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Is there a cause-and-effect relationship between αααα-synuclein fibrillization and Parkinson’s disease?

Matthew S. Goldberg and Peter T. Lansbury Jr

The first gene to be linked to Parkinson’s disease encodes the neuronal protein αααα-synuclein. Recent mouse and Drosophila models of Parkinson’s disease support a central role for the process of αααα-synuclein fibrillization in pathogenesis. However, some evidence indicates that the fibril itself may not be the pathogenic species. Our own biophysical studies suggest that a structured fibrillization intermediate or an alternatively assembled oligomer may be responsible for neuronal death. This speculation can now be experimentally tested in the animal models. Such experiments will have implications for the development of new therapies for Parkinson’s disease and related neurodegenerative diseases.

arkinson’s disease is a common age-associated degenerative disorder char-acterized by rigidity, difficulty in initi-

ating movements and a resting tremor1,2. Atautopsy, the brain is characterized by loss ofbasal ganglia neurons, predominantlydopaminergic neurons of the substantianigra. The underlying cause of this degener-ation is unknown, so current treatments forthe disease are based on ameliorating themotor symptoms, either by replacement ofthe deficient neurotransmitter (for example,by L-DOPA therapy) or, less commonly, bydirect surgical perturbation of the affectedneuronal circuitry. A possible clue to theaetiology of Parkinson’s is the presence insome of the nigral neurons that remain atautopsy of fibrillar cytoplasmic inclusionsknown as Lewy bodies3. In contrast, Lewybodies in cortical neurons characterize dif-fuse Lewy body disease (DLBD), a relativelycommon dementia often mistaken forAlzheimer’s disease4. Whether Lewy bodiesare a cause or a consequence of neuronaldeath in Parkinson’s disease and DLBD, orare an unrelated epiphenomenon, has yet tobe determined. This question is reminiscentof the unresolved role of fibrillar deposits(amyloid plaques and nuclear inclusions,respectively) in the aetiology of Alzheimer’sand Huntington’s diseases5,6.

A clue from familial Parkinson’s The genetic contribution to idiopathic late-onset Parkinson’s disease is difficult todetermine because of the existence of a sig-nificant presymptomatic phase that hasbeen revealed by positron emission tomo-graphic (PET) imaging; neuropathologicalexamination shows that loss of around 60%of the nigral dopaminergic neurons canapparently be tolerated. It is, however, clearfrom studies of identical twins that cases inwhich one twin is diagnosed before the ageof 50 have a significant genetic component7.

The first Parkinson’s disease gene to be iden-tified was linked to a rare, autosomal-domi-nant, early-onset form of the disease (FPD;other genes have been reported subse-quently, see Box 1)8,9. This gene encodes theprotein α-synuclein, which is widelyexpressed in the brain. Subsequent to

reports of the genetic linkage,immunohistochemical10,11 and biochemical12

studies of Lewy bodies from idiopathic casesshowed that wild-type α-synuclein is amajor component of the insoluble fibrillarportion. The fact that the disease-associatedgene encodes the fibrillar protein is again

P

Table 1 Comparison of human Parkinson’s disease with–synuclein transgenic and knockout animals.α-synuclein- null mouse*

* Ref. 19.

Transgenicmouse†

† Ref. 20.

Transgenic Drosophila‡

‡ Ref. 21.

Human Parkinson’s disease

Anatomical features

Phenotypic restriction to the nervous system

Yes Yes Yes Yes

Anatomic specificity within the nervous system

Yes§

§ Although α-synuclein is highly expressed in the hipppocampus, α-synuclein null mice do not show altered long-term potentiationof glutaminergic synapses in hippocampal slice cultures. By contrast, nigrostriatal slice cultures from α-synuclein null mice showaltered dopamine release upon stimulation with paired electrical pulses relative to wild-type controls.

Yes Yes Yes

Neuronal pathology

Loss of dopaminergic neurons

No¶

¶ α-Synuclein null mice show normal brain architecture, cell morphology and normal numbers of neurons and synapses.

No#

# No difference was observed in the number of dopamine neurons in transgenic and control mice; however, α-synuclein transgenicmice show degeneration of dopamine nerve terminals. The threshold for detecting a loss of dopamine neurons may be lower inDrosophila than in mice, because of their anatomic dispersion in the former.

Yes# Yes

Degeneration of dopaminergic nerve terminals

No Yes# Yes Yes

α-Synuclein immuno-reactive neuronal inclusions

No Yes**

** These mice show α-synuclein-positive nuclear inclusions. Whereas other neurodegenerative diseases, such as Huntington’sdisease, are characterized by inclusions within cell nuclei, inclusions are rarely found within the nuclei of neurons in Parkinson’sdisease.

Yes Yes

Lewy-body-like fibrillar α-synuclein

No No Yes Yes

Behavioural or biochemical alterations

Decreased striatal dopamine

Yes Yes NA Yes

Age-dependent onset of phenotype

No Yes Yes Yes

Locomotor impairment No Yes Yes††

†† A30P mutant transgenic Drosophila showed a more rapid onset of the reported behavioural phenotype compared to fliesexpressing similar, but not identical, levels of wild-type or A53T mutant α-synuclein. This difference may be due to differences inthe level of transgene expression that are difficult to quantify but may be significant (two- to fourfold). NA, not applicable.

Yes

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reminiscent of other neurodegenerative dis-eases, including Alzheimer’s and Hunting-ton’s (Box 2), and suggests that a gain oftoxic function may be linked to α-synucleinfibrillization.

An αααα-synuclein knockout mouseα-Synuclein is expressed throughout thebrain at high levels13, but there is little infor-mation on its normal function. Unlike mostproteins of 140 amino acids, neither wild–type α-synuclein nor the two disease–linkedmutants fold into structured, globularforms in vitro; the protein is intrinsticallydisordered or ‘natively unfolded’14,15. Thisunusual behaviour raises the question ofhow an unstructured cytoplasmic proteinevades rapid degradation. It may be that α-synuclein exists in vivo as a complex withanother protein or proteins; one possiblecandidate has been identified11. α-synuclein

is a presynaptic protein that appears to beassociated with vesicular structures16,17 andhas been linked to learning, developmentand plasticity18. Thus, despite apparentlyconvergent evidence that points to a toxicgain of α-synuclein function being respon-sible for Parkinson’s disease, the possibilitythat disease results, in full or in part, fromthe loss of functional α-synuclein must beinvestigated. Knockout mice, in which theα-synuclein gene has been removed, repre-sent one loss–of–function model. Thesemice appear to develop normally andexhibit slightly altered stimulus–dependentdopamine release, suggesting that α-synu-clein is a negative regulator of dopaminerelease19. The brains of the knockout mice,like those of Parkinson’s patients, are char-acterized by reduced levels of striataldopamine, but, unlike Parkinson’s brains,they do not contain Lewy bodies, nor are

they characterized by neuronal or synapticloss (Table 1). Although it is possible thatthese differences may be related to the slowprogression of the disease relative to thelifespan of the mouse, it is more likely thatsome or all of the features of Parkinson’sdisease are a consequence of α-synuclein–dependent toxicity, casting further suspi-cion on the process of fibrillization.

αααα-synuclein-expressing transgenicsThe gain of toxic function scenario inspiredtwo recent animal models of Parkinson’sdisease – transgenic mice20 and Drosophila21

(see Table 1). These animal models recreatecertain features of the disease by expressionof human wild–type α-synuclein (in thecase of the flies, both mutant proteins havesimilar effects). In both cases, cytoplasmicinclusions that resemble Lewy bodies, todifferent degrees, were detected. Bothreports note a rough correlation betweenthe transgene expression level and inclusionformation; in the mouse, the correlation iswith the number of inclusions, in the flies,with the rate at which fibrillar inclusionsappear. In the mouse model, the α-synu-clein inclusions have no detectable fibrillarsubstructure, unlike those in the ‘sympto-matic’ fly (see below) and those in post–mortem Parkinson’s and DLBD brains.Thus the mouse inclusions, as well as dif-fuse α-synuclein deposits detected in ‘pre-symptomatic’ fly brains, may represent aprefibrillar stage. Importantly, both themice and the flies have age-dependent path-ological and behavioural dopaminergicabnormalities; synaptic loss in the mice andcell loss in the flies – and motor deficienciesin both organisms (Table 1). The onset of amotor phenotype was in both cases corre-lated to inclusion formation; in the case ofthe mice, the expression level of wild-typeα-synuclein correlated with the frequencyof inclusions and with the extent of the phe-notype. Similarly, in the case of the flies, theappearance of inclusions parallelled theonset of motor abnormalities, consistentwith α-synuclein fibrillization being criticalfor disease.

αααα-synuclein oligomerizationThe transformation of α-synuclein from itsnormal soluble state to the disease-associatedfibrillar state involves coupled changes in itsconformation (increased β-sheet content)and its quaternary structure (oligomeriza-tion and subsequent fibrillization). There isno evidence for a stable, structured α-synu-clein monomer; that is, the β-sheet confor-mation is not long-lived in the absence ofoligomerization (this also seems to be thecase for many other fibrillogenic proteins)22.The FPD-linked mutations do not signifi-cantly alter the conformation of α-synuclein,suggesting that they may instead influencethe intermolecular interactions that driveformation of the ordered fibrillar form15. Our

Figure 1 Images obtained by atomic force microscopy (AFM) of several discrete αααα-synuclein oligomers; placed in a pathway that is consistent with the time–course of αααα-synuclein fibrillization and parallels the pathway of Aββββ fibrillization, which has been extensively studied25. The first step involves the conversion of natively unfolded monomeric αααα-synuclein to an apparently spherical (height is 3–5 nm) oligomeric form that is rich in ββββ–sheet structure (by circular dichroism, J-C. Rochet and P.L., unpublished). This is the step that seems to be sensitive to both mutations linked to Parkinson’s disease27. It is not difficult to imagine how age-associated or toxin-induced2 deficits in energy metabolism could lead to increased concentrations of cytoplasmic synuclein (the proteasome is ATP-dependent) and/or the inability to prevent oligomerization (many chaperones are ATP-dependent). Subsequent steps seem to involve a series of sphere–sphere annealing steps, leading to chain-like assemblies (height is 3–5 nm) that can either fuse head-to-tail to produce an annular protofibril (height is 3–5 nm), or can anneal side-to-side to produce, by a cooperative mechanism, large fibrils (height. is around 8 nm and length is much greater than chain protofibrils). The blue scale bar = 250 nm.

α-synuclein

Sphericalprotofibril

Annularprotofibril

Chainprotofibril

Fibril

β-Sheet formation

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own studies of in vitro fibril formation by α-synuclein, and also by the Aβ protein ofAlzheimer’s disease, demonstrate that it doesnot follow a simple one-step transition, butrather a complex process that involves one ormore discrete intermediates, termed protofi-brils (Fig. 1)15,23–26. The possible existence ofthese prefibrillar species in vivo offers a sim-ple explanation for the fact that fibrillar α-synuclein is not detected in ‘symptomatic’transgenic mice20; that is, a protofibril,rather than the fibril itself, may be patho-genic. This scenario (Fig. 2) is also consist-ent with the in vitro behaviour of the A30Pvariant of α-synuclein, which is linked toearly–onset Parkinson’s disease9. A30Paccumulates in an oligomeric form, as itforms fibrillar species more slowly thandoes wild-type α-synuclein27. We havecharacterized several α-synuclein oligomersthat are structurally related to the fibril inthat they, like fibrils but unlike the nativelyunfolded monomer, contain β–sheet struc-ture (J-C. Rochet and P.T.L., unpublishedobservations). These oligomers are muchsmaller than fibrils and appear early in thefibrillization process (Fig. 1)26,27.

The proposal that a prefibrillar interme-diate or an alternative assembly state of α-synuclein is toxic also explains two observa-tions made on pathological examination ofhuman brains. The first is that the substan-tia nigra dopaminergic neurons containingLewy bodies appear to be ‘healthier,’ bymorphological and biochemical criteria,than neighbouring neurons28,29; the secondis that it is not uncommon to observe ‘inci-dental’ Lewy bodies at autopsy of aged indi-viduals who had no symptoms ofParkinson’s or other neurodegenerativediseases30. Thus, fibrillar inclusions maysequester toxic species and/or divert α-synuclein from toxic assembly pathways.Careful studies of pathogenesis in animalmodels of related neurodegenerative dis-eases also support the notion that a nonfi-brillar species, the formation of which islinked to fibrillization, is pathogenic. First,two transgenic mouse models of Alzhe-imer’s disease exhibit Alzeimer’s-likeabnormalities before fibrillar plaques can bedetected31,32. Second, at the time of birth, atransgenic mouse model of Creutzfeldt-Jakob disease contains abnormal forms ofthe prion protein (PrP), albeit not the formcorrelated with full-blown disease (PrPSc)33.As the animals age, neurologic abnormali-ties can be detected and, subsequent to thattime, PrPSc appears in the brains of theseanimals. Third, several cellular and animalmodels of Huntington’s disease and othertriplet-expansion diseases have demon-strated that experimental perturbations thatreduce the number of nuclear inclusionsthat are detectable by light microscopyincrease the severity of disease-relatedabnormalities (see Box 2). Unfortunately,direct detection of the prefibrillar interme-

diates that may be responsible for theseeffects has proved extremely difficult,because the Aβ- and α-synuclein-derivedoligomeric species formed and character-ized in vitro (Fig. 1) are transient, too smallto be reliably detected in tissue by light andelectron microscopy, and relatively unsta-ble to dilution and other conditions used toextract abnormal protein from tissue.

The missing link The mechanism by which abnormal α-synuclein oligomers may cause dysfunc-tion and death of dopaminergic neurons isthe missing link in the model of the Par-kinson’s disease pathogenic cascadedepicted in Fig. 2 (ref. 2). It is important toremember that the toxic pathway was notnecessarily optimized by evolution, butmay be a survivor of a selective process thatwould disfavour its interference withreproduction5. Thus, the cell-type selectiv-

ity of Parkinson’s disease degeneration, ahallmark of the disease, may merely be aconsequence of the fact that dopamine is,itself, neurotoxic, so that any disruption ofits packaging and secretion could result incell death. Alternatively, it may be that thedopaminergic cytoplasm presents the opti-mal environment for α-synuclein oli-gomerization or that the target of the toxicspecies is most highly expressed in theseneurons. Structured α-synuclein oligom-ers could exhibit potent interactions withcertain targets that present repeating inter-acting motifs, such as membrane surfaces,proteoglycans and other oligomeric pro-teins. These binding partners will be diffi-cult to identify, because the popularmethods for identifying protein-proteininteractions in pathways (for example,yeast two-hybrid assay) are designed todiscover monomer-monomer complexes.Although it is not necessary to understand

Figure 2 A scenario, based on the actual species shown in Fig. 1, that offers one explanation for the imperfect correlation between fibrils/Lewy bodies and Parkinson’s disease. This scenario represents one extreme version of a group of related possible mechanisms, in that it holds that the fibrils are inert. In fact, a low level of toxicity (per αααα-synuclein molecule) may be associated with the fibrillar state. Two critical steps that could be targeted by small drug-like molecules are highlighted in red and green. Inhibition of the initial formation of ββββ-sheet containing spherical protofibril (red step) might be expected to decrease the number of Lewy bodies and delay onset of Parkinson’s-like symptoms. In contrast, inhibition of the protofibril-to-fibril transition (green step) might be expected to decrease the number of Lewy bodies while accelerating onset of disease symptoms, by causing accumulation of a toxic species. Finally, the effect of inhibition of protofibril circularization (blue step) may help to determine whether the annular protofibril is the toxic species or a harmless off-pathway reservoir for toxic protofibrillar forms.

Parkinson'sdisease

?

??

Lewybody

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the mechanism of oligomer toxicity inorder to devise effective therapies for Par-

kinson’s disease, as once the toxic specieshas been identified, oligomerization itself

can be targeted, elucidation of this path-way will provide new downstream targetsfor therapeutic intervention.

Aetiology of transgenic animals The animal models of Parkinson’s diseaseallow the timing of α-synuclein oligomeri-zation and fibrillization to be compared todisease-associated phenomena and testableaetiological models of disease to be con-structed. The promise of these models isthat they can be used to distinguish causeand effect experimentally, by observing theconsequences of various perturbations onthe progression of the disease and its asso-ciated phenomena. If it is possible to‘uncouple’ a particular phenomenon (forexample, α-synuclein fibrillization) fromdisease progression, then it is unequivo-cally an epiphenomenon. The repeated fail-ure to do so suggests, but does not prove, adirect involvement of that phenomenon inthe pathway.

Two classes of perturbations can beenvisioned. The first are endogenous per-turbations from altered expression ofexisting or mutant genes, and the secondare exogenous perturbations from drug-like compounds. Using classical Dro-sophila genetic methods, mutants can beselected on the basis of their ability to sup-press or enhance disease in the fly34. Sup-pressor genes are candidate protectivefactors, and the products of these genes arepotential protein therapeutics. Alterna-tively, using classical screening methods ofmedicinal chemistry, drug-like moleculescan be selected on the basis of their abilityto modify a particular disease-associatedphenomenon in vitro. For example, mole-cules could be selected that inhibit α-synu-clein fibrillization, while allowingoligomerization (Fig. 2). According to thespeculative model presented above, thesemolecules should accelerate disease onsetand progression by causing accumulationof the toxic oligomer. Molecules of thisclass would, however, inhibit appearanceof fibrillar Lewy-body-like inclusions. Asimilar effect may account for the effects ofcertain proteins that inhibit the appear-ance of visible inclusions on experimentaltriplet repeat disease (see Box 2).

Therapeutics and diagnosticsThe rapid progression from identificationof the first Parkinson’s disease gene in1997 to animal models in early 2000 holdsgreat promise that, in the next few years,the pathogenesis of the disease might beelucidated to the extent that target pro-teins and processes can be identified andnew therapeutic approaches developed.The successful implementation of anyapproach based on the earliest pathogenicevents, such as α-synuclein oligomeriza-tion, may, however, depend on the detec-tion of very early preclinical disease. The

An analogue to Huntington’s disease? The strictly inherited neurodegenerative movement disorders such as Huntington’s disease and the spinocerebellar ataxias (types 1, 2, 3, 6 and 7) involve expansion of pre-existing polyglutamine tracts in otherwise unrelated proteins. All these diseases are characterized by the existence of nuclear or cytoplasmic fibrillar inclusions, which consist of the product of the expanded gene, in the post-mortem brain. A correlation exists between the length of the polyglutamine expansion and the age of onset of the disease on the one hand, and the rate of in vitro model protein fibrillization on the other38–40, suggesting that fibrillization of the expanded mutant protein may be pathogenic. Several studies have attempted to test this correlation by perturbing cellular models of these related diseases and observing the effect on cell death and inclusion formation. Three studies demonstrated that a reduction in aggregates detectable by light microscopy produces similar41 or increased levels of cell death42,43. However, two other studies demonstrated that a reduction in detectable aggregates parallels a reduction in cell death or apoptosis44,45. Taken together, these studies suggest that detectable aggregates are either unrelated to cell death or are a linked epiphenomenon. In the latter case, the perturbations may produce different effects by inhibiting different steps in the fibrillization pathway; interference at an early step in oligomerization could block the formation of toxic oligomers, whereas interference

at a late step may lead to accumulation of toxic oligomers (see Fig. 2).

Animal models of these diseases, in which polyglutamine-expanded peptides are expressed, can also be interpreted in the light of the toxic oligomer model presented here. In Caenorhabditis elegans, progressive loss of neuronal function precedes the appearance of cytoplamic inclusions or morphological abnormalities46. In Drosophila, retinal degeneration and inclusion formation were temporally indistinguishable, but second-site suppressors of this neurodegenerative phenotype did not significantly effect the density of inclusions in the eye, again uncoupling detectable fibrillar inclusions and cell death34. Finally, a conditional transgenic mouse in which the expression of an expanded huntingtin fragment could be turned on and off demonstrated that continuous expression of the mutant protein was necessary to maintain inclusions and Huntington’s-like symptoms and to drive progressive neurodegeneration47. This result can be interpreted in light of the model presented here as evidence for the toxicity of a transient prefibrillar intermediate, which is rapidly converted to the stable fibrillar form and therefore must be continuously produced in order for the disease to progress. One obvious therapeutic approach would be to prevent the first step of the fibrillization pathway, possibly by imitating molecular chaperones such as Hsp40 and Hsp70, which prevent inclusion formation and cell death34,44,45,48.

Two other familial Parkinson’s disease genes Two genes other than that for α-synuclein have been linked to rare early-onset Parkinson’s disease. They are the genes for ubiquitin C-hydrolase-L1 (UCH-L1)35 and parkin36. The latter was first linked to a juvenile-onset form of parkinsonism that is not characterized by Lewy bodies, possibly as a result of its rapid onset (it will be interesting to see if cases of late-onset Parkinson’s are found to be linked to parkin mutations and, if so, if these patients have Lewy bodies). Both these gene products

may have a role in the ubiquitin-dependent pathway of protein degradation. Defective protein degradation, resulting from these mutations or from ageing, could lead to an increase in the cytoplasmic α-synuclein concentration above its critical concentration for oligomerization and fibrillization6. Alternatively, very slight increases in the concentrations of all cytoplasmic proteins could promote α-synuclein fibrillization by a ’molecular crowding’ phenomenon37.

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possibility of identifying Parkinson’s dis-ease presymptomatically is likely to resultfrom rapid advances in two distinct areasof research. First, genetic screening mayallow the identification, and eventuallythe detection, of susceptibility factors forthe disease, and thus allow the identifica-tion of individuals who are at increasedrisk of contracting it. Second, imagingmethods such as PET that are already ableto detect presymptomatic dopaminergicdeficits will become more practical andless expensive, allowing secondary screensof the at-risk populations. Although eco-nomic considerations may prevent wide-spread screening for presymptomaticParkinson’s disease, these advances arelikely to be critical for the development ofnovel therapeutics.Matthew S. Goldberg and Peter T. Lansbury Jr are at the Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, The Department of Neurology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USAe-mail: lansbury@cnd.bwh.harvard.edu

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ACKNOWLEDGEMENTS

The authors thank the NINDS for their support in the form of a

Morris K. Udall Parkinson’s disease research center of excellence

at Brigham and Women’s Hospital. The continuing support of

the Foundation for Neurologic Diseases (Newburyport,

Massachusetts) is also gratefully acknowledged. P.L. thanks the

Alzheimer’s Association for a 1999 Zenith award. M.S.G.

acknowledges the NIH for support in the form of a postdoctoral

traineeship in molecular biology of neurodegeneration.

We also thank the following individuals for suggestions

concerning the manuscript: Mel Feany, Michael Schlossmacher,

Matthew Frosch, Jie Shen, Anne Hart, Ethan Signer and

members of the Lansbury laboratory. We also thank Parsa

Kazemi–Esfarjani and Mel Feany for sharing their unpublished

results and Tomas Ding for supplying the AFM images in Fig. 1.

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