Mutant �-synuclein-induced degeneration isreduced by parkin in a fly model of Parkinson’sdisease

Annika F.M. Haywood and Brian E. Staveley

Abstract: Parkinson’s disease (PD) patients show a characteristic loss of motor control caused by the degeneration ofdopaminergic neurons. Mutations in the genes that encode α-synuclein and parkin have been linked to inherited formsof this disease. The parkin protein functions as a ubiquitin ligase that targets proteins for degradation. Expression ofisoforms of human α-synuclein in the Drosophila melanogaster nervous system forms the basis of an excellent geneticmodel that recapitulates phenotypic and behavioural features of PD. Using this model, we analysed the effect of parkinco-expression on the climbing ability of aging flies, their life span, and their retinal degeneration. We have determinedthat co-expression of parkin can suppress phenotypes caused by expression of mutant α-synuclein. In the developingeye, parkin reduces retinal degeneration. When co-expressed in the dopaminergic neurons, the ability to climb is extendedover time. If conserved in humans, we suggest that upregulation of parkin may prove a method of suppression for PDinduced by mutant forms of α-synuclein.

Key words: parkin, α-synuclein, Drosophila melanogaster, model of Parkinson’s disease.

Résumé : Les patients atteints de la maladie de Parkinson souffrent d’une perte caractéristique de contrôle moteur causéepar une dégénérescence des neurones dopaminergiques. Des mutations dans les gènes qui codent pour la synucléine αet la Parkine sont associées aux formes héréditaires de cette maladie. La Parkine est une ubiquitine ligase qui cible lesprotéines pour la dégradation. L’expression d’isoformes de la synucléine α humaine dans le système nerveux duDrosophila melanogaster forme l’assise d’un excellent modèle génétique qui reproduit les caractéristiques phénotypiqueset comportementales de la maladie de Parkinson. A l’aide de ce modèle, les auteurs ont analysé l’effet de la co-expressionde la Parkine sur la capacité de mouches âgées à grimper sur les murs, sur la longévité des mouches et sur la dégéné-rescence de la rétine. Les auteurs ont déterminé que la co-expression de la Parkine pouvait supprimer les phénotypesdécoulant de l’expression de formes mutantes de la synucléine α. Dans l’œil en développement, la Parkine réduit ladégénérescence. Lorsque co-exprimée dans les neurones dopaminergiques, la capacité à grimper est prolongée. Si cesobservations valent également chez les humains, les auteurs suggèrent que l’augmentation de l’expression de la Parkinepourrait supprimer les symptômes induits par les formes mutants de la synucléine α.

Mots clés : Parkine, synucléine α, Drosophila melanogaster, modèle pour la maladie de Parkinson.

[Traduit par la Rédaction] Haywood and Staveley 510

Introduction

The neurodegenerative disorder Parkinson’s disease (PD)is characterized by the progressive loss of motor control,difficulty initiating and sustaining movements, and the selectiveloss of dopaminergic neurons from the substantia nigraregion of the brain (Dawson 2000; Parkinson 1817; Spaceyand Wood 1999). Generally, by the time the disease is diag-nosed as many as 80% of these neurons are lost; between50% and 60% of the nigral neurons can be lost with no

obvious clinical consequence (Lansbury and Brice 2002).Although the majority of PD cases are apparently sporadic,approximately 5%–15% have been determined to have aninherited basis (de Silva et al. 2000; Mizuno et al. 2001).Recently, mutations in a number of genes have been identifiedas causes of PD, and many of these genes are associatedwith the ubiquitin–proteasome protein degradation pathway.

One of the first identified genetic causes of PD is mutationin α-synuclein (Polymeropoulos et al. 1996, 1997). Theα-synuclein protein is found in the pre-synaptic region ofneurons (Clayton and George 1999; Jakes et al. 1994; Kahleet al. 2000) and appears to be involved in the biosynthesis ofdopamine (Baptista et al. 2003; Perez et al. 2002). Threeindependent point mutations in α-synuclein, each of whichgives rise to amino acid substitutions, have been identified tocause PD (Kruger et al. 1998; Polymeropoulos et al. 1996,1997; Zarranz et al. 2004). Alteration of the primary structureof the α-synuclein protein may enhance oligomerization andfibril formation (Conway et al. 1998). Whether these fibrilsare a cause of toxicity or act in a mechanism to sequester

Genome 49: 505–510 (2006) doi:10.1139/G06-011 © 2006 NRC Canada

505

Received 12 October 2005. Accepted 15 January 2006.Published on the NRC Research Press Web site athttp://genome.nrc.ca on 6 June 2006.

Corresponding Editor: A.J. Hilliker.

A.F.M. Haywood and B.E. Staveley.1 Department ofBiology, Memorial University of Newfoundland, St. John’s,NL A1B 3X9, Canada.

1Corresponding author (e-mail: bestave@mun.ca).

toxic oligomers of α-synuclein is an unresolved question. Astriplication of α-synuclein has been shown to lead to thedevelopment of the disease (Singleton et al. 2003), both thecomposition and the dosage of α-synuclein appear to playroles in the development of PD.

A number of point mutations and deletions of the parkingene cause autosomal recessive juvenile Parkinson’s disease(ARJP) (Kitada et al. 1998; Nisipeanu et al. 1999). Theparkin gene encodes a 465 amino acid protein (Kitada et al.1998), which functions as one of a number of E3 ubiquitinprotein ligases (Shimura et al. 2000). Ubiquitin protein ligasesmediate ubiquitination (i.e., the sequential attachment of anumber of ubiquitin monomers) of damaged, misfolded, andshort-lived proteins. These proteins are subsequently escortedto the proteasome for degradation (Hershko and Ciechanover1998; Pickart 2001). In vitro ubiquitination assays demon-strate that the parkin protein can ubiquitinate a glycosylatedform of α-synuclein (Shimura et al. 2001). Elevated expres-sion of parkin protects neuronal explants from the toxicityassociated with expression of mutant α-synuclein (Petrucelliet al. 2002). The loss of parkin may lead to an accumulationof its protein substrates and endoplasmic reticulum stress(Imai et al. 2000, 2001), which may then trigger the activa-tion of cellular suicide mechanisms (Rao et al. 2002). Therole of parkin appears to be the protection of cells from thetoxic effects of inappropriate protein behaviour.

The conditional expression of human α-synuclein in trans-genic Drosophila melanogaster provides an excellent modelof PD (Feany and Bender 2000). Pan-neuronal expression ofboth wild-type and mutant α-synuclein leads to prematureloss of climbing ability, a feature that control flies retain intoold age (Feany and Bender 2000). In addition, the expres-sion of α-synuclein in the developing eye results in preco-cious degeneration of the retina. Expression of Drosophilamelanogaster parkin can suppress the PD-like phenotypescaused by directed expression of wild-type α-synuclein(Haywood and Staveley 2004). These features recapitulatethe main behavioural and pathological phenotypes of PD andprovide an excellent model system to study the biologicalbasis of the disease.

The A30P mutant form of α-synuclein, which substitutesthe alanine at position 30 with a proline, causes autosomaldominant PD in humans (Kruger et al. 1998). In Drosophilamelanogaster, expression of A30Pα-synuclein in a pan-neuronaland dopaminergic fashion has a deleterious effect on climbingability without affecting lifespan (Feany and Bender 2000).A30Pα-synuclein expression in a dopaminergic neuronalmanner causes loss of these neurons, which is unaffected bythe co-expression of β-galactosidase (Auluck et al. 2002).Recently, we have demonstrated that co-expression of parkinwith wild-type α-synuclein counteracts the Parkinson’s likeeffects of α-synuclein expression (Haywood and Staveley2004). From the literature it is unclear as to whether mutationsin α-synuclein inhibit its ability to be targeted for degrada-tion by parkin in vivo. Should the A30P mutation preventparkin from mediating α-synuclein ubiquitination then theloss of climbing ability and retinal degeneration caused bymutant α-synuclein would not be inhibited by co-expressionof parkin. To determine if parkin has the ability to suppressthe deleterious effects of mutant α-synuclein, we have co-expressed parkin with A30Pα-synuclein and compared the

flies climbing ability and retinal degeneration with flies thatexpress mutant α-synuclein alone. We demonstrate thatparkin can counteract the effects of mutant α-synuclein-in-duced retinal degeneration and improve the flies climbingactivity. This is the first demonstration that a directed in-crease in parkin expression can counteract the effects of mu-tant α-synuclein in an experimental organism.

Materials and methods

Fly stocks and cultureDr. M. Feany (Harvard Medical School) generously provided

the UAS-A30Pα-synuclein flies (Feany and Bender 2000);similarly, Dr. J. Hirsh (University of Virginia) provided theDdc-Gal44.36 stock (Li et al. 2000). The GMR-Gal4 flies(Freeman 1996) were obtained from the Bloomington DrosophilaStock Center of Indiana University at Bloomington. The 2independent transgenic lines, UAS-parkin1.1 and UAS-parkin2.1,were derived through standard Drosophila techniques as pre-viously reported (Haywood and Staveley 2004). These lineswere used to generate double transgenic lines of the geno-types UAS-A30Pα-synuclein/CyO;UAS-parkin1.1/TM3 andUAS-A30Pα-synuclein/CyO;UAS-parkin2.1/TM3 using standardtechniques. To drive expression of the transgenes, Ddc-Gal44.36

(for expression in the dopaminergic neurons) or GMR-Gal4(for expression in the eye) homozygous females of theselines were crossed to UAS-A30Pα-synuclein males with orwithout UAS-parkin1.1 or UAS-parkin2.1. All flies were culturedon standard cornmeal – yeast – molasses – agar media at25 °C.

Climbing ability assayFlies were assayed for their ability to climb using a stan-

dard method (Feany and Bender 2000). Every 5 d, 80 maleflies from a cohort were assayed for their ability to climb6 cm in 18 s in a sterile plastic vial. Twenty trials werecarried out for each time point. Flies were tested for up to90 d. This experiment was repeated and the results compiledand statistically analysed using Prism 4.02 software (GraphPadSoftware Inc., San Diego, Calif.). Initially the data wasgraphed with the SEM and subjected to a 1-way analysis ofvariance (ANOVA), this was followed up by linear regres-sion curve-fit analysis with 95% confidence intervals. Co-expression of UAS-lacZ with UAS-A30Pα-synuclein doesnot alter the neuronal loss caused by the latter to indicatethat there is no competition effect in the expression of 2transgenes with the Ddc-Gal44.36 driver (Auluck et al. 2002).

Aging analysisAdult males were aged in small groups (n ≤ 20 individuals)

in vials upon standard media at 25 °C and scored for viabilityevery 2–3 d as described previously (Staveley et al. 1990).The total numbers of individuals aged were: 218 UAS-A30Pα-synuclein/+; Ddc-Gal44.36/+ flies, 182 UAS-A30Pα-synuclein/+; UAS-parkin1.1/Ddc-Gal44.36 flies, and 245 UAS-A30Pα-synuclein/+; UAS-parkin2.1/Ddc-Gal44.36 flies. Thedata was subjected to a log rank test and the mean life spanswere also compared using Prism 4.02 software.

© 2006 NRC Canada

506 Genome Vol. 49, 2006

© 2006 NRC Canada

Haywood and Staveley 507

Fig. 1. Expression of parkin increases climbing ability of flies expressing α-synuclein. (A) The life spans of flies that express A30Pα-synuclein with and without parkin are shown. Genotypes are as follows: �, w1118;UAS-A30Pα-synuclein /+;Ddc-Gal44.36/+; �,w1118;UAS-A30Pα-synuclein/+;UAS-parkin1.1/Ddc-Gal44.36; �, w1118;UAS-A30Pα-synuclein/+;UAS-parkin2.1/Ddc-Gal44.36. Logrank testcomparison of flies that express mutant α-synuclein with and without parkin1.1 are not significantly different, p = 0.1106. However,comparison flies that express mutant α-synuclein with and without parkin2.1 are significantly different, p = 0.0001. (B) Aged flies thatexpress parkin and mutant α-synuclein climb significantly better than flies that express mutant α-synuclein. The percentage of fliesthat climbed successfully was subtracted from 100 and the results were subjected to a non-linear regression curve fit. The genotypesare marked the same as in A. The error bars show the standard error of the mean of 20 trials at each point. Note the error bars aremostly within the symbols.

Histological examination of adult Drosophila melanogasterretinas

Nine adult flies of each genotype were aged (1 or 30 dafter eclosion), fixed in Karnovsky’s fixative, and embeddedin TAAB 812 epoxy embedding resin (Canemco andMarivac, Lakefield, Que.) as previously described (Feanyand Bender 2000). Tangential retinal sections were preparedat a thickness of 0.5 µm and stained with toluidine blue, ex-amined by light microscopy, and photographed at 800× mag-nification. Eight eye slices from separate 30-d-old flies wereanalysed by counting the number of ommatidia that had ab-normal ommatidial arrangement and presented this as a per-centage of the total number of ommatidia in that slice. Thesenumbers were collated and an average percentage of dis-rupted ommatidia calculated and statistically analysed usingPrism 4.02 software.

Results

Aging assays were carried out in tandem with the climbingassays described above to account for changes in climbingability as a result of premature senescence. Comparison ofsurvival curves indicates that there is little difference betweenthe longevity of flies that express A30Pα-synuclein with andwithout the UAS-parkin1.1 transgene (Fig. 1A). A logranktest showed that the 2 curves were not significantly different.The survival curve of flies expressing A30Pα-synuclein andUAS-parkin2.1 showed a slight decrease in median survivalage compared with flies that express A30Pα-synuclein alone(Fig. 1A).

Flies that express mutant α-synuclein with and withoutparkin in their dopaminergic neurons were assayed forclimbing ability. Flies that express both A30Pα-synucleinand parkin transgenes, specifically in the dopaminergic neu-rons under the control of Gal4, showed an extension ofclimbing ability at later ages compared with flies thatexpress mutant α-synuclein alone (Table 1; Fig. 1B). Thissuggests that parkin could act to prevent any deleteriouseffects mutant α-synuclein expression may have and certainlyacts to improve the climbing ability of these flies over theirlifespan. Apparently the improved climbing ability in laterlife was not due to increased mean life span.

Ectopic expression of parkin prevents premature wild-typeα-synuclein-induced degeneration in the Drosophila melano-gaster eye (Feany and Bender 2000; Haywood and Staveley2004). To determine if parkin has the similar ability tosuppress mutant α-synuclein-induced premature degeneration,A30Pα-synuclein and parkin were co-expressed in theDrosophila eye. Cross-sections of the retinas of 1-d-old fliesthat express A30Pα-synuclein or both mutant α-synucleinand parkin appeared intact and normal (Figs. 2A and 2B).As previously described, the retinas of 30-d-old flies that

express mutant α-synuclein show signs of premature degen-eration (Feany and Bender 2000), such as disintegration ofthe outer ring of the ommatidia and distortion of the normallyclose arrangement of rhabdomeres observed in the presentstudy (Fig. 2C, black arrows). On average, approximately40% of ommatidia had a defect including degeneration ofthe outer ring of the ommatidia or separation of the normallyclose arrangement of rhabdomeres (Fig. 2E). In contrast,30-d-old flies that express both A30Pα-synuclein and parkinappeared to maintain an intact ommatidial array (Fig. 2D),with only 5% of ommatidia showing any defect (Fig. 2E).Retinal deterioration can be observed by examining an opticaleffect termed the pseudo-pupil, which is lost in aged fliesthat express mutant α-synuclein (Feany and Bender 2000).When 20-d-old flies co-expressing mutant α-synuclein andparkin were examined, there appeared to be retention of thisoptical effect compared with flies that express mutant α-synuclein alone (data not shown). Overall, expression of parkinsuppressed degeneration of the ommatidial array caused bythe expression of mutant α-synuclein.

Discussion

Our experiments demonstrate that the directed expressionof parkin in the developing eye negates the retinal defectsresulting from mutant α-synuclein expression. In addition,we demonstrate that increased parkin expression in thedopaminergic neurons extend the climbing ability of agedflies that express mutant α-synuclein. This suggests that parkincan suppress the degeneration resulting from mutant α-synucleinexpression in spite of the amino acid substitution present inthe mutant form of α-synuclein that is thought to lead to aconformational change in the protein (Conway et al. 1998).Although the exact mechanism of phenotype suppression isnot clear, it does indicate that mutant α-synuclein is likely atarget of parkin’s ubiquitin-ligase activity.

Co-immunoprecipitation studies have suggested thatthe parkin protein does not interact with or ubiquitinateunmodified α-synuclein (Chung et al. 2001) but willubiquitinate O-glycosylated α-synuclein (Shimura et al. 2001).We have established the suppression of mutant α-synuclein-induced retinal degeneration by the ectopic expression ofparkin. Therefore, we believe that mutant α-synuclein proteinis modified in Drosophila melanogaster in a manner thatwill enable it to be ubiquitinated by the parkin ubiquitinprotein ligase then targeted to the proteasome for degrada-tion.

Transgenic Drosophila melanogaster that express eitherthe wild type or mutant form of α-synuclein in their centralnervous systems, via the pan-neural elav-Gal4 transgene,have shown an age-dependent reduction in climbing abilitywhen compared with control flies (Feany and Bender 2000).

© 2006 NRC Canada

508 Genome Vol. 49, 2006

Genotype SlopeStandarderror 95% CI

UAS-A30Pα-synuclein/+; Ddc-Gal4/+ 0.05 0.0009 0.050–0.054UAS-A30Pα-synuclein/+; Ddc-Gal4/UAS-parkin1.1 0.08 0.0012 0.074–0.078UAS-A30Pα-synuclein/+; Ddc-Gal4/UAS-parkin2.1 0.07 0.0016 0.070–0.076

Table 1. Comparison of the non-linear fitted curves for climbing ability showing slope, standard error, andconfidence interval (CI).

Notably, flies that express mutant α-synuclein under thecontrol of elav-Gal4 show a greater reduction in climbingability. Although flies that express wild-type α-synuclein intheir dopaminergic neurons show a marked premature lossof climbing ability (Haywood and Staveley 2004), expressionof mutant α-synuclein results in only a slight premature lossof the ability to climb (Auluck et al. 2002). Regardless,over-expression by both of the parkin transgenes had theeffect of extending the climbing ability of flies expressingA30Pα-synuclein when compared with the controls. Thusthe premature loss of climbing ability arising from a mutantform of α-synuclein that is known to cause PD in humanswas prevented by the directed expression of parkin.

Over-expression of parkin suppresses the PD-like symptomsinduced in Drosophila melanogaster by wild-type (Haywoodand Staveley 2004) and mutant α-synuclein with no apparentadverse consequences. We suggest that the manipulation ofthe ubiquitin–proteasome degradation pathway in such aspecific manner acts to remedy the toxicity of the accumulationof α-synuclein. Activation of parkin may be a viable treat-ment for PD caused by increased levels or mutant forms ofα-synuclein and we suggest that the selection of therapeuticstrategies should be directed towards this end.

Acknowledgements

The authors wish to acknowledge E. Lloyd Smith andBernard Healy (Faculty of Engineering, Memorial Universityof Newfoundland) for the design and production of the

climbing assay device. We thank Lisa Lee and Roy Ficken(Department of Biology, Memorial University of Newfound-land) for help with SEM and photography, respectively, andKate Williams (Faculty of Medicine, Memorial Universityof Newfoundland) for help with sectioning. This researchwas funded by the Natural Sciences and Engineering Re-search Council of Canada and the Parkinson Society CanadaFriedman Pilot Project Grant to B.E.S. A.F.M.H. was par-tially funded by a Graduate Student Demonstratorship and theSchool of Graduate Studies at Memorial University of New-foundland.

References

Auluck, P.K., Chan, H.Y., Trojanowski, J.Q., Lee, V.M., and Bonini,N.M. 2002. Chaperone suppression of alpha-synuclein toxicityin a Drosophila model for Parkinson’s disease. Science (Wash-ington, D.C.), 295: 865–868.

Baptista, M.J., O’Farrell, C., Daya, S., Ahmad, R., Miller, D.W.,Hardy, J., Farrer, M.J., and Cookson, M.R. 2003. Co-ordinatetranscriptional regulation of dopamine synthesis genes by alpha-synuclein in human neuroblastoma cell lines. J. Neurochem. 85:957–968.

Chung, K.K., Zhang, Y., Lim, K.L., Tanaka, Y., Huang, H., Gao, J.,et al. 2001. Parkin ubiquitinates the alpha-synuclein-interactingprotein, synphilin-1: implications for Lewy-body formation inParkinson disease. Nat. Med. 7: 1144–1150.

Clayton, D.F., and George, J.M. 1999. Synucleins in synaptic plastic-ity and neurodegenerative disorders. J. Neurosci. Res. 58: 120–129.

© 2006 NRC Canada

Haywood and Staveley 509

Fig. 2. Expression of parkin suppresses α-synuclein-induced retinal degeneration. (A and B) Tangential sections measuring 0.5 µmfrom 1-d-old flies; (C and D) 0.5 µm tangential sections from 30-d-old flies. The genotypes in A and C are w1118;UAS-A30Pα-synuclein/GMR-Gal4; in B and D, they are w1118;UAS-A30Pα-synuclein/GMR-Gal4;UAS-parkin1.1/+. Black arrows indicate degenerationof outer edges of ommatidia. Scale bar is 15 µm. (E) The distorted ommatidia in a single section from 8 separate 30-d-old fly eyeswere counted and displayed as a percentage of total ommatidia.

Conway, K.A., Harper, J.D., and Lansbury, P. 1998. Accelerated invitro fibril formation by a mutant alpha-synuclein linked toearly-onset Parkinson disease. Nat. Med. 4: 1318–1320.

Dawson, T.M. 2000. New animal models for Parkinson’s disease.Cell, 101: 115–118.

de Silva, H.R., Khan, N.L., and Wood, N.W. 2000. The genetics ofParkinson’s disease. Curr. Opin. Genet. Dev. 10: 292–298.

Feany, M.B., and Bender, W.W. 2000. A Drosophila model ofParkinson’s disease. Nature (London), 404: 394–398.

Freeman, M. 1996. Reiterative use of the EGF receptor triggersdifferentiation of all cell types in the Drosophila eye. Cell, 87:651–660.

Haywood, A.F., and Staveley, B.E. 2004. parkin counteracts symptomsin a Drosophila model of Parkinson’s disease. BMC Neurosci. 5:14.

Hershko, A., and Ciechanover, A. 1998. The ubiquitin system.Ann. Rev. Biochem. 67: 425–479.

Imai, Y., Soda, M., and Takahashi, R. 2000. Parkin suppressesunfolded protein stress-induced cell death through its E3ubiquitin-protein ligase activity. J. Biol. Chem. 275: 35661–35664.

Imai, Y., Soda, M., Inoue, H., Hattori, N., Mizuno, Y., and Takahashi,R. 2001. An unfolded putative transmembrane polypeptide, whichcan lead to endoplasmic reticulum stress, is a substrate of Parkin.Cell, 105: 891–902.

Jakes, R., Spillantini, M.G., and Goedert, M. 1994. Identificationof two distinct synucleins from human brain. FEBS Letters, 345:27–32.

Kahle, P.J., Neumann, M., Ozmen, L., Muller, V., Jacobsen, H.,Schindzielorz, A., et al. 2000. Subcellular localization of wild-type and Parkinson’s disease-associated mutant alpha-synucleinin human and transgenic mouse brain. J. Neurosci. 20: 6365–6373.

Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura,Y., Minoshima, S., Yokochi, M., Mizuno, Y., and Shimizu, N.1998. Mutations in the parkin gene cause autosomal recessivejuvenile parkinsonism. Nature (London), 392: 605–608.

Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel,S., et al. 1998. Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease. Nat. Genet. 18: 106–108.

Lansbury, P.T., Jr., and Brice, A. 2002. Genetics of Parkinson’s dis-ease and biochemical studies of implicated gene products. Curr.Opin. Cell Biol. 14: 653–660.

Li, H., Chaney, S., Roberts, I.J., Forte, M., and Hirsh, J. 2000.Ectopic G-protein expression in dopamine and serotonin neu-rons blocks cocaine sensitization in Drosophila melanogaster.Curr. Biol. 10: 211–214.

Mizuno, Y., Hattori, N., Mori, H., Suzuki, T., and Tanaka, K. 2001.Parkin and Parkinson’s disease. Curr. Opin. Neurol. 14: 477–482.

Nisipeanu, P., Inzelberg, R., Blumen, S.C., Carasso, R.L., Hattori,N., Matsumine, H., and Mizuno, Y. 1999. Autosomal-recessive

juvenile parkinsonism in a Jewish Yemenite kindred: mutationof Parkin gene. Neurology, 53: 1602–1604.

Parkinson, J. 1817. An essay on the shaking palsy. In A manual ofdiseases of the nervous system. Edited by W.R. Gowers. Blakiston,Philadelphia, Penn. pp. 6366–6657.

Perez, R.G., Waymire, J.C., Lin, E., Liu, J.J., Guo, F., and Zigmond,M.J. 2002. A role for alpha-synuclein in the regulation of dopa-mine biosynthesis. J. Neurosci. 22: 3090–3099.

Petrucelli, L., O’Farrell, C., Lockhart, P.J., Baptista, M., Kehoe,K., Vink, L., et al. 2002. Parkin protects against the toxicityassociated with mutant alpha- synuclein: proteasome dysfunc-tion selectively affects catecholaminergic neurons. Neuron, 36:1007–1019.

Pickart, C.M. 2001. Mechanisms underlying ubiquitination. Annu.Rev. Biochem. 70: 503–533.

Polymeropoulos, M.H., Higgins, J.J., Golbe, L.I., and Nussbaum,R.L. 1996. Mapping of a gene for Parkinson’s disease tochromosome 4q21-q23. Science (Washington, D.C.), 274: 1197–1198.

Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide, S.E., Dehejia,A., Dutra, A., et al. 1997. Mutation in the alpha-synuclein geneidentified in families with Parkinson’s disease. Science (Wash-ington, D.C), 276: 2045–2047.

Rao, R.V., Peel, A., Logvinova, A., del Rio, G., Hermel, E.,Yokota, T., et al. 2002. Coupling endoplasmic reticulum stressto the cell death program: role of the ER chaperone GRP78.FEBS Lett. 514: 122–128.

Shimura, H., Hattori, N., Kubo, S., Mizuno, Y., Asakawa, S.,Minoshima, S., et al. 2000. Familial Parkinson disease geneproduct, parkin, is a ubiquitin-protein ligase. Nat. Gen. 25: 302–305.

Shimura, H., Schlossmacher, M.G., Hattori, N., Frosch, M.P.,Trockenbacher, A., Schneider, R., et al. 2001. Ubiquitination ofa new form of alpha-synuclein by parkin from human brain:implications for Parkinson’s disease. Science (Washington, D.C.),293: 263–269.

Singleton, A.B., Farrer, M., Johnson, J., Singleton, A., Hague, S.,Kachergus, J., et al. 2003. alpha-Synuclein locus triplicationcauses Parkinson’s disease. Science (Washington, D.C.), 302:841.

Spacey, S.D., and Wood, N.W. 1999. The genetics of Parkinson’sdisease. Curr. Opin. Neurol. 12: 427–432.

Staveley, B.E., Phillips, J.P., and Hilliker, A.J. 1990. Phenotypicconsequences of copper–zinc superoxide dismutase over-expression in Drosophila melanogaster. Genome, 33: 867–872.

Zarranz, J.J., Alegre, J., Gomez-Esteban, J.C., Lezcano, E., Ros,R., Ampuero, I., et al. 2004. The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia. Ann.Neurol. 55: 164–173.

© 2006 NRC Canada

510 Genome Vol. 49, 2006