Parkinsonism & Related Disorders Parkinsonism and Related Disorders 7 (2001) 185±191
www.elsevier.com/locate/parkreldis
C. elegans: a novel pharmacogenetic model to study Parkinson's disease R. Nass a,c, D.M. Miller III b,c, R.D. Blakely a,c,* a
Department of Pharmacology, Vanderbilt University School of Medicine, MRBII, Room 419, Nashville, TN 37232-6600, USA b Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-2175, USA c Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, TN 37232-6420, USA
Abstract Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Although the use of vertebrate and tissue culture systems continue to provide valuable insight into the pathology of the neurodegeneration, the molecular determinants involved in the etiology of the disease remain elusive. Because of the high conservation of genes and metabolic pathways between invertebrates and humans, as well as the availability of genetic strategies to identify novel proteins, protein interactions and drug targets, genetic analysis using invertebrate model systems has enormous potential in deducing the factors involved in neuronal disease. In this article, we discuss the opportunities for the use of the nematode Caenorhabditis elegans (C. elegans) for gaining insight into the molecular mechanisms and pathways involved in PD. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Dopamine; Transporter; Neurodegeneration; Nematode; Genetics; Necrosis; Apoptosis; 6-OHDA; MPP 1
Parkinson's disease (PD) is a slowly progressive, neurodegenerative disorder characterized by the irreversible loss of over 80% of the nigrostriatal dopaminergic neurons. Although the pathogenesis of the disease appears to be multifactorial, correlative evidence supports the role of oxidative stress and mitochondrial dysfunction [1]. PD patients have reduced levels of mitochondrial complex I and glutathione activity, as well as increased levels of superoxide dismutase activity, lipid peroxidation and iron in the substantia nigra [1,2]. This evidence suggests that the generation of reactive oxygen species (ROS), such as the superoxide ion and hydrogen peroxide, plays a signi®cant role in dopamine (DA) neuronal death. Current animal model systems of PD rely on inducing nigrostriatal damage in monkeys and rodents with the neurotoxins 6-hydroxydopamine (6-OHDA), 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) or 1-methyl-4phenylpyridinium ion (MPP 1 [the active metabolite of MPTP]) [1]. Toxin exposure causes DA neuronal death and the animals exhibit many of the same symptoms as PD. The speci®city of the toxin for the DA neurons lies in their af®nity for the Na 1- and Cl 2-dependent dopamine transporter (DAT), a presynaptic membrane protein that terminates DAergic transmission by the active reuptake of DA into presynaptic neurons [3±9]. Transfections of DATs into nonneuronal cells confer sensitivity to MPP 1, and 6* Corresponding author. Tel.: 11-615-936-1691; fax: 11-615-936-0212. E-mail address:
[email protected] (R.D. Blakely).
OHDA and MPP 1 toxicity can be blocked with DAT antagonist in vivo [4±7]. Despite decades of research, the primary insults and mechanisms leading to the degeneration of the nigrostriatal DA neurons and the increase in production of ROS in PD is unknown. Nonhuman primates do not naturally develop PD, so these model systems are unavailable to study the etiology of the disease. Current animal and tissue culture systems are able to mimic some of the pathology and morphology of idiopathic PD. However, identifying the molecular determinants involved in PD without any a priori knowledge of the mechanism of the neurodegeneration is signi®cantly hindered due to the lack of a de®nitive model system. An approach to identifying the molecular determinants involved in PD without any prior knowledge of their function could be through forward genetics [10]. In this classical method of genetic analysis a well-de®ned phenotype is modi®ed by mutagenizing the genomic DNA. The progenies that have the modi®ed trait are further analyzed to determine the location of the altered loci (this is the opposite of reverse genetics in which the goal is to identify animals that have a speci®c gene mutated) [10]. In order for forward genetics to be successful in studying DAergic neurodegeneration in an animal, either an aspect of the behavior should depend on normal DA neurotransmission or the morphology of the DA neurons should be easily accessible. Furthermore, the organism should provide an ef®cient strategy to map and identify the mutated gene. Because of the above criteria, as well as
1353-8020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 1353-802 0(00)00056-0
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Table 1 C. elegans genes involved in catecholamine biosynthesis, metabolism and transport Gene protein cat-4 cat-2 bas-1 cat-1 dat-1 R13G10.2 C48D5.1 a
Reference GTP cyclohydrolase I Tyrosine hydroxylase Aromatic l-amino acid decarboxylase Vesicular monoamine transporter Dopamine transporter Monoamine oxidase a NHR, Nurr1 homolog a
Loer and Kenyon, 1993 [18] Lints and Emmons, 1999 [19] Loer and Kenyon, 1993 [18] Duerr et al., 1999 [20] Jayanthi et al., 1998 [21] Sulston et al., 1992 [22] BLAST Search [27]
Predicted gene product; NHR, nuclear hormone receptor.
their relatively long life-span and maintenance cost, mammals are not amenable for these studies. Forward genetics in the nematode Caenorhabditis elegans offers a powerful tool for dissecting the components involved in mammalian neurodegenerative disorders. The high conservation of the genome and metabolic pathways between nematodes and vertebrates, the ease of screening a large number of worms following mutagenesis for neural defects and the ability to identify quickly the modi®ed gene (the whole genome has essentially been sequenced) should allow for the rapid discovery of proteins involved in neuropathologies [11]. Because C. elegans reproduces rapidly (approximately 3 days at 258C), is relatively small (approximately 1 mm) and generates about 300 worms from a single hermaphrodite, genetic screens involving large numbers of animals can be performed within a short period of time [12,13]. C. elegans is also transparent, with virtually every cell identi®able under a light microscope. Furthermore, the entire 302-cell nervous system (approximately one-third of the total somatic cells in the hermaphrodite) has been mapped by serial three-dimensional electron microscopic reconstruction, allowing for the identi®cation of virtually all synaptic connections [14]. Multicolor reporters, using derivations of the green ¯uorescent protein (GFP) from the jelly®sh Aequorea victoria, also allow for the detailed visualization of cells, intracellular compartments or protein localization in the living animal [15±17]. Methods for generating transgenic animals (obtainable within 4 days) and production of gene knockouts (minimum of about 1 week) are also well developed. A signi®cant advantage of the worm over vertebrate systems is that sexual reproduction can be achieved by self-fertilization and therefore is not disrupted by mutations that would otherwise perturb mating behavior. The similarity between the worm and the mammalian nervous system suggests that the fundamental interactions that occur during DA neurotransmission and pathogenesis in vertebrates may also occur in C. elegans. Most of the known molecular components involved in DA signaling in mammals are also present in the nematode (Table 1) [18± 22]. Furthermore, analysis of the C. elegans genome has
revealed a high conservation of ion channels, neurotransmitter synthesis enzymes (including tyrosine hydroxylase [TH] and aromatic amino acid decarboxylase [AAAD]), synaptic vesicle and presynaptic terminal proteins (including syntaxins, synaptotagmin, synaptobrevin), neurotransmitter receptors (including glutamate, acetylcholine, GABA, amine, and peptide) and neurotransmitter transporters (including acetycholine [VChAT], the vesicular monoamine transporter [VMAT], GABA, glutamate [EAAT], serotonin [SERT], dopamine [DAT]) (Table 1) [23]. A mammalian nuclear hormone receptor, Nurr1, which is expressed in DA neurons and provides a measure of neuroprotection against MPTP toxicity, also has signi®cant homology with the worm gene C48D5.1 [24±27]. However, to date, the cellular expression pattern of C48D5.1 has not been de®ned. An important distinction to make between the two nervous systems at the molecular level is the redundancy of the genomes; vertebrates will often have several similar versions of a particular gene, while C. elegans may have a single locus [23]. This feature of the nematode genome provides an enormous advantage relative to vertebrate systems because it decreases the probability that a mutant effect will be masked by a genome redundancy. Finally, the genetic basis of cell death is highly conserved between the worm and vertebrates, and C. elegans provides an opportunity to explore genes involved in neuronal cell death [28]. The C. elegans hermaphrodite contains eight dopaminergic neurons which include six head DA neurons (four CEPs and two ADEs), and two neurons which are located in posterior lateral positions (PDEs) [29]. All of these neurons contain ciliated endings which terminate in sensory organs embedded in the cuticle that surrounds the animal. Laser ablation and genetic studies have revealed that these cells provide mechanosensory function during foraging and movement (and may also modulate pharyngeal pumping and egg laying behaviors) [20,29±31]. The male contains another three pairs of DA-containing neurons in the tail that are involved in mating and four additional DA-containing socket cells of the mating spicules [18,19,29,32,33]. To provide an opportunity for genetic and pharmacological evaluation of DAT and genes involved in altered DA neurotransmission, we cloned the C. elegans dopamine transporter (CeDAT) [21]. CeDAT is highly conserved with the mammalian catecholamine transporters with an amino acid identity of approximately 45%. The gene encodes a 615 amino-acid protein with 12 putative transmembrane domains. Mammalian cells expressing CeDAT exhibit saturable and high-af®nity Na 1- and Cl 2-dependent DA transport
Km 1:2 mM and are sensitive to the mammalian DAT antagonist GBR 12909 and cocaine and to the substrate d-amphetamine at potencies comparable to their actions on human DAT in transfected cells [21,34,35]. CeDAT is also inhibited by the tricyclic antidepressant SERT antagonist imipramine and the NET-selective antagonist nisoxetine [21].
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Fig. 1. (a) A three-dimensional confocal reconstruction; and (b) differential interference contrast (DIC) image of the six DA neurons located in the head of a hermaphrodite expressing GFP driven by the endogenous CeDAT promotor. Arrows identify the CEP and ADE DA neurons.
Mammalian DAT proteins are exclusively expressed in dopamine neurons. Evidence of a conservation of this pattern in the worm would strengthen the argument that CeDAT is an evolutionary homolog of the mammalian DAT [36,37]. Following the initial observation that a genomic fragment comprising part of the CeDAT gene (2.1 kilobase pair [kb] fragment upstream of the start ATG to 0.7 kb downstream of the ATG) was suf®cient to drive reporter expression in DA cell bodies (Ishihara, personal communication; see Hope, worm expression pattern for gene T23G5.5 [38]), we created an integrated transgenic line expressing a CeDAT promotor±GFP fusion (PCeDAT::GFP) containing 717 bp immediately upstream of the start ATG. This line displays intense GFP expression in all eight DA neurons within the hermaphrodite, but is not evident in other cells (Fig. 1). The axons and dendrites in these transgenic animals are clearly visible and can be easily observed as the animal moves under a ¯uorescent dissecting scope. In order to set up a screen for molecules involved in DA neurodegeneration in C. elegans, one should have a well-
de®ned behavior which is dependent on the normal DAergic function or be able to visualize the morphology of the DA neurons. Since the DA-mediated behaviors in the worm are relatively subtle, and screening a large number of worms based on the modi®cation of these behaviors could be tedious, a more ef®cient and robust screen may be to select directly for mutants within the transgenic lines expressing GFP that have altered DA neuronal morphology. 6-OHDA and MPP 1 provide cell-speci®c lesions reminiscent of the selective pathology of PD and have received considerable attention as tools to probe the mechanism of environmentally triggered DAergic neural degeneration [1,39,40]. If the worm's DA neurons can also be selectively degraded by these neurotoxins by entering the cell via CeDAT, then one should expect a speci®c loss of the GFP expression in the transgenic lines following exposure to the toxin. Based on these results, one could propose a genetic screen for the retention of GFP in DA neurons following toxin exposure as in Fig. 2. Following mutagenesis and toxin exposure, any retention of GFP within the DA neuron
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Fig. 2. Genetic screens to identify CeDAT mutants, CeDAT regulators and determinants of toxin sensitivity (EMS, ethylmethanesulfonate).
must be due to either the inability of the toxin to enter the cell (for example via CeDAT), suppression of some mediator of toxin sensitivity or inhibition of neuronal degradative pathway(s). It is unlikely that we will recover mutations in the transcriptional fusion which could affect GFP expression since there are probably multiple copies of the fusion construct within the integrated array in the genome [41]. To establish this screen, one can envision the use of our integrated line carrying the transcriptional reporter PCeDAT::GFP. Following mutagenesis of the last larval stage of the worm (the L4 stage, when gametogenesis is initiated in the hermaphrodite), one would subject the F2 generation (second generation of the mutagenized worm) to 6-OHDA or MPP 1 and test for loss of toxin sensitivity. Since most mutations would be recessive, screening in F2s would allow them to reach homozygosity and therefore increase the likelihood of a modi®ed phenotype. All mutants recovered would be physically mapped using two-factor linkage analysis, and the genetic map interval de®ned by three-factor crosses [42]. Once the area of the mutated allele has been identi®ed, candidate genes can be tested through microinjection of cosmid spanning the gene. One would then test whether DA neuron-speci®c expression of the wild-type gene rescues sensitivity to 6-OHDA. Finally, one could explore the impact of coexpression of CeDAT
or mammalian DAT with the identi®ed protein or its mammalian homolog (if one has been identi®ed) following toxin exposure in mammalian cells where biochemical studies are more easily performed. So what types of mutant would one expect to ®nd in such a screen? One should be able to identify CeDAT mutants and CeDAT regulators. Since DATs are required for toxin accumulation in mammalian DA neurons, we should be able to recover an allelic series of mutations in CeDAT which would modify its activity, as well as regulators of CeDAT. A variety of CeDAT mutants would likely be identi®ed, which would limit the toxin's ability to enter the cell (and hence alter GFP expression), including those affecting transporter activity, stability and localization. These mutants would be especially valuable because the structural basis for DA transport and DAT regulation are poorly de®ned. Furthermore, the availability of DATs at the nerve terminals has been proposed to play a role in the vulnerability of DAergic neurons to PD, and this screen should assist in delineating its role in the neurodegeneration [40]. High-af®nity antibodies raised against CeDAT should be able to partition these mutants into their proper complementation groups. Finally, since DAT plays a principle role in DA homeostasis and abnormal DA neurotransmission has been indicated in a variety of other neuropsychiatric disorders, including schizophrenia, drug addiction, Tourette Syndrome and attention de®cit hyperactivity disorder, novel regulators pulled from this screen could aid in understanding pathophysiology of these diseases [9,40]. One should also be able to identify molecular determinants of toxin sensitivity. 6-OHDA and MPP 1 appear to confer DA neuronal death via impairing cellular ROS homeostasis, although their mechanism of action in vivo remains unclear [1,39]. Both toxins inhibit complexes in the mitochondrial transport chain (6-OHDA, complex I and IV, MPP 1, complex I) [4,43]. 6-OHDA is also easily oxidized to generate hydroxyl radicals and the superoxide ion and can covalently interact with cysteinyl groups to inhibit protein function [44,45]. It is debatable whether MPP 1 confers toxicity via inhibition of mitochondrial function since a cell which does not have mitochondria is also sensitive to the toxin [46]. The screen that we have proposed should be able to identify genes involved directly or indirectly in the increased generation of ROS (loss-of-function mutants), as well as those which protect the cell from oxidation (gain-offunction mutants). Since DA itself may contribute to DA neurotoxicity (see below), we may expect to ®nd mutations within the DA synthesizing (e.g. TH, AAAD), metabolizing (e.g. monoamine oxidase) and vesicle sequestration (e.g. VMAT, vacuole H 1-ATPase [V-ATPase]) pathways (Table 1) [40]. Novel targets that would normally induce neuronal degeneration, independent of oxidative pathways and those involved in DA metabolism and sequestration, would also be identi®ed. If DAergic neuronal degeneration is dependent on the cell death or a necrotic pathway, then mutants within these
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pathways would also be identi®ed. Recent investigations suggest both neurotoxins may rely on products of programmed cell death pathways (see below), though to what degree for each toxin is debated. 6-OHDA has been reported to cause shrinkage of DAergic cells, nuclear condensation and DNA fragmentation (apoptotic characteristics) in vitro, while MPP 1 does not induce these events [47]. Morphological and biochemical correlates of MPP 1 toxicity in vitro are more consistent with necrosis rather than apoptosis displaying mitochondrial swelling and scattered heterochromatin [47]. Interestingly, the anti-apoptotic protooncogenes in the Bcl-2 class can protect against both MPP 1 and 6-OHDA toxicity in vitro and in vivo, suggesting overlap between the two degradation pathways [48,49]. Apoptosis and necrotic death are both well-de®ned events in C. elegans, with apoptosis causing cytoplasmic shrinkage, mitochondrial distortion and rapid phagocytosis, while necrosis produces vacuoles, cell swelling and membrane whorls [24,50,51]. Presently, little is known about the genes involved in the necrotic pathway, and considering that necrosis can follow neuronal insult independent of activation of the apoptotic pathway, novel genes involved in necrosis will likely be identi®ed [52]. Could C. elegans be a useful animal model system to study the effects of these neurotoxins on DA neuronal degradation independent of genetic screens? If the DA neurons are sensitive to the toxin, the answer is a resounding yes. There exists a wealth of C. elegans mutants which can be used to address some of the questions raised in this paper regarding contributions of various pathways and genes to DA neurotoxicity [53]. For example, PD may be the result of excess DA in the cytoplasm due to the weakening of the ion gradients required for DA sequestration in vesicles. To determine if endogenous DA levels play a role in toxinmediated neural degeneration, we could cross our reporter line PCeDAT::GFP to worm lines de®cient in DA synthesis, such as the TH (cat-2) or AAAD (bas-1) null mutants, and test for sensitivity to 6-OHDA. There is also evidence that the endogenous VMAT levels may predict the sensitivity of the DA neurons to the toxins: the lower the amount of VMAT, the greater the level of DA in the cytoplasm and therefore the greater the likelihood of neural degeneration [40]. This possibility could be addressed by crossing the GFP reporter line into the VMAT null strain (cat-1) and then testing for toxin-induced neurodegeneration. Finally, reserpine, an agent which blocks VMAT and inhibits DA vesicular storage (which causes a reduction in intracellular DA [both in humans and worms]), could be applied to WT worms prior to toxin exposure to determine the role of VMAT on neurotoxicity [20,29,40]. C. elegans also provides an opportunity to explore genes involved in cell death. C. elegans was the ®rst organism in which the factors which regulate cell death were identi®ed on a genetic level. Mutants in these genes are available for evaluating their roles in DA neurodegeneration [28]. For example, CED-3 is a member of the ICE-family of cysteine
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proteases or caspases, which are known to function in both nematode and mammalian apoptosis. CED-3 activity is required for programmed cell death and a ced-3 (lof) mutation blocks all programmed cell death in the nematode [54]. CED-4 is required for CED-3 activation. CED-9 is homologous to mammalian Bcl-2 and negatively regulates CED-3 through interactions with CED-4. Gain-of-function mutations in ced-9 also block apoptosis [55]. Crossing our PCeDAT::GFP line into the ced mutants could test directly whether apoptosis is involved in 6-OHDA or MPP 1 neurotoxicity. Our preliminary studies reveal that the C. elegans DA neurons are vulnerable to mammalian DA neuronal toxins [56]. We ®nd a loss of reporter expression in DA neurons following 6-OHDA treatment. A CeDAT-de®cient line has been established (Duerr and Rand, personal communication). It will be important to validate that the sensitivity to 6-OHDA is lost in this strain. Furthermore, one would expect that CeDAT antagonist will reduce or eliminate toxin sensitivity. If the 6-OHDA action truly re¯ects DA neuron toxicity, one may also expect to see behavioral de®cits like those seen after laser and genetic ablations of DA neurons [19,20,30]. Finally, based on high similarities between the human and worm nervous systems at the molecular level, our CeDAT-directed screens should yield molecular insight into presynaptic DAT regulation and DA neurodegeneration, which are likely to be of general relevance. The development of this model may bring a new paradigm to the study of DAergic neuronal vulnerability that could have a signi®cant impact on PD.
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