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0166-2236/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.doi:10.1016/S0166-2236(03)00238-8

a-Synuclein oligomerization: a role for lipids?

Kevin Welch and Junying Yuan

Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA

a-Synuclein is a core component of the proteinaceous

aggregates observed in several neurodegenerative

diseases. A central role of a-synuclein in neurodegen-

eration was demonstrated by the discovery of missense

a-synuclein mutations in familial Parkinson’s disease.

However, the specific mechanism by which a-synuclein

contributes to these diseases remains unclear. A recent

study by Sharon et al. linked the presence of specific

fatty acids to the appearance of a-synuclein oligomers

in vivo. a-Synuclein oligomers might be a first step in

the formation of a-synuclein aggregates present in a

number of neurodegenerative diseases, although their

cytotoxicity remains to be directly demonstrated.

a-Synuclein has notoriety for being implicated in manyneurodegenerative diseases. A central role for this proteinin Parkinson’s disease has been established with thediscovery of two Parkinson’s disease familial mutations.a-Synuclein is a primary component of Lewy bodies, aswell as of abnormal proteinaceous aggregates present inParkinson’s disease, dementia with Lewy bodies, multiplesystem atrophy and other diseases [1]. However, thespecific mechanism by which a-synuclein contributes tothese diseases remains unclear.

Oligomerization of a-synuclein

a-Synuclein has been found in insoluble aggregates inneurodegenerative diseases. One notion gaining support isthat the primary insult leading to the deaths of relevantcell populations is formation of a-synuclein prefibrillaroligomers, rather than formation of the insoluble fibrilsthat are the hallmarks of many neurodegenerativediseases [2]. This phenomenon might not be confined toa-synuclein but, rather, common to soluble oligomers ingeneral: for example, b-amyloid (Ab) soluble oligomers inAlzheimer’s disease [3], and even soluble oligomers formedby non-disease-associated proteins, have been shown to beinherently cytotoxic [4].

Evidence supporting this notion comes mostly fromin vitro experiments. The formation of oligomeric species ofa-synuclein in vitro was found to parallel that of Ab, non-fibrillar oligomers of which are toxic in cell culture [2] andcan disrupt cellular processes in vivo [5]. Further in vitroexperiments showed that the Parkinson’s-disease-related

a-synuclein mutations (A30P and A53T) promote theformation of fibrillar and non-fibrillar aggregates, and 20–25 molecules of a-synuclein were found to form oligomerswith pore-like morphologies [6]. Noting that the A30P andA53T mutations cause an earlier appearance of thesestructures than that seen with wild-type a-synuclein,Volles and Lansbury [7] posit that such a structure mightbe responsible for permeabilization of membranes by pre-fibrillar a-synuclein, in in vitro assays and possibly inin vivo conditions leading to the disease. Although this isintriguing, the formation of a-synuclein pre-fibrillaraggregates resembling pores has yet to be demonstratedin a cellular context. Gosavi et al. [8] provide correlativeevidence of the disruption of Golgi complex with theappearance of a-synuclein aggregates, but the nature ofthe a-synuclein aggregates is unclear and the role ofmonomeric a-synuclein, or larger a-synuclein aggregates,cannot be ruled out.

The formation of a-synuclein oligomers can beaffected by neurological injuries. Oxidative injuries,which are linked to many neurodegenerative diseases,can affect a-synuclein in several ways, including thenitration of tyrosine residues (nitrated a-synuclein hasbeen observed in Lewy bodies) or the formation of adopamine–a-synuclein adduct [9,10]. Consistently,both of these phenomena have effects on the formationof a-synuclein oligomers in vitro. Nitration inducesthe formation of a-synuclein oligomers and stabilizesa-synuclein polymers [9], although it can lead to theaccumulation of oligomerized but pre-fibrillar speciesofa-synuclein[11],whereasdopaminereducestheamountoffibrillara-synuclein inacell-freesystem,possiblystabilizingapre-fibrillarspeciesofa-synucleinbyformingadopamine–a-synuclein adduct [10].

a-Synuclein–lipid associations

The in vitro association between a-synuclein and lipidmembranes, and particularly small vesicles, is wellestablished. Although a-synuclein is generally cytosolicin its distribution, it is enriched in synaptosomal fractionsof mouse and human brain, and similarities between theN terminus of a-synuclein and the lipid-binding domainsof some apolipoproteins suggest a role for a-synucleininteractions with lipid membranes [12]. Further data insupport of a lipid–a-synuclein interaction were observedCorresponding author: Junying Yuan (jyuan@hms.harvard.edu).

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by Sharon et al. [13], who reported lipid-binding protein-like domains in the N-terminal and C-terminal regions ofa-synuclein, and observed high-molecular-weight a-synu-clein complexes following delipidating treatments ofcytosolic fractions from mouse brain and from a mesence-phalic neuronal cell line (MES cells). Sharon et al. [14]have provided evidence that these high-molecular-weightreactive bands are in fact a-synuclein multimers, ratherthan a-synuclein in a heterogeneous protein complex,using 2D gel electrophoresis and size-exclusion chroma-tography of extracts from a-synuclein-expressing MEScells and mouse brains. Furthermore, in a comparison ofMES cells stably expressing wild-type and A53T a-synu-clein, Sharon et al. [14] found that the A53T mutationcaused the appearance of oligomeric a-synuclein signifi-cantly earlier than the appearance of equivalent amountsof a-synuclein multimers in cells expressing wild-typea-synuclein. The apparent larger multimers they observedare strongly reminiscent of the high-molecular-weighta-synuclein-reactive bands seen in brain tissue of individ-uals afflicted with synucleinopathies such as Parkinson’sdisease, multiple system atrophy and possibly Alzheimer’sdisease [15,16]. Sharon et al. concluded that delipidationrevealed pre-existing a-synuclein oligomers, although itremains to be rigorously ruled out that removal of lipidsdid not promote oligomerization by exposing previouslyhidden epitopes. The requirement of high temperature(378 C) treatment with Lipidex 1000 is intriguing becausesuch treatment at 08 C is sufficient to bind free lipids,whereas at 378 C Lipidex 1000 binds both free and protein-bound lipids [17]. The presence of other proteins in thesehigh-molecular-weight complexes cannot be discounted.

Effects of lipids on a-synuclein oligomerization

Multimerization of recombinant a-synuclein in vitro canbe promoted by the treatment of long- chain polyunsatu-rated fatty acids (PUFAs) [18]. In keeping with theobservations of Perrin et al. [18], Sharon et al. [14]cultured MES cells stably expressing human wild-typeand mutant a-synuclein in the presence of a variety of fattyacids, and found that the formation of a-synuclein multi-mers correlated well with the length and degree ofsaturation of fatty acids in a time-dependent manner.They observed that long chain PUFAs promoteda-synucleinmultimerization, whereas saturated fatty acids decreasedthe levels of a-synuclein multimers; mono-unsaturatedfatty acids had no discernable effect. In what could be acapsule summary of a-synuclein aggregation in Parkin-son’s disease, the authors observed the formation ofa-synuclein oligomers as early as one hour after addingPUFAs to MES cell culture media, with amounts ofoligomers increasing in a time-dependent fashion, untilat later time-points the levels of a-synuclein oligomersdiminished again, as very high-molecular-weight and gel-excluded material began to appear.

How might PUFAs promote a-synuclein oligomeriza-tion? In the study by Perrin et al. [18], a concentration ofarachidonic acid in excess of the critical micelle concen-tration (CMC) was necessary to detect a significantincrease in a-synuclein multimerization. Possibly a-synu-clein requires a micellar or vesicular surface to provide a

focal point for the initiation of oligomerization. However,Sharon et al. [14] observed a-synuclein multimerization inthe presence of PUFA concentrations as low as half theCMC, questioning a requirement for a micellar orvesicular surface for a-synuclein oligomerization. Howa-synuclein oligomerization might be mediated by freefatty acids is unclear. Longer and increasingly unsatu-rated fatty acids, even in free and non-micellar form, mightpromote an a-synuclein conformation that is more favor-able for oligomerization with other a-synuclein molecules.

What is the significance of the effect of PUFA ona-synuclein oligomerization? If the accumulation ofa-synuclein oligomers correlates with cytosolic PUFAlevels in brains of patients with synucleinopathy, wecouldspeculate thatPUFA-inducedformationofa-synucleinoligomers might be a key nucleation step that leads tothe subsequent formation of pre-fibrillar and fibrillara-synuclein. If this is so, it might be possible to manipulatefatty acids in the brain in such a way as to reduce theformation of toxic a-synuclein species.

Consistent with their in vitro observation, Sharon et al.[14] report that the expression of human a-synuclein intransgenic mice leads to a-synuclein multimerization inolder animals. The age-related accumulation of humana-synuclein multimers exposed by delipidation in trans-genic mice is suggestive of a role for a-synuclein inneurodegeneration. It is tempting to interpret this age-related accumulation of a-synuclein oligomers asrecapitulating human Parkinson’s disease. However, thepresence of a-synuclein multimers in normal mice asyoung as two-months old has been demonstrated using acommercial lysis buffer [19]. It will be interesting tocompare the effects of these different extraction buffersside by side and determine crucial factors that affectthe detection of a-synuclein oligomers.

Finally, Sharon et al. [14] detected multimerica-synuclein in normal human brain and in human brainsamples from individuals afflicted with Parkinson’s dis-ease or dementia with Lewy bodies. As was the case fora-synuclein transgenic mice, the detection of a-synucleinoligomers was enhanced by delipidating treatment.Furthermore, Sharon et al. detected a twofold increasein the ratio of a-synuclein dimers to monomers in brainsamples from individuals with Parkinson’s disease ormultiple system atrophy relative to the ratio in controlbrains. These data suggest that some of the wild-typesoluble a-synuclein in the brains of the diseased individ-uals might have already oligomerized, or might have ahigher tendency to oligomerize than the a-synuclein ofnormal human brain.

Concluding remarks

Although the possible existence of a-synuclein oligo-mers in vivo is intriguing, we still do not know howcytotoxic they are or whether they form protofibrils inthe same way as those described in in vitro systems [2,6,7].The possible toxicity of a-synuclein oligomers does notpreclude a toxic insult from monomeric a-synuclein, alarger a-synuclein homomultimer, or from a-synuclein incomplex with other proteins. The presence of a toxicprotein complex of a-synuclein and 14-3-3 has been

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suggested by Xu et al. [20], who described dopamine-dependent a-synuclein-induced apoptosis in dopaminergicneurons. No a-synuclein protofibrils were observed in thepresence, or absence, of dopamine in their cell lines. Itmight be interesting to re-investigate the possiblepresence of a-synuclein oligomers with Lipidex 1000treatment in the system used by Xu et al. [20] – this mightallow examination of functional roles of a-synucleinoligomers and the impact of dopamine metabolism onthe potential cytotoxicity of a-synuclein oligomers. It isinteresting to speculate that the selective death ofdopaminergic neurons in Parkinson’s disease is causedby a combination of cellular stress from dopaminemetabolism and a-synuclein oligomers that might includeother protein complexes as constitutive components.

References

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3 Kayed, R. et al. (2003) Common structure of soluble amyloid oligomersimplies common mechanism of pathogenesis. Science 300, 486–489

4 Bucciantini, M. et al. (2002) Inherent toxicity of aggregates implies acommon mechanism for protein misfolding diseases. Nature 416,507–511

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7 Volles, M. and Lansbury, P. (2002) Vesicle permeabilization byprotofibrillar a-synuclein is sensitive to Parkinson’s disease-linkedmutations and occurs by a pore-like mechanism. Biochemistry 41,4595–4602

8 Gosavi, N. et al. (2002) Golgi fragmentation occurs in the cells with

prefibrillar a-synuclein aggregates and precedes the formation offibrillar inclusion. J. Biol. Chem. 277, 48984–48992

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10 Conway, K. et al. (2001) Kinetic stabilization of the a-synucleinprotofibril by a dopamine–a-synuclein adduct. Science 294,1346–1349

11 Yamin, G. et al. (2003) Nitration inhibits fibrillation of humana-synuclein in vitro by formation of soluble oligomers. FEBS Lett. 542,147–152

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13 Sharon, R. et al. (2001) a-synuclein occurs in lipid-rich high molecularweight complexes, binds fatty acids, and shows homology to the fattyacid-binding proteins. Proc. Natl. Acad. Sci. U. S. A. 98, 9110–9115

14 Sharon, R. et al. (2003) The formation of highly soluble oligomersof a-synuclein is regulated by fatty acids and enhanced in Parkinson’sdisease. Neuron 37, 583–595

15 Lippa, C. et al. (1998) Lewy bodies contain altered a-synuclein inbrains of many familial Alzheimer’s disease patients with mutations inpresenilin and amyloid precursor protein genes. Am. J. Pathol. 153,1365–1370

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17 Glatz, J. and Veerkamp, J. (1983) A radiochemical procedure for theassay of fatty acid-binding proteins. Anal. Biochem. 132, 89–95

18 Perrin, R. et al. (2001) Exposure to long chain polyunsaturated fattyacids triggers rapid multimerization of synucleins. J. Biol. Chem. 276,41958–41962

19 Papay, R. et al. (2002) Mice expressing the a1B-adrenergic receptorinduces a synucleinopathy with excessive tyrosine nitration butdecreased phosphorylation. J. Neurochem. 83, 623–634

20 Xu, J. et al. (2002) Dopamine-dependent neurotoxicity of a-synuclein: amechanism for selective neurodegeneration in Parkinson’s disease.Nat. Med. 8, 600–606

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