The role of α-synuclein in Parkinson's disease: insights from animal models

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NATURE REVIEWS | NEUROSCIENCE VOLUME 4 | SEPTEMBER 2003 | 727

The role of α-synuclein in the aetiology and pathogenesisof neurodegenerative diseases, especially Parkinson’s disease (PD), has been extensively studied since 1997,when it was discovered that a MISSENSE MUTATION in the gene for α-synuclein caused familial PD in a smallcohort. Since 1997, hundreds of papers on the role ofα-synuclein in neurodegenerative diseases have beenpublished.Whereas this information has generated cluesas to the aetiology of PD, the development and character-ization of α-synuclein-based animal models might becrucial to a complete understanding of pathogenesis andthe mechanisms of cell death, and provide a frameworkto test therapeutic strategies.We present a comprehensiveoverview of the information that has been derived fromthese animal models, and how this information mighthelp in the development of therapeutic strategies.

Establishing the modelThe genetics of human PD. Of the ten genes that havebeen found to be associated with inherited PD, only four have been characterized1–4: α-synuclein, parkin,ubiquitin carboxy-terminal hydrolase L1(UCHL1) and,most recently, a protein termed DJ1. α-Synuclein is a140-amino-acid protein that is found mainly in neuronsand is present in Lewy bodies — intracytoplasmic inclusions that are characteristic of both sporadic

and inherited forms of PD5. Two rare mutations in α-synuclein have been discovered in families with anautosomal dominant, early-onset form of PD. In theA53T mutation, threonine is substituted for alanine atresidue 53; in the A30P mutation, phenylalanine is substituted for alanine at residue 30.

Mutations in the parkin gene (PRKN) were found in autosomal recessive, juvenile-onset Parkinsonism(ARJP)2. Parkin is an E3 ubiquitin ligase that is involvedin the degradation of several substrates. One study sug-gests that parkin might ubiquitinate a glycosylated formof α-synuclein6. The mutation of parkin in ARJP abolishes the activity of this enzyme, and results in the death of nigral dopamine (DA) neurons withoutLewy bodies5.

UCHL1 was first linked to PD when mutations ofthis enzyme were found in two siblings from a familywith autosomal dominant PD that showed incompletePENETRANCE3. Lansbury and collaborators have recentlyfound in vitro evidence that UCHL1 can have two enzy-matic activities: a beneficial hydrolase activity and a partly detrimental ligase activity7. Variations in the activity of each enzyme could confer resistance orsusceptibility to PD7.

Two recessive mutations in DJ1 were recently foundto cause early-onset PD in two European families4.

THE ROLE OF α-SYNUCLEIN INPARKINSON’S DISEASE: INSIGHTSFROM ANIMAL MODELSEleonora Maries*, Biplob Dass‡, Timothy J. Collier‡, Jeffrey H. Kordower‡ andKathy Steece-Collier‡

The abnormal accumulations of fibrillar α-synuclein in Lewy bodies and the mutations in the genefor α-synuclein in familial forms of Parkinson’s disease have led to the belief that this protein has acentral role in a group of neurodegenerative diseases known as the synucleinopathies. Ourunderstanding of the biology of α-synuclein has increased significantly since its discovery in1997, and recently developed animal models of the synucleinopathies have contributed to thisunderstanding. The information gleaned from animal models has the potential to provide aframework for continuing the development of rational therapeutic strategies.

MISSENSE MUTATION

A mutation that results in thesubstitution of an amino acid ina protein.

PENETRANCE

The probability that anindividual with a particulargenotype manifests a givenphenotype. Completepenetrance corresponds to thesituation in which everyindividual with the same specificgenotype manifests thephenotype in question.

*Department ofNeuroscience, The ChicagoMedical School, 3333 GreenBay Road, N. Chicago,Illinois 60064, USA.‡Department ofNeurological Sciences, RushPresbyterian–St. Luke’sMedical Center, 2242 W. Harrison Street, Chicago,Illinois 60012, USA.Correspondence to K. S.-C.e-mail: [email protected] doi:10.1038/nrn1199

728 | SEPTEMBER 2003 | VOLUME 4 www.nature.com/reviews/neuro

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binds other proteins such as synphilin-1 (REF. 13), parkin14

and the anti-apoptotic chaperone 14-3-3 (REF. 15).Lewy bodies and Lewy neurites containing α-synucleinare features of disorders other than PD. These synucle-inopathies include the Lewy-body variant of Alzheimer’sdisease, dementia with Lewy bodies, neurodegenerationwith brain iron accumulation type 1, and multiple-system atrophy1,16–18. The potential role of α-synucleinin the formation of Lewy bodies and the pathogenesis ofPD has been compared to the role of β-amyloid andamyloid plaques in Alzheimer’s disease.

One hypothesis for Lewy body formation is that α-synuclein monomers become oligomers (protofibrils),which coalesce into fibrils and then aggregate into Lewybody inclusions19,20 (FIG. 1). Some authors believe thatthis is the means by which cells attempt to packageexcess cytoplasmic debris into a less noxious form. Themonomers and oligomers are soluble, whereas the fibrilsand Lewy bodies are insoluble in the neuronal cytoplasm.

The roles of the various physical forms of α-synucleinin PD pathogenesis are controversial. Some researchersconsider Lewy bodies to be neurotoxic21,22, while othershave deemed them to be protective5,23. A recent hypo-thesis states that the protofibrillary intermediates,and not the fibrils or the Lewy bodies themselves,are damaging to DA neurons. At the centre of this controversy is the in vitro finding that fibril formation is accelerated by the A53T PD mutation24, which mightunderlie the toxicity of this mutation in some animalmodels. By contrast, the A30P mutation, which also causes autosomal dominant PD, results in a slower rate of fibril formation24. However, both muta-tions lead to accelerated protofibril formation in vitro. These findings, which should be confirmed in vivo, indicate that the presence of the oligomericprotofibrils is more detrimental than their conversion to fibrils24.

A possible mechanism of protofibril toxicity hasbeen proposed by Lansbury and co-workers25,26, whoshowed that α-synuclein protofibrils can form ellipticalor circular amyloid pores. These pores are similar to those of bacterial toxins that can puncture cell membranes, resulting in the release of cellular contents and cell death (FIG. 2). It is interesting to consider that there could be a similar mechanism for α-synuclein protofibrils.

α-Synuclein and neurodegeneration. Although α-synu-clein is extensively expressed in the central nervous system, neuronal death and Lewy body formation in PDare mostly restricted to the substantia nigra. This mightbe explained by evidence that interactions between certain α-synuclein conformations and DA metabolismcause selective DA neuron degeneration in PD.For example, although wild-type α-synuclein causedapoptosis in a human DA neuronal cell line, it was neuroprotective for the non-DA cell line that was examined27. Similarly, viral transfection of human α-synuclein into the substantia nigra of rats selectivelydamaged the nigrostriatal DA system, despite high levelsof transgene expression in non-DA neurons28.

One mutation causes a homozygous deletion of DJ1and the other (L166P) might lead to the production of insufficient amounts of the protein8. Although the roles of DJ1 have not been determined, one possiblefunction, which has been proposed on the basis of studieson human endothelial cells and on the analysis ofthe crystal structure, is a compensatory role duringoxidative events8–11. The mechanism by which the loss offunction of this protein results in toxicity in geneticforms of PD remains to be elucidated.

Mutations in these genes are considered to affect thestructure and/or clearance of α-synuclein, supporting the idea that α-synuclein is crucial in the pathophysiologyof PD.

The biology of α-synuclein. α-Synuclein is an abundantcomponent of Lewy bodies, which are found in thesoma of nigral DA neurons in the brains of patientswith PD12. It can assume various conformations —from monomeric to fibrillar, from unfolded in solutionto α-helical in the presence of lipid-containing vesiclesto a β-pleated sheet or amyloid structure in the fibrilsthat compose Lewy bodies. The β-pleated conformation

a cb

d e f

Figure 1 | αα-Synuclein conformations. One hypothesis of the mechanism of Lewy bodyformation is that α-synuclein monomers join to generate oligomers (protofibrils) (a; electronmicrographs in b). Lansbury and collaborators have recently demonstrated that protofibrils canform elliptical or circular amyloid pores resembling bacterial toxins25,26 (b). α-Synucleinprotofibrils coalesce into fibrils (c,d), which have the β-pleated conformation of amyloid. Thefibrils can aggregate into Lewy body inclusions (e,f), structures that are diagnostic features ofidiopathic and familial PD. Lewy bodies are eosinophilic cytoplasmic intraneuronal inclusions,the architecture of which is characterized by an electron-dense core and a lighter surroundingring (f). They contain α-synuclein (α-synuclein immunohistochemistry is indicated by the arrowin e), and other proteins such as ubiquitin, proteasomal proteins, synphilin, neurofilaments andmicrotubule-associated proteins42. Ultrastructurally, the dense core is granular, whereas theouter ring of the Lewy body is formed by α-synuclein fibrils that radiate from the centralcore49,50. Reproduced, with permission, from REF. 91 American Association for theAdvancement of Science (2001) (a,c), Nature REF. 26 Macmillan Magazines Ltd (2002) (b),Nature Medicine REF. 19 Macmillan Magazines Ltd (1998) (d), REF. 92 Springer (2003) (e)and REF. 49 Lippincott Williams & Wilkins (1996) (f).


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This proposed toxic interaction might be related tothe oxidative environment of DA neurons. During DAmetabolism, reactive oxygen species (ROS), which canactivate apoptotic cascades leading to neuronal death5,are formed in high concentrations. ROS have beenshown to accelerate the aggregation of α-synuclein intoprotofibrils in vitro29,30, a phenomenon that could leadto the formation of more ROS31,32. DA itself stabilizesthe potentially toxic protofibrillary form of α-synuclein.So, in the oxidative environment of a DA neuron, thebiology of α-synuclein is potentially altered to generatea negative cycle, leading to neuronal death31.

The physiology of αα-synucleinSeveral animal models have been used to investigate therole of α-synuclein in normal neurons. Early studiesusing the zebra finch indicated that the expression ofα-synuclein in neuron terminals is related to synapticplasticity33. Song learning, which occurs only at a specific time early in life, coincided with the upregulationof the messenger RNA of α-synuclein33 (then calledsynelfin). Cell-culture studies show that α-synucleinalso accumulates rapidly in damaged axons and mightbe involved in regenerative sprouting34.

α-Synuclein-knockout mice, first developed byAbeliovich and collaborators, are viable and have normal synaptic architecture and brain morphology35.However, the knockout animals differ from wild-typemice in an important functional aspect of DA neuro-transmission — paired-stimulus depression (PSD).PSD occurs when a DA neuron is stimulated with twopaired electrical pulses, causing it to release DA aftereach pulse. Normally, the amount of DA released is less after the second stimulus than after the first one.The DA neurons of the α-synuclein-knockout micereleased more DA than normal after a second electrical pulse and had a faster recovery to the initial levels ofDA release. So, these animals had reduced PSD, indi-cating that α-synuclein might be a negative regulator of activity-dependent DA release.

α-Synuclein-knockout mice also have decreased striatal DA,but have normal DA metabolism.Speculationsuggests that the loss of DA in knockout mice could becaused by deficient neurotransmitter synthesis or storage,or be a consequence of the altered DA efflux that wasobserved following paired stimuli. Behavioural findingssupported both hypotheses, as the knockout animals hada decreased locomotor response to amphetamine35,a drug that acts through the DA system to cause hyper-kinesia in normal mice. These results require further evaluation, however, as other α-synuclein-knockout micehave not shown any differences in amphetamine-inducedlocomotor behaviour relative to wild-type animals36. Theresults of Abeliovich and colleagues35 indicated that α-synuclein might be a presynaptic regulator of DArelease, synthesis or storage, as well as a regulator of a specific form of synaptic plasticity, PSD.

Although these ideas have yet to be proved, studieson the overexpression of α-synuclein support them.Overexpression of α-synuclein significantly reduces theactivity of tyrosine hydroxylase (TH) both in vitro and

VMATMPP+MPP+

MPP+

Mitochondrion

DAT

MPP+

MPP+

12

3

b α-Synuclein–/–

DA efflux (–60%)

Normal DA efflux

Oligomers

Toxic (?)

α-Synuclein(–)Lewy bodiesNon-toxic (?)

Fibrils

O2–•

VMATMPP+

MPP+

Mitochondrion

ROS

DA re-uptake

DA re-uptake

DAT

MPP+

MPP+

a α-Synuclein+/+

c

Cell or mitochondrialmembrane

α-Synuclein DA, Ca2+, H+, cytochrome c

Oligomers/protofibrils

Figure 2 | Mechanisms of αα-synuclein and MPTP toxicity. a | In normal animals (α-synuclein+/+),the parkinsonian effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) are due primarilyto inhibition of mitochondrial complex I, which causes generation of reactive oxidative species(ROS) in the cell and leads to cell death. ROS activate apoptotic cascades that lead to the demiseof dopamine (DA) neurons5. They are also believed to accelerate the process of α-synucleinaggregation and potentially lead to formation of more ROS through the toxic protofibrils, thereforegenerating a cycle of death for the DA neurons of the substantia nigra pars compacta31. DA itself,when oxidized, stabilizes the protofibrillary form of α-synuclein during the process of fibrillization,increasing the concentration of protofibrils and, therefore, of ROS present in the DA neurons93.DAT, dopamine transporter; VMAT, vesicular monoamine transporter. b | The DA neurons of α-synuclein-null mice (α-synuclein–/–) are resistant to MPTP38. This observation might beaccounted for by reduced MPTP transport through the DAT (1), increased vesicular sequestrationof the drug (2), or the lack of α-synuclein (3) if this protein is required for MPTP toxicity38.c | Recently, α-synuclein protofibrils have been suggested to be neurotoxic through a mechanismother than the induction of ROS formation. They can form elliptical or circular amyloid pores,resembling bacterial toxins. These pores have the ability to puncture the membrane and releasethe cellular contents, leading to cell death25,26.


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MPTP. In the 20 years since Langston and Irwin foundthat MPTP produced parkinsonism in humans43, theMPTP-treated monkey has become the gold standardfor the motor phenotype of PD.

The parkinsonian effects of MPTP are due primarilyto the loss of ATP in nigrostriatal neurons, secondary toinhibition of mitochondrial complex I. This loss resultsin elevated concentrations of ROS, which can cause celldeath. Decreased complex I activity and increasedoxidative stress are particularly relevant in a model of PDbecause both are hallmarks of post-mortem pathologyin the brains of patients with PD44–47. α-Synuclein aggre-gation is accelerated in the presence of ROS29,30, whichresults in an increased number of potentially toxicprotofibrils48. In turn, overexpression of α-synucleinitself can produce ROS31,32. So, MPTP treatment leads toROS formation, which can result in acceleratedprotofibril formation and aggregation that, in turn,cause further ROS production. This feedforward processis expected to potentiate neuronal degeneration.A crucial interaction between MPTP and α-synuclein is supported by the inability of MPTP to induce neurodegeneration in α-synuclein-knockout mice38.

The relationship between MPTP-induced neuro-toxicity and idiopathic PD is undefined. Lewy bodies,which are characteristic of idiopathic PD, are believed tobe products of proteasome function and to sequesterpotentially toxic proteins into innocuous aggregates.Lewy bodies are eosinophilic cytoplasmic intraneuronalinclusions, the architecture of which is quite specific.They contain an electron-dense core and a lighter surrounding ring (FIG. 1f). In addition to α-synuclein(FIG. 1e), Lewy bodies contain ubiquitin, proteasomalproteins, synphilin, neurofilaments and microtubule-associated proteins42. Ultrastructurally, the dense core isgranular, whereas the outer ring is formed by 10–16-nmα-synuclein fibrils that radiate from the central core49,50.These fibrils have the β-pleated sheet conformation ofamyloid, which can be visualized by staining with thefluorescent dye thioflavin S.‘True’ Lewy bodies with thiscomplex concentric architecture and radiating β-fibrilshave only been seen in the human disease.

In rare cases, MPTP-treated animal models havedeveloped α-synuclein inclusion bodies, but theseaggregates usually fail to reproduce the structure ofLewy bodies. For example, baboons treated with MPTPcan develop intracytoplasmic, fine granular α-synucleinaccumulations and some larger deposits, but theseinclusions do not adopt the distinctive architecture ofLewy bodies51. In mice, chronic exposure to MPTPincreases the number of DA neurons that contain diffuse α-synuclein immunoreactivity52, and results inelevated levels of α-synuclein mRNA and protein in thesubstantia nigra52,53. However, only one study has produced evidence of inclusion bodies or aggregation.Mice that were treated for three weeks with MPTP andprobenecid, a drug that delays MPTP metabolism and clearance, had small granular, non-fibrillary inclusions in the substantia nigra that ultrastructurallyresembled the dense core of a Lewy body54. After 24 weeks, the substantia nigra and cortex contained

in vivo, and produces a corresponding reduction in DAlevels that is not caused by an increase in DA catabolismor efflux37.

Other experiments using α-synuclein-knockout animals have investigated its role in the pathogenesis ofPD by investigating whether the absence of α-synucleinalters nigrostriatal degeneration in the presence oftoxins that produce PD-like symptoms. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is convertedto MPP+ by monoamine oxidase-B and is selectivelytaken up by DA neurons through the DA transporter(DAT) (FIG. 2a). Rotenone is a non-selective mitochondrialcomplex I inhibitor that penetrates cell membranes.Both compounds are selectively toxic to nigral DA neurons in wild-type animals. Although both acute andchronic MPTP treatment reduced the number of DAneurons in wild-type mice, neither protocol induced theloss of DA neurons in α-synuclein-knockout mice38.Primary neuronal cultures from these mice were alsoresistant to MPP+ (REF. 38). However, unlike MPTP,rotenone was toxic in cultures from knockout mice.Although both MPTP and rotenone inhibit complex I,they apparently have different mechanisms of actionprior to reaching complex I. So, MPTP resistance in theknockout mice might be caused by an inability of MPP+

to access complex I in the absence of α-synuclein. Theauthors suggest that such upstream alterations couldinvolve reduced MPP+ transport by DAT or an increasein vesicular sequestration of MPP+ in the knockout mice38

(FIG. 2b). Another study using α-synuclein-knockoutmice showed them to be partially protected againstacute exposure to MPTP39. However, these investigatorsalso found that mice carrying a spontaneous deletion inthe α-synuclein gene are not protected39. On the basis of their results, they suggest that α-synuclein is notobligatorily coupled to MPTP sensitivity, but can influ-ence MPTP toxicity in some genetic backgrounds. Thisillustrates the need for extensive controls when describ-ing the effects of mouse knockouts on MPTP sensitivity.

αα-Synuclein and intoxication models of PDThe effective study of the relationship of α-synuclein toPD requires reliable and accurate animal models. Anideal model of PD-related synucleinopathies wouldexhibit the cardinal signs of idiopathic PD: bradykinesia,akinesia, postural impairment, resting tremor, gait dis-order, rigidity40,41, adult onset, a relatively selective loss ofDA neurons of the substantia nigra pars compacta, agood response to DA replacement early in the diseaseprocess42, and the presence of Lewy bodies and Lewyneurites. Other desirable features would include areduced activity of mitochondrial complex I and a highconcentration of ROS42.

Several models using neurotoxins have been used toinvestigate the functions of α-synuclein, in particular itspotential role in the pathogenesis of PD (TABLE 1). Recentdata have been obtained from several laboratoriesstudying MPTP and rotenone in rodent and primatemodels. These studies have investigated how oxidativestress and metabolic challenges alter the structure,expression and aggregation of α-synuclein.


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Table 1 | Main characteristics of animal models of synucleinopathies

Model (reference) DA neuron loss Motor deficits LB-like inclusions Other important findings

Knockout mice

(35) – ↓ Amphetamine-induced – ↑ DA release with pairedlocomotion electrical stimuli

(38) + (In presence – – In vitro, mutant DA neurons of MPTP) resistant to MPTP but sensitive

to rotenone;↓ DA efflux in knockouts

Intoxication models

MPTP-treated primates + Rigidity and bradykinesia; Intracytoplasmic, (51) variable tremor not LB-like

MPTP-treated mice + Rigidity and bradykinesia – ↑ Striatal α-syn mRNA (52) and protein

MPTP and probenecid-treated + ND Granular, similar to ↓ Striatal α-syn mRNAmice (55) dense core of LB

Rotenone-treated rats (62) + Ataxia, hypokinesis, hunching, +rigidity, shaking limbs

Transgenic Drosophila

Pan-neuronal promoter-Hu WT, + ↓ Negative geotactic response +A30P, A53T α-syn (65,67) in A30P and WT flies

Flies co-expressing Hsp70 and – ND +Hu α-syn through DOPA-decarboxylase gene promoter (64)

Human αα-synuclein transgenic mice

PDGFβ promoter-WT ↓ Striatal DA ↓ Rotarod performance Atypical inclusionsα-syn (63) terminals

Thy1 promoter-WT and A53T – ↓ Rotarod performance; Non-fibrillar inclusions Detergent-soluble andα-syn (77) progressive deterioration insoluble α-syn species

over time were identified

TH promoter:

A30P α-syn – – Non-fibrillar inclusions(80)

A30P and A53T α-syn – ND Non-fibrillar inclusions(79)

WT and hm2 α-syn ↓ DA Hm2 α-syn mice: – Abnormal axons and terminals(81) ↓ amphetamine-induced in hm2 α-syn mice;↓ locomotion

locomotion; motor deterioration after MPTP in WT and hm2 α-synwith advancing age mice

PrP promoter:

A53T α-syn – Severe locomotor disability + Axonal degeneration; astrocytic(82) and death gliosis. Insoluble and soluble

α-syn species

WT, A30P and A53T α-syn ND A53T α-syn mice: severe A53T mice: fibrillar A53T brains: ↑ insoluble(83) locomotor disability and inclusions, not LB-like; α-syn; astrocytic gliosis

death A30P mice: diffuseinclusions

Rats overexpressing human αα-synuclein

AAV-A30P α-syn + Amphetamine-induced Non-fibrillar inclusions Abnormal axons(86) rotationand ↓ rotarod

performance (no statisticalsignificance)

AAV-WT and A53T α-syn + Apomorphine rotation and LB-like inclusions but(87) impaired paw-reaching test ubiquitin negative

(if > 60% DA depletion)

Lentivirus-WT, A30P and A53T + ND Non-fibrillar inclusions ↓ Striatal TH expression in WTα-syn (28) A30P α-syn animals

Viral overexpression of αα-synuclein in primates

AAV-WT or A53T α-syn + Bias in head position in some Granular Surviving cells with diffuse (89) animals intracytoplasmic α-syn immunoreactivity had

inclusions a normal appearance

AAV, adeno-associated virus; DA, dopamine; hm2 α-syn, doubly mutated α-synuclein; Hsp70, heat-shock protein 70; LB, Lewy bodies; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; ND, not determined; PDGF, platelet-derived growth factor; PrP, prion-related promoter; α-syn, α-synuclein; TH, tyrosine hydroxylas; Thy1, thymus cellantigen; WT, wild-type.


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toxin and develop the appropriate syndrome. Despitethese limitations, these models show that inhibition of complex I results in the accumulation of excess α-synuclein and selective degeneration of DA neurons.The animal models have also shown that, at least in the case of MPTP, α-synuclein can influence toxicity in DA neurons.

αα-Synuclein and genetic models of PDThe discovery of mutations associated with PD inspiredthe development of genetic animal models of the synucleinopathies (TABLE 1). These models involve the overexpression of wild-type or mutant human α-synuclein, either in transgenic animals or throughviral expression. Recent studies have reported variableoutcomes from such models, which might result fromfactors such as the degree of gene expression and therelationship between toxicity and other variables, suchas environment and age.

Drosophila. One of the first attempts to overexpress α-synuclein was made in the fruitfly Drosophilamelanogaster, an organism that contains no endogenousα-synuclein or synuclein-like genes64–66. Feany andBender overexpressed human wild-type and mutant(A53T and A30P) α-synuclein in the fruitfly using theGAL4-responsive pUAST pan-neuronal expression vector65. This model has been useful when examiningthe effects of α-synuclein genotypes on DA survival,synuclein aggregation and behaviour. The developmentof neural and non-neural tissue was normal in thetransgenic flies. However, they began to show selectivedepletion of DA neurons at 30–60 days of age64,65.Behavioural analysis of the transgenic flies showed lossof motor function. Normal young flies have a negativegeotactic response, characterized by their ability toclimb to the top of a vial; this ability is reduced in fliesover 45 days of age. A loss of climbing ability wasobserved in both wild-type and A30P transgenic flies,but it manifested earlier (at 23 days) in the A30P α-synuclein mutants, perhaps because A30P α-synucleinis more toxic than the wild-type protein to Drosophila65.The motor impairments in transgenic Drosophila werefully reversed by the administration of levodopa,pergolide, SKF38393 or bromocriptine67, underscoringthe validity of this animal model for the replication of thepathology and therapeutic response of PD.

Takahashi and colleagues showed that the degen-erating DA regions of transgenic Drosophila containedα-synuclein-positive inclusions. The inclusions werecomposed of filaments and granules, and their size andlocation was reminiscent of the Lewy bodies of patientswith PD. The α-synuclein in the inclusion bodies wasphosphorylated at serine 129, one of the molecularmarkers of human synucleinopathies66. α-Synucleinbecame phosporylated after it aggregated, and the concentration of phosphorylated α-synuclein increasedwith age66. However, inclusions were found in relativelyfew neurons compared to the widespread distributionthat occurs in human synucleinopathies68, and they didnot contain insoluble α-synuclein69. A53T transgenic

filaments and granules that stained positive for both α-synuclein and ubiquitin. Although the aggregates didnot resemble true Lewy bodies, they were similar to the cortical inclusions found in PD. Specifically, theseaggregates were round bodies composed of filamentsintermixed with granules, which lacked the concentricradial architecture of Lewy bodies55. Interestingly,α-synuclein expression was downregulated, consistentwith post-mortem data from patients with PD56.Downregulation of the α-synuclein gene might repre-sent a feedback response to the presence of α-synucleinaggregates in the brains of patients with PD or in thoseof MPTP-treated rodents55. On the basis of the findingthat α-synuclein inhibits TH activity, such downregula-tion of the α-synuclein gene, or a decrease in proteinlevels by aggregation, could increase DA synthesis withan accompanying increase in toxic DA metabolites37.

Although the appearance of increased levels ofcytoplasmic α-synuclein and inclusion bodies indicatesthat similar cellular mechanisms might operate in theanimal models and the human disease, reproducing the exact pathology is desirable. It is possible that thetime required for the organization of the elaboratearchitecture of true Lewy bodies has precluded their formation in many animal models. A further caveat tofinding such pathology in mice might be related to alack of ‘permanent’ DA toxicity in this model afterMPTP treatment. Perhaps continued examination of pathology following more protracted treatmentschedules in non-human primates will eventually revealtrue Lewy body architecture, although it is conceivablethat Lewy bodies are unique to humans.

Rotenone. Rotenone-treated rodents have emerged asanother promising animal model of synucleinopathiesand PD. Epidemiological studies have revealed a correla-tion between the onset of PD and exposure to pesticidesand insecticides such as rotenone57,58. In vitro, rotenoneinduces degeneration of DA neurons59,60 by inhibitingcomplex I and generating ROS, which can cause oxidative damage to proteins and DNA59,60, leading toapoptotic death of DA neurons.

In the rotenone model of PD, rotenone is infusedchronically into the jugular vein of Lewis rats61,62. Abouthalf of the surviving animals showed specific DA neurondegeneration and some of these animals developedsevere nigrostriatal degeneration. These rats exhibitedataxia, hypokinesia, hunched posture, and in somecases, marked rigidity and shaking of the forelimbs,resembling resting tremor in PD. These animals alsodeveloped intraneuronal cytoplasmic inclusions thatwere positive for α-synuclein and ubiquitin. Some ofthese aggregates were composed of fibrils that werearranged around an electron-dense core, resemblingLewy bodies more closely than any other animal model63.

The MPTP and rotenone models of PD are valuablebecause they exhibit DA neurodegeneration and a patternof motor impairment that is characteristic of PD.However, the MPTP-lesioned animals do not alwayshave α-synuclein aggregates, and only a percentage of rotenone-treated rats survive treatment with the


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a significant decrease in chaperone concentration couldbe responsible for cell death71. Regardless of whichhypothesis proves to be true (FIG. 3), these Drosophilastudies indicate that increasing chaperone activity in the brain could be a useful therapeutic approach tosynucleinopathies such as PD.

Drosophila that overexpress α-synuclein have alsobeen used to investigate the function of parkin.Yang andcolleagues co-expressed human parkin and α-synucleinin Drosophila. The presence of parkin protected about50% of DA neurons from death when compared to flies that expressed only α-synuclein. In addition, fliesexpressing parkin showed no α-synuclein granularinclusions, and reduced ubiquitin in DA neurons72.These results indicate that parkin might reduce the for-mation of protein accumulations in synucleinopathiesand is neuroprotective, at least in this Drosophila model.

Deletion of parkin in Drosophila reduced the lifespanof the flies, induced sterility in males, and decreasedflight and climbing ability73. The defects in locomotorbehaviour were not due to degeneration of the DA system, but to abnormal muscle fibre structure. In fact,DA neurons of parkin-null Drosophila were mostly normal in number and TH intensity, although cell bodiesin the dorsomedial DA cluster were shrunken and haddecreased neuritic TH immunostaining.

The Drosophila parkin knockout did not reproducethe DA cell loss that occurs in humans with ARJP, wholack parkin. One possible explanation for the lack of DAcell loss in the parkin-null flies is that Drosophila do nothave the glycosylated 22-kDa form of α-synuclein,which is one of the substrates for parkin6. This solubleα-synuclein species accumulates in ARJP and might betoxic to nigral neurons. As the flies cannot accumulatethis protein, deletion of parkin would not be expectedto be deleterious to DA neurons in Drosophila.

The Drosophila model has strengths and limitations.Its limitations are the lack of glycosylated α-synuclein,and that the nervous system of the fly is relatively simpleso that only a narrow range of locomotor behaviourscan be tested. The value of this model is that Drosophilagenes are highly homologous to their human counter-parts, and entire genetic pathways have been conservedduring evolution74. Further, the mechanisms ofneurodegeneration in flies and humans are similar.Specifically, high concentrations of α-synuclein in thebrain lead to the loss of DA neurons, the formation of inclusions with the morphology of Lewy bodies and deficient motor output, which are reversible following treatment with anti-parkinsonian medica-tions. Despite the fact that Drosophila normally lack α-synuclein, it is interesting that processing of the protein seems to be similar between this model and humans, and that the specificity of toxicity to DAneurons is maintained.

Transgenic mice. Transgenic rodent models have alsobeen used in an attempt to elucidate the pathogenesis ofthe synucleinopathies. The most numerous of thesemodels are α-synuclein transgenic mice created usingvarious gene promoters.

flies contained more phosphorylated α-synuclein thanother transgenic animals66. This could be explained bythe more rapid formation of fibrils by the A53T mutantprotein24, which would make more fibrils available for phosphorylation. The finding that chaperones —molecules that aid protein folding and the formation ofcorrect tertiary structure — can prevent protein misfolding in Drosophila models of polyglutamate disease70 has generated interest in these proteins aspotential neuroprotective agents. In support of this idea,co-expression of human heat-shock protein 70 (Hsp70)alleviated the toxicity of α-synuclein in transgenic flies64.Conversely, when the constitutive Drosophila Hsp70chaperone Hsc4 was rendered defective in flies over-expressing α-synuclein, the flies had 50% fewer DA neurons at birth and accelerated α-synuclein-inducedDA cell loss in adulthood64. Increasing chaperone activity by geldanamycin treatment delayed DA neurondegeneration in wild-type and mutant (A30P andA53T) α-synuclein transgenic flies64.

There are several mechanisms by which chaperonemolecules could mitigate the deleterious effects ofα-synuclein. First, although they do not affect the morphology of the inclusion bodies, chaperones mightreduce the toxicity of potentially toxic protofibrils oroligomers by altering their protein structures.Additionally, by modulating the structure of α-synucleinproteins, chaperones might inhibit their interactionwith other neurotoxic proteins that can activate celldeath pathways71. Alternatively, chaperone moleculescould be sequestered in Lewy bodies and Lewy neurites,leading to chaperone depletion in the cells.As chaperonesdirect the folding and function of many cellular proteins,

α-Synucleinprotofibrils(oligomers)

Lewy bodies

Chaperone

Non-toxic α-synucleinconformations

Interaction with potentialneurotoxic proteins

Chaperonedepletion

3

1

2

23

Dopamine neuron cell death

Motor impairment

Figure 3 | Relationship between chaperone molecules and αα-synuclein toxicity. It hasbeen proposed that chaperones might alter the conformation of highly toxic protofibrils oroligomers — the intermediates of fibril formation — and reduce their neurotoxicity throughchanges in protein structure (1). By modulating the structure of α-synuclein, chaperones inhibittheir interaction with other neurotoxic proteins that would activate cell death pathways (2). In thesynucleinopathies, chaperone molecules become sequestered in Lewy bodies and Lewy neurites,leading to chaperone depletion, which might be directly responsible for cell death (3) (REF. 71).


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animals also showed reduced sensitization to repeateddoses of amphetamine, had increased spontaneouslocomotor activity, and exhibited progressive motordeterioration. Although the double-mutant α-synucleindid produce some nigrostriatal abnormalities, it did notproduce a parkinsonian condition characterized by DAcell loss and Lewy-like bodies. Treatment of the doubletransgenic mice with MPTP led to motor impairmentand increased DAT similar to that in transgenic animalsexpressing wild-type α-synuclein.

The doubly mutated (A30P and A53T) α-synucleintransgenic mice were the only line created using the THpromoter that showed age-related (13–23 months)motor impairment. This finding supports the idea thatoverexpression of wild-type or single-mutant α-synucleinalone might not be responsible for nigrostriatal neurotoxicity, at least in mice. Additional factors —environmental, age-related or other — probably interactwith α-synuclein to cause parkinsonism.

Use of the mouse prion-related protein (PrP) promoter results in uniform expression of the α-synuclein transgene throughout the brain82,83.A53T α-synuclein transgenic mice showed a markedand ultimately fatal motor phenotype with advancingage82,83. Their motor neurons had areas of axonal degen-eration near fibrillary α-synuclein inclusions. Theseinclusions stained positive for neurofilament, thioflavin Sand ubiquitin82. Although it is unclear whether thesestructures had the morphology that is typical of Lewybodies83, it is possible that at least some of the inclusionswere true Lewy bodies, especially as they containedinsoluble A53T α-synuclein83. The transgenic brains alsocontained α-synuclein dimers and trimers83. Althoughthe association between soluble α-synuclein and diseasesupports the protofibrillary hypothesis of PD pathogen-esis, the data do not exclude the possibility that the fibrillary form is responsible for neurodegeneration.

Mice with the wild-type transgene under the controlof the PrP promoter developed no motor pathology forup to 24 months, and showed no α-synuclein accumu-lations83. A30P transgenics developed with the PrP promoter exhibited no cell death and only diffuse α-synuclein aggregations that did not contain ubiquitin83.By contrast, A53T transgenic animals generated with thePrP promoter showed severe motor impairments andα-synuclein inclusions, supporting the idea that A53T isthe most neurotoxic α-synuclein mutation in themouse82,83. However, as both idiopathic and A30P α-synuclein-mediated PD are characterized by motordisability and Lewy bodies, these wild-type and A30P α-synuclein transgenic mice were not representative ofthe human disease. By contrast, the A53T transgenicmice are a potentially useful model for elucidating themechanisms of α-synuclein pathogenicity.

A promising avenue for future research using transgenic animals is the identification of the mecha-nisms that protect against α-synuclein-induced neurodegeneration. Co-expression of human α- and β-synuclein in transgenic mice limited the developmentof motor deficits and the number of synuclein-positiveinclusions, and increased striatal TH immunoreactivity,

Transgenic mice overexpressing wild-type α-synucleinusing the platelet-derived growth factor-β (PDGFβ) promoter had electron-dense inclusion bodies localizedto the brain structures that are affected in PD, and wereimmunoreactive for α-synuclein and ubiquitin63.However, these inclusions were composed of fine granules interspersed with clear vacuolar structures, andmany were present in atypical locations, such as therough endoplasmic reticulum and the nucleus, insteadof the cytoplasm. Among these animals, those with thehighest levels of α-synuclein expression exhibitedreduced striatal DA terminals and motor impairmenton the ROTAROD TEST. None of the animals showed DA cellloss in the substantia nigra. This indicates that a strongpromoter might be needed to obtain a high level ofα-synuclein expression that facilitates the induction of Lewy body-type inclusions, loss of DA neurons and motor impairment63.

To achieve reproducible, high-level, neuron-specificgene expression, several groups have used the mousethymus cell antigen 1 (Thy1) regulatory cassette75,76.A53T and wild-type α-synuclein transgenic animalscreated using this promoter showed motor impairmentsin rotarod testing, with progressive deterioration77.However, these transgenic mice exhibited pathology in the anterior spinal cord, and not in the nigral neurons. Pathology in the spinal cord was in the form ofubiquitin-positive, granular α-synuclein inclusions,astrocytic gliosis, activation of microglial cells andaxonal degeneration77. Similar to the transgenic mice in which expression of α-synuclein is under the controlof the PDGF promoter, animals in which expression is driven by Thy1 did not exhibit true Lewy bodies.However, the brains of these transgenic animals contained insoluble α-synuclein species similar to thosefound in fibrils and Lewy bodies78.

In contrast to the Thy1 promoter, the DA-specificTH promoter can drive robust transgene expression incatecholamine-containing neurons79,80, and was used todevelop A30P α-synuclein transgenic mice80. These animals exhibited intense α-synuclein immunoreactivityin the substantia nigra and ventral tegmentum, but thenumber of DA neurons and catecholamine concentra-tions were unaltered. Locomotion was also unaffected,indicating that the level of A30P α-synuclein might nothave been sufficiently high to be neurotoxic80. Similarly,A53T α-synuclein overexpression driven by the TH promoter had no impact on the number of DA neuronsor on the concentration of striatal DA79. Unlike theA30P α-synuclein transgenic mice, A53T mice did haveα-synuclein aggregates in DA neurons79, but these werenot immunoreactive for ubiquitin or thioflavin S,indicating that they were not true Lewy bodies.

The rat TH promoter has also been used to express adouble-mutant form of α-synuclein that containedboth the A30P and A53T mutations81. Overexpression of the double-mutant α-synuclein failed to induce theformation of any α-synuclein inclusions, but was associ-ated with abnormal medial forebrain bundle axons andsynaptic terminals, lower levels of DA and its metabolites,and higher concentrations of DAT. These transgenic

ROTAROD TEST

Motor test that probes the abilityof rodents to keep their balanceon a cylinder that rotatescontinuously at a slow speed,commonly 5–6 revolutions perminute.


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the neurotoxic effects of human A53T α-synuclein. Theremaining DA neurons contained granular α-synucleinaggregates that lacked ubiquitin immunoreactivity andthe fibrillary structure of true Lewy bodies28. In thisstudy, no behavioural tests were performed.

In spite of the excitement that was initially generatedby the rodent models of synucleinopathies, they show agreat deal of variability in the degree of neuropathologicalchanges and motor impairment (TABLE 1). Only twotransgenic mouse models showed nigrostriatal degener-ation: animals generated with the PDGF promoter63,and mice created with the TH promoter to expresswild-type or doubly mutant α-synuclein81. The inconsistent results that have been obtained with these animals might be due to variations in the levels of α-synuclein expression and to species-specific differences in the processing of this protein. These models offer a great deal of promise, but require furtherdevelopment if they are to be useful in developing a better understanding of the pathogenic mechanisms ofα-synuclein.

Vector-mediated expression in monkeys. Recently, viraldelivery of α-synuclein has been used in non-humanprimates. AAV expressing wild-type or mutant A53T α-synuclein was unilaterally administered to the substantia nigra pars compacta of three common marmosets89. Signs of degeneration were found innigrostriatal neurons transduced with either the wild-type or mutant α-synuclein. A 30–60% loss of DAneurons was observed on the injected side, and wasaccompanied by decreased striatal DA innervation.Intracytoplasmic α-synuclein-immunoreactive inclu-sions and granular deposits, as well as swollen axonsand fragmented neurites, were evident in some nigralneurons and along the nigrostriatal pathway. A sub-population of surviving DA neurons that were diffuselyimmunoreactive for α-synuclein had a normal appear-ance, indicating that diffuse α-synuclein accumulationsmight be harmless. The study did not documentthioflavin S or ubiquitin staining of the inclusions.

Disappointingly, these animals showed no unilateralmotor dysfunction when treated with amphetamine. Insome animals, a bias in head position was noted sixweeks after the administration of the α-synuclein AAV,and it persisted until the animals were killed 16 weeksafter administration89. So, overexpression of α-synucleinby AAV generated moderate nigral degeneration, butdid not create motor dysfunction in a manner similar toMPTP treatment. However, the appearance of a bias in posture might indicate that degeneration occurred at a much slower rate than is found after MPTP admin-istration. The use of a higher titre of AAV or of astronger viral expression system might increase thedegree of DA cell loss and lead to motor disability.

This study used only three animals transfected withAAV–α-synuclein (one injected with wild-type and twowith A53T α-synuclein). So, the results must be repli-cated before the use of vector-mediated expression ofα-synuclein in primates can be regarded as a validmodel of synucleinopathies.

compared to mice overexpressing α-synuclein alone84.These data can be explained by the recent finding thatβ-synuclein reduced the in vitro aggregation of A53T α-synuclein into protofibrils and fibrils in a dose-dependent manner85. β-Synuclein alone did not formamyloid pores, and formed protofibrils more slowlythan either α- or γ-synuclein85. The interaction ofβ-synuclein with α-synuclein might confer neuro-protection by preventing the formation of α-synucleinprotofibrils. So far, no studies have reported co-precipi-tation of α- and β-synuclein after their expression in cells. Therefore, the mechanisms by which the inter-actions between these two molecules might occur in vivo are still unclear.

Vector-mediated overexpression in rats. Delivery ofα-synuclein by viral vectors has three advantages overtransgenic expression. First, overexpression of a particulargene can be site-specific. Second, animals other than mice can be used to express the gene of interest,allowing a more thorough behavioural analysis. Third,overexpression can be achieved in adulthood, withoutaffecting development or raising issues of compensation.

ADENO-ASSOCIATED VIRUSES (AAV) have been used tointroduce A30P α-synuclein by unilateral injection intothe rat substantia nigra86. This manipulation resulted ina significant loss (53%) of DA neurons and formationof α-synuclein aggregations in nigral neurons. However,these inclusions did not stain for ubiquitin or thioflavin S.The treated animals tended to show amphetamine-induced rotations ipsilateral to the injection site and to fall off a rotating rod in a shorter time than controlrats, although these effects did not attain statistical significance86.

Rats expressing wild-type or A53T α-synucleinintroduced using AAV also showed intraneuronal,ubiquitin-negative α-synuclein inclusions, some ofwhich comprised a core surrounded by an α-synuclein-positive ring87. In contrast to the rats overexpressingA30P α-synuclein, rats expressing wild-type or A53T α-synuclein exhibited only transient alterations in DAphenotype and nigrostriatal axonal pathology87.Although no significant gross motor impairment wasevident in the wild-type or A53T α-synuclein rats,statistically significant deficits in a paw-reaching test andan increased number of apomorphine-induced rotations contralateral to the injection site were found inanimals with more than 60% striatal DA depletion.A similar threshold must be reached to induce the

development of symptoms in patients with PD40,41.A LENTIVIRAL VECTOR has also been used to transfect rats

with human wild-type or mutant A53T or A30P α-synuclein28. In each case, there was about a 30% lossof nigral TH-positive neurons 5 months after treatment.Interestingly, striatal TH expression was decreased bywild-type and A30P α-synuclein, but not by A53T α-synuclein. So, in contrast to transgenic mice, theA53T mutation was not more neurotoxic in transfectedrats. This finding could be explained by the presence ofthreonine at position 53 of rat α-synuclein88, whichmight interfere with the human protein and diminish

ADENO-ASSOCIATED VIRUSES

A group of viruses that requireco-infection with an adenovirusor a herpesvirus for theirreplication. If no helper virus ispresent, the genome of adeno-associated viruses can beintegrated into the host DNA,resulting in latent infection.

LENTIVIRUSES

A group of retroviruses thatincludes HIV.Virus derivativesthat are engineered to bereplication-defective can be usedas expression vectors. Lentiviralvectors have advantages overretroviral vectors because oftheir ability to infect non-dividing human cells,particularly neurons.


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models are advantageous because they uniformly showDA neurodegeneration and a pattern of motor impair-ment that is characteristic of PD, whereas the geneticmodels exhibit significant variability in these parameters.

The rotenone-treated rat is the only model that replicates all of the key features of synucleinopathiessuch as PD. However, expression of the syndrome is variable, occurring in fewer than 50% of animals treated,and maintenance of the animals is labour-intensive.α-Synuclein transgenic flies are also excellent geneticmodels of PD; their disadvantages are the limited rangeof locomotor behaviour that can be tested in flies, andthe fact that flies represent a simpler system than rodentsand primates.

None of the transgenic mice models show DA celldeath, only exhibiting loss of DA or DA terminals. Thereason for this remarkable resistance of DA neurons tothe toxicity of transgenic α-synuclein is unclear. Viralmodels have been more successful in reproducing thespecific DA neuron death that is characteristic of PD.Although the results from the α-synuclein transgenicmice are highly variable, it is instructive to consider thefactors behind this variability. These differences mightbe explained by the amount of α-synuclein expressionattained in each model, the location of gene expression inthe brain, and the duration of the experiment. Anotherimportant caveat is that endogenous α-synuclein mightinterfere with Lewy body formation. The combinationof mouse and human α-synuclein prevents α-synucleinfibril formation, owing to an inhibitory substitution atasparagine 87 (REFS 24,88,90). Rat α-synuclein, which contains a threonine at position 53, might also interferewith toxicity after overexpression of human A53T α-synuclein. To circumvent this problem, α-synuclein-knockout mice could be used in combination with viraloverexpression of human α-synuclein. These knockoutsare viable, can breed, and have normal DA neuron function35. It is therefore possible that they could betransduced with human α-synuclein.

Animal models of the synucleinopathies are essentialfor the study of both idiopathic and hereditary PD,diseases that are characterized by the presence of wild-type or mutant (A30P and A53T) α-synuclein, respec-tively. Future development of these models should focuson using viral expression systems to transduce rodentsand primates with the α-synuclein gene. Specifically, the

ConclusionsAlthough no animal model of the synucleinopathiesshows the complete spectrum of PD features, the modelsdescribed in this review complement each other andhave helped elucidate various elements of disease patho-genesis. Animal models of the synucleinopathies werecreated using neurotoxins that inhibit complex I (MPTPor rotenone), or by overexpressing α-synuclein (FIG. 4). Inboth cases, α-synuclein accumulates in neurons, leadingto the formation of protofibrils, fibrils and occasionalLewy body-like inclusions.

There are marked differences between the intoxica-tion and genetic α-synuclein models with regard toselective DA neuron loss, presence of motor deficits andLewy body development (TABLE 2). The intoxication

MPTPRotenone

Wild-type α-synucleinoverexpression

A30P/A53T α-synucleinoverexpression

Complex 1inhibition

Mitochondrion

ROS

Dopamine neuron death Cell survival

Lewy body-likeinclusions

α-Synucleinoligomers

α-Synucleinfibrils

α-Synucleinmonomers

ApoptosisCytotoxicity

+ +

?

?

Figure 4 | Generation of animal models of the synucleinopathies. Animal models of thesynucleinopathies have been created using neurotoxins (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or rotenone) or by overexpressing α-synuclein. In both cases, α-synuclein accumulates in the cells, leading to formation of protofibrils, fibrils, and occasionalinclusion bodies. Overexpression of A30P or A53T α-synuclein results in an increasedconcentration of α-synuclein oligomers. Overexpression of wild-type α-synuclein leads to anincreased concentration of α-synuclein monomers, which then undergo oligomerization and formfibrils. When inhibitors of mitochondrial complex I such as MPTP and rotenone are used, radicaloxidative species (ROS) are formed, and apoptotic or cytotoxic cascades are activated, ultimatelyleading to death of dopamine neurons. The ROS also induce α-synuclein aggregation andprotofibril formation29,30. The protofibrils can then lead to further ROS production31,32, generating a feedforward process that could potentiate neuronal degeneration.

Table 2 | Intoxication and genetic models of synucleinopathies

Characteristics of PD Intoxication models αα-Synuclein genetic models

MPTP-treated Rotenone-treated Drosophila Rodents Primates

DA neuron loss + + + variable +

Motor deficits + + + + variable

Lewy bodies Intracytoplasmic + + variable Intracytoplasmicgranular inclusions granular inclusions

Time of onset Immediate Immediate Advanced age Advanced age Immediate(transgenics); (viral vectors)immediate (viral vectors)

Complex I inhibition + + ? ? ?

DA, dopamine; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson’s disease.


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As the glycosylated form of α-synuclein accumulates inautosomal recessive PD, leading to juvenile onset of thedisease, it might be more detrimental than wild-type α-synuclein. The use of appropriate transgenics and cellcultures will establish wether the glycosylated conforma-tion is more neurotoxic than the wild-type protofibrils.

In addition to exploring whether there is differentialneurotoxicity between the various forms of α-synuclein,research is needed to identify the mechanisms oftoxicity and to understand the role of α-synuclein in PD.This information could then be used to design therapeu-tic approaches directed at reducing the concentration ofthe toxic α-synuclein conformation in the brain.Strategies that either inhibit the oligomerization processor accelerate the formation of fibrils might prove usefulin the clinical setting of both sporadic and inherited PD.

viral vectors will need to generate a high level of proteinexpression and to be placed stereotactically in the substantia nigra. The transduced animals should beevaluated in long-term studies, as Lewy bodies mightrequire months or years to develop. New animal modelsof synucleinopathies could be used in studies investigat-ing the presence and quantities of the various forms of α-synuclein, ranging from oligomeric to fibrillaryand Lewy bodies. By blocking the conversion frommonomers to protofibrils, from protofibrils to fibrils,and from fibrils to Lewy bodies in these animals, thestructure of the pathogenic α-synuclein conformationsand the mechanism of toxicity could be elucidated.

It would also be instructive to analyse whether the tox-icity of different soluble α-synuclein conformations (thewild-type and the glycosylated 22-kDa proteins) varies.

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Online links

DATABASESThe following terms in this article are linked online to:LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/GAL4 | PDGFβ | PRKN | PrP OMIM: http://www.ncbi.nlm.nih.gov/Omim/Alzheimer disease | ARJP | β-amyloid | dementia with Lewybodies | DJ1 | Parkin | Parkinson disease | α-synuclein | β-synuclein | γ-synuclein | ubiquitin | UCHL1

FURTHER INFORMATIONEncyclopedia of Life Sciences: http://www.els.net/Adeno-associated virusesAccess to this interactive links box is free online.

The role of α-synuclein in Parkinson's disease: insights from animal models

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