rate-limiting enzyme for synthesizing the TH-regulating co-factor tetrahydrobiopterin (BH4), and catecholamines-up (Catsup) for negatively regulating DA synthesis in Drosophila (Stathakis et al., 1999). Although Catsup mutants, similar to DJ-1b mutants, conferred enhanced sensitivity to H2O2, flies displayed enhanced survival and later onset of mobility deficits and DA neuron death following paraquat exposure than wild-type animals. This phenomenon might be due to the elevated catalase activity that Catsup mutants presented with, which would promote scavenging capacity. However, the finding that catalase activity in Catsup mutants, compared with wild-type controls were indistinguishable (Chaudhuri et al., 2007), prompted the authors to suggest that, alternatively, the delayed onset of paraquat-induced damage in the Catsup mutants may be due to elevated DA pools resulting from loss of the negative regulatory function of Catsup. This could potentially maintain DA-dependent functions, which allows these mutants to survive longer than wild-type controls. Moreover, Catsup mutation itself had no effect on the number of DA neurons under non-toxic conditions. However, following 24 h of paraquat ingestion, a dramatic reduction in numbers of DA neurons was observed in Catsup mutant heterozygotes, suggesting that functional Catsup protein fulfills a neuroprotective role (Chaudhuri et al., 2007). The study offered several insights into the role of the remaining endogenous DA pools in PD, including the finding that mutations which alleviate DA function offer protection against paraquat, while mutations that diminish DA levels increase susceptibility for developing a PD phenotype. The study data furthermore agreed

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with epidemiological data, showing that males are more at risk for developing human PD than females (Van Den Eeden et al., 2003), by revealing that male flies exhibit PD-like symptoms earlier than female flies (Chaudhuri et al., 2007). The evidence given by Chaudhuri et al. (2007) suggests that the paraquat Drosophila model is a useful tool for dissecting the gene–environment interactions that appear to play a major role in the onset and progression of human PD. Sirtuins are members of the histone deacetylase family of proteins, which have been implicated in regulating ageing and longevity in a variety of organisms (Imai, 2007). Also, sirtuin 2 (Sirt2) proteins have been implicated as key mediators of longevity induced by calory restriction (Canto´ and Auwerx, 2009). In neurodegenerative disease, AGK2 and AK-1, two potent inhibitors of SIRT2, protected against aSYN-mediated toxicity and rescued dorso-medial neurons in a dose-dependent manner in a Drosophila model of PD (Outeiro et al., 2007). Although the molecular mechanism by which these effects are produced remains unclear, these compounds may function by promoting the production of neuroprotective cellular inclusion-bodies (Garske et al., 2007). 3.3. Anuran models of PD Although used less often than nematodes, Drosophila and zebrafish, anuran species, including frogs and toads, are also valuable in neuroscience research. Since Xenopus laevis is the most widely used anuran amphibian research organism, the recent completion of its genome has provided a valuable resource. Moreover, a draft genome sequence assembly of X. tropicalis has recently been made public (Hellsten et al., 2010), revealing that its genome contains more than 20,000 protein-coding genes, including orthologs of at least 1,700 human disease genes. The authors furthermore reported that the X. tropicalis genome exhibits substantial shared synteny with humans across major parts of large chromosomes, broken by lineage-specific chromosome fusions and fissions, predominantly in the mammalian lineage. The ease with which frogs are raised, their ability to produce large, single-celled eggs, external development, huge, easily manipulatable embryos, and transparent tadpoles, equip them with several practical experimental advantages (Brown, 2004). Moreover, amphibian phylogenetics allows for drawing comparisons to other vertebrates, having diverged from mammals, birds and reptiles approximately 360 million years ago, providing an opportunity for elucidating on the dynamics of tetrapod chromosomal evolution. Immunohistological and morphological evidence showed that the basal ganglia of anuran species are largely homologous to mammals (Marin et al., 1998). This includes the presence of a striatum proper, with g-aminobutyric acid (GABA-), enkephalinand substance P-immunoreactive neurons that project to a structure deemed homologous to the mammalian globus pallidus (Endepols et al., 2004a). Anuran brains also contain a group of posterior tuberculum DA neurons which equates to the mammalian SNpc which, similar to mammals, innervates both the striatum and globus pallidus (Marin et al., 1997). Furthermore, Xenopus DA neurons in the mesencephalic interpeduncular nucleus project to the nucleus accumbens, the fiber tracts being regarded as homologous to the mammalian ventral tegmental area (Smeets et al., 2000). Compared to human brains, anurans lack a pronounced isocortex (Westhoff and Roth, 2002). However, this difference allows for studying thalamic input to the striatum, separate from the influence of intermingled cortical afferents. Anurans represent an acute toxin-induced model of PD. They recover rapidly from 6-OHDA lesions, without developing exces-

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sive thirst, even under conditions of body water depletion (adipsia) or the inability to swallow effectively (aphagia), which often limits the use of mammalians for modeling aspects of PD (Ungerstedt, 1971; Schwarting and Huston, 1996). Female gray treefrogs (Hyla versicolor) bilaterally injected with 6-OHDA into the telencephalic ventricles (Endepols et al., 2004b) revealed difficulty initiating movements as well as cognitive symptoms, shown as greater distraction by irrelevant stimuli, supporting the notion that the functions of the basal ganglia is conserved between anurans and mammals. Similar to humans and rodents, the anuran parkin homolog is expressed abundantly in the cortex, hippocampus, basal ganglia and cerebellum (Horowitz et al., 1999, 2001a; Shimura et al., 1999). Immunoblot analyses identified three distinct parkin isoforms in the adult Xenopus brain, ranging between 50 and 65 kDa, which seemingly reflects post-translational modifications, possibly including phosphorylation (Horowitz et al., 2001b). Although similar modifications have previously been suggested in rodent brains, evidence for a modification of parkin remains inconclusive (Horowitz et al., 2001b). Understanding the role of PARK2, such as for regulating protein ubiquitination, is imperative for developing therapeutic options for treating PD. Since parkin is absent from the premetamorphic tadpole, but present in adult frog brains, this suggests that parkin induction may occur independently of the development of the nigrostriatal pathway (Horowitz et al., 2001a). Whether the expression of parkin is also developmentally regulated in mammals, remains to be determined by further studies. Injection of Rana pipiens with paraquat or MPTP causes a range of behavioral abnormalities, including rigidity, akinesia and tremor, remiscent of human PD (Barbeau et al., 1985). In addition, MPTP, but not paraquat, evoked depleted brain DA levels and increased skin pigmentation (Sethi, 2002). PD patients often develop cognitive deficits that manifest as difficulty to switch attention from one stimulus to another (Brown and Marsden, 1990; Cools et al., 2001). Proposed mechanisms include disturbances in prefrontal cortico-striatal projections (Cools et al., 2001), as well as suboptimal filtering of irrelevant information (Hayes et al., 1998). In many frog species, males produce acoustic signals that female frogs respond to by moving towards the source of the advertisement call (for a review, see Gerhardt and Huber, 2002). DA depletion disrupts the ability to process sensory input thereby preventing sensorimotor integration, hence resulting in greater than normal attention given to irrelevant stimuli. Since this behavioral phenomenon, termed phonotaxis, can also be elicited by synthetic sounds, mate choice and call discriminations are convenient outcome measures for determining the effects of DA lesions in frogs. In particular, since phonotaxis was deemed to correlate broadly with cognition, serving as a mechanism by which animals acquire, process, filter, store and act upon information derived from the physical and social milieu (Shettleworth, 1998), lesioning the DA system with 6OHDA allows for investigating the effects of DA deficiency on cognition. A delay in initiating movement in response to phonotaxis that correlated with the dose of 6-OHDA the frogs received was reported in a study by Endepols et al. (2004a). Phonotaxis scores also correlated positively with the number of TH-immunoreactive neurons in the posterior tuberculum. Anurans exposed to 6-OHDA, followed by phonotaxic behavioral assessment, revealed an interesting implication for DA deficiency on sensory-guided behavior (Endepols et al., 2004a). The dark pigment neuromelanin is found abundantly in SN neurons of humans and other primates, as well as in anurans (Prota, 1992). Since rodents, guinea pigs and rabbits either completely lack neuromelanin, or only express low levels, anurans may be an ideal model for studying the role of neuromelanin in PD.

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Mitochondrial toxins that bear structural similarity to MPTP are known as b-carbolines (Neafsey et al., 1995). Increased levels of b-carbolines in the cerebrospinal fluid of PD patients (Kuhn et al., 1996), and its metabolites in the SN of PD-affected brains compared to a control brain region, highlights their potential role in PD pathology (Matsubara et al., 1993). Since b-carbolines are abundant in the every day environment, e.g. in cooked food, coffee, alcoholic beverages and tobacco smoke (Bosin and Faull, 1988; Herraiz, 2002), concerns have been raised that exposure to this class of toxins may increase the risk for developing PD. ¨ stergren et al. (2004) found a selective uptake Interestingly, O and retention of radiolabelled b-carbolines in pigmented anuran brain neurons. Further research using frogs suggests that selective binding of b-carbolines to melanin-containing neurons in the SN may lead to an accumulation of these compounds and their methylated metabolites in the SN. This study highlights the need for further investigations, since retention of these compounds in the pigmented human SN may induce idiopathic PD. The rationale for this pertains to the ability by b-carboline to act as potent mitochondrial toxin, by inhibiting NAHD-coenzyme Q reductase of the mitochondrial respiratory chain (Albores et al., 1990). Morona and Gonza´lez (2010) provided a comprehensive comparative description of the distribution of neurons and fibers that contain the calcium-binding proteins calbindin and/or calretinin in the brainstem of two species of anuran (X. laevis and Rana perezi) and two urodele amphibians (Pleurodeles waltl and Ambystoma tigrinum). The results revealed distinct and consistent distribution patterns of calbindin and calretinin in the amphibian brainstem. Interestingly, the present work refuted previous assumptions that neurons containing these proteins primarily associate with the GABAergic phenotype (Ga´briel et al., 1998; Verney et al., 2001) by also detecting them in cholinergic and catecholaminergic neuronal subpopulations along the brainstem of amphibians. This result reinforces the notion that these proteins are unrelated to any neurochemical subtype. 3.4. Nematode models of PD The nematode Caenorhabditis elegans offers several advantages as a tool for studying PD, including its relatively short lifespan, lasting 20 days on average, and low costs in growing and maintaining large colonies (Link, 2006). Genetic, genomic and chemical mutant screens are more easily performed in C. elegans than in most other experimental species. Specifically, RNA interference (RNAi) is particularly easy in nematodes, since worms that can be grown on agar plates where they feed on bacteria that express small interfering RNA (siRNA) to downregulate expression of distinct target genes (knock-down) (Link, 2001). Compound screening using C. elegans is also less time-consuming, due to fast reproduction and a high progeny number (Schmidt et al., 2007). The basic cell biology and biochemistry of nematodes overlap with mammals, including similar ion channels, neurotransmitters (e.g. DA, serotinin, acetylcholine, GABA, etc.), vesicular transporters, receptors and synaptic components (Bargmann, 1998; Chalfie and White, 1986). The wiring diagram of the nematode nervous system consists of a defined set of 302 neurons, of which 8 are dopaminergic (Gitler et al., 2009). The discovery that application of exogenous DA inhibits locomotion and egg laying behavior in this species (Schafer and Kenyon, 1995; Se´galat et al., 1995) led to developing C. elegans animal models for PD. Mutant worms that lack TH, and are therefore unable to synthesize DA, show a deficit in the ‘‘basal slowing response’’, a food-foraging behavior that depends on specific dopaminergic neurons termed ADE, PDE and CEP neurons. Laser-assisted targeting of the dopaminergic system disrupted area-restricted searching behaviors employed by nematodes in

locating food, thereby demonstrating that this behavior depends on dopaminergic signalling (for a review, see Schmidt et al., 2007). Transgenic worms that overexpress wild-type and mutant forms of human SNCA (A30P and A53T) in DA neurons caused an accumulation of aSYN in these neurons. Moreover, worms expressing mutant forms of human SNCA failed to modulate the locomotor rate in response to the availability of food, a function normally attributed to dopaminergic neurons. This behavioral abnormality was accompanied by a reduction in neuronal DA levels, which was rescued by administering DA (Kuwahara et al., 2006). In addition, worms expressing human SNCA under control of the promoter for DAT displayed age- and dose-dependent dopaminergic neurodegeneration (Cao et al., 2005; Hamamichi et al., 2008). A role for mitochondrial dysfunction in PD was highlighted by a transcriptional study performed in worms that overexpressed either wild-type or the human SNCA mutant, A53T. This approach identified 433 upregulated and 67 downregulated genes that represented the functional categories of development and reproduction, catalytic activity and histone groups. Interestingly, amongst the upregulated genes, more than 50% had a known mitochondrial-related function, raising the possibility that this upregulation represents a compensatory mechanism for combating impaired mitochondrial function in Parkinsonism (Vartiainen et al., 2006). C. elegans combined with siRNA technology is considered to be a particularly powerful tool for identifying proteins involved in stress pathways. Accumulating evidence suggests that the unfolded protein response is impaired, with key regulators such as the chaperone GRP78 and the transcription factors ATF6 and XBP-1 being well conserved between mammals and worms (Calfon et al., 2002; Shen et al., 2001; Urano et al., 2002). Using RNAi knock-down, almost 900 candidate genetic targets were systemically screened for association with PD. Depletion of 20 gene products reproducibly enhanced misfolding of aSYN that correlated with ageing of the worms (Hamamichi et al., 2008). Another RNAi study identified 80 genes that, when knocked-down, resulted in a premature increase in numbers of aSYN-containing inclusions (Van Ham et al., 2008). Quality control and vesicletrafficking genes expressed in the ER/Golgi complex and in vesicular compartments were overrepresented, indicating a specific role for these processes in aSYN inclusion formation. Suppressors included ageing-associated genes, such as the sirtuin homogue sir-2.1/SIRT1 (Van Ham et al., 2008). Finally, another RNAi screen identified 10 genes, amongst a total of 1673 genes that, when knocked-down, caused severe growth and motor abnormalities in SNCA transgenic worms (Kuwahara et al., 2008). Although the precise physiological role of LRRK2 and PINK1 remains to be fully elucidated, a number of studies support a functional association between these two putative kinases. Mutations in both genes suppress the phenotype observed in their respective single mutants. Further, loss of PINK1 function in the nematode is traceable to mitochondrial dysfunction, consistent with studies using PINK1 KO mice displaying defects in complexes I, II and IV of respiratory chain (Gautier et al., 2008). It has been reported that PD-associated dominant mutations in LRRK2 result in a progressive reduction of neurite length and branching in cultured mammalian cells (MacLeod et al., 2006). Defects in axonal outgrowth were also detected in canalassociated neurons of C. elegans PINK1 mutants. However, when lrk1 was deleted, PINK1-mediated axonal outgrowth was suppressed. Together, these findings support the notion that PINK1 and LRRK1 exert opposing actions when subjected to oxidative stress. Moreover, the study validates previous findings that expression levels or activity of human LRK1/LRKK2 is critical for regulating optimal cellular functions (Park et al., 2006).

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Use of C. elegans provided evidence for an antagonistic effect between PINK1 and LRK1 (Sa¨mann et al., 2009). Intriguing results reported that a mutation in the C. elegans homologue of PINK1 reduced mitochondrial cristae length, produced defects in axonal outgrowth of canal-associated neurons, while PINK1 mutants displayed increased sensitivity to paraquat exposure. In contrast, LRK1 mutants appeared not to be affected by paraquat-induced oxidative stress, suggesting that wild-type PINK1 protects C. elegans against the effects of ROS, similar to what has been reported in D. melanogaster and other vertebrates (Park et al., 2006; Exner et al., 2007). Furthermore, PINK1 seemingly provided advanced protection against the ER stressor tunamycin, compared to lrk1. Nematode DA neurons exposed to 6-OHDA undergo apoptosis within 6 h of toxin exposure (Cao et al., 2005; Nass et al., 2002), with complete neuronal destruction detected after 72 h and larval arrest was also seen following rotenone administered to C. elegans (Schmidt et al., 2007), while treatment with MPTP evoked decreased motility and degenerated DA neurons (Braungart et al., 2004). All these Parkinsonian toxins are used in rodent and non-human primate models, and, therefore, the results, such as discussed above, obtained in models other than such traditional animal models of PD offer encouragement. Yet, despite these promising results, nematodes do present with important caveats. For instance, the anatomical and functional connectivity in its nervous system does not recapitulate the complex features of mammalian DA neurons, nor are they capable of mimicking the precise behavioral deficits that associates with their loss. In addition, Tischler et al. reported that genetic interactions identified in yeast, unlike gene functions or protein interactions, are not highly conserved in nematodes, calling into question whether genetic interactions can be regarded representative of simple redundancy between genes or pathways (Tischler et al., 2008). Such reports call for caution when reviewing the data emerging from the use of nematodes in PD research, but, at the same time an awareness of the significant contributions they potentially stand to make. 3.5. Yeast models of PD The use of baker’s yeast (Saccharomyces cerevisiae) for modeling several aspects of neurodegenerative disease came to the forefront with the finding that at least 30% of its genes are conserved with human genes, and that yeast therefore displays considerable evolutionary conservation with many human cellular pathways. The utility of yeast as a genetic organism in which to study agerelated neurodegenerative disease has also been illustrated by resolving in yeast several molecular pathways that fulfill a critical regulatory role in normal ageing (Kaeberlein et al., 1999). Several examples suffice to demonstrate the potential offered by genome-wide genetic yeast screens for providing insight into the molecular mechanisms underlying human PD. These comprise of libraries containing either the deletion or overexpression of single yeast genes. A yeast screen revealed that the ER-Golgi regulator Rab1, suppresses aSYN toxicity, by rescuing the toxic phenotype elicited by aSYN overexpression in mammalian neurons (Cooper et al., 2006), providing further testimony that the underlying processes implicated in PD pathology are evolutionary conserved. Also, Gitler et al. (2009) reported a strong functional interaction between aSYN toxicity and ATP13A, the yeast ortholog of PARK9. It is significant that modifiers of aSYN toxicity identified from the application of genetic yeast screens, show overrepresentation in vesicle-mediated transport and lipid metabolism. This suggests that these disease-related proteins perhaps cause toxicity via alternative disease mechanisms, rather

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than via aSYN aggregation, as previously suggested (Van Ham et al., 2009). Transgenic expression of the human disease protein aSYN in yeast has led to several novel insights regarding both its intact biology and the pathogenic implications resulting from its misfolding. An aSYN-GFP fusion was created and the construct was integrated into the genome under the control of a galactoseinducible promoter (Outeiro and Lindquist, 2003), the method offering an advantage over mammalian cells, which are subject to proteolysis (McLean et al., 2001). The GFP fusion proteins localized intensely at the plasma membrane level, while a smaller percentage was detected within the cytoplasm, as seen in in vitro models also (Jo et al., 2000). The majority of yeast cells retain the ability to form colonies after 12 h. This offers a convenient time-lag for studying the protein’s biological effects, prior to toxicity developing. The investigators took advantage of this feature of yeast to confirm that aSYN inhibits phospholipase D (PLD) in vivo, following the report that the protein acts as a potent and selective inhibitor of mammalian PLD in vitro (Jenco et al., 1998). Following on from previous work reporting aSYN’s tendency to accumulate on the lipid monolayers surrounding triglyceride-rich lipid droplets (Cole et al., 2002), the study furthermore determined the effects of aSYN on lipid metabolism. Although mutant A53T SNCA produced a similar effect; this was not observed with mutant A30P (Outeiro and Lindquist, 2003). Immunohistochemistry, confirmed by electron microscopy, revealed that wild-type SNCA and the mutant A53T resulted in the accumulation of discrete lipid droplets, whereas this effect was not seen in cells expressing mutant A30P. This differs from the pattern of accumulation for A53T and A30P, the two SNCA point mutants associated with rare forms of early-onset familial PD. When expressed in yeast, each SNCA did mutant accumulated at similar sites than wild-type SNCA. However, their cellular distributions were strikingly different. Similar to wild-type SNCA, A53T concentrated at plasma membranes, while A30P was widely dispersed throughout the cytoplasm, similar to that seen in in vitro mammalian PLD (Jenco et al., 1998). The yeast cell model also offers a convenient tool for studying the effects aSYN has on vesicular bodies. Outeiro and Lindquist (2003) investigated this by monitoring internalization of the membrane-binding fluorescent dye FM4-64. Whereas in cells not expressing aSYN, FM4-64 was rapidly endocytosed and accumulated at the vacuolar membrane, cells expressing wild-type, A53T or A30P aSYN showed defective dye localization. One of the most notable results from this study was that a mere two-fold difference in the expression levels of aSYN was sufficient to induce nucleated polymerization and allowed for proteins that had previously associated with membranes to cytoplasmic inclusions, to be recruited. This striking find suggests that a toxic gain of aSYN function, concomitant with a loss of normal physiology, could result from even a slight disturbance of the ubiquitin–proteasomal system. Thus, the use of this model system allows for reconciling two previously opposite explanations for PD etiology, namely a gain of toxic function and a loss of intact function. Yeast models of PD, as simple model system, provide several advantages to elucidate on fundamental pathological elements identified as being important in PD pathology. However, due to inherent restriction as to how much can be derived from the use of yeast models, molecular mechanisms, i.e. protein aggregation and aSYN toxicity, indentified as potentially valuable in neurodegeneration, will require validation in neuronal and other animal models. As a unicellular eukaryote, yeast cells lack several cellular processes directed towards intercellular communication. An

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example of where this could potentially limit use of this model is seen in the in vivo study that showed aSYN localizes nearly exclusively to synaptic termini, while the protein was suggested to play a role in neuronal plasticity, synaptogenesis and neurotransmitter release (Fortin et al., 2004). Also, whereas the A30P mutation, abolishes synaptic localization of aSYN (Fortin et al., 2004), its presence initiated loss of DA neurons in the PD brain (Tofaris and Spillantini, 2007). However, a severe aSYN-induced toxicity remains to be described in yeast. Moreover, Tischler et al. (2008) recently demonstrated that genetic interactions from yeast do not directly predict genetic interactions in higher eukaryotes, including humans. Such findings serve to illustrate that novel findings made in the yeast model are merely indicative and should be confirmed in higher eukaryotic systems and eventually be validated in the context of the diseased human brain. However, once validated, yeast models can be used to accelerate the identification of both novel therapeutic targets and compounds with therapeutic potential. 4. Concluding remarks With ever more experimental results indicating that findings generated from the use of small organisms and even yeast can be extrapolated to human neurons, researchers are turning increasingly towards non-rodent and non-primate species as experimental tools for studying aspects relevant to PD pathology and/or its treatment. In this review, we have provided an overview of how inexpensive species such as fish, anurans, flies and worms can serve to complement the expensive primate and rodent models in the search for neuroprotective and disease-modifying anti-PD drugs. It is anticipated that new ways of modeling PD will help determine how differential gene and protein expression contributes to neurodegeneration in PD (David et al., 2005). It is also hoped that, once firmly established, these models will assist in elucidating on the sequence of molecular events that ultimately lead to neuronal impairment and the clinical features that characterize human PD, while, at the same time, they will be instrumental in identifying novel drug targets for treating PD patients. Conflict of interest statement The authors of this manuscript have no conflicts of interest, financial or otherwise, to disclose. Acknowledgements IP’s research receives financial support from the UK and South African Medical Research Councils. The financial contribution made by the National Research Foundation towards her research is hereby also acknowledged. The authors wish to thank Dr. Luis Sanchez-Pulido and Prof. Paul Bolam, both from the University of Oxford, for their help rendered in compiling the figure used here, and for critical reading of the article. References Albores, R., Neafsey, E.J., Drucker, G., Fields, J.Z., Collins, M.A., 1990. Mitochondrial respiratory inhibition by N-methylated beta-carboline derivatives structurally resembling N-methyl-4-phenylpyridine. Proc. Natl. Acad. Sci. U.S.A. 87, 9368– 9372. Anachtchik, O., Sallinen, V., Peitsaro, N., Panula, P., 2006. Distinct structure and activity of monoamine oxidase in the brain of zebrafish (Danio rerio). J. Comp. Neurol. 498, 593–610. Anichtchik, O.V., Kaslin, J., Peitsaro, N., Scheinen, M., Panula, P., 2004. Neurochemical and behavioral changes in zebrafish. Danio rerio after systemic administration of 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine. J. Neurochem. 88, 443–453.

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