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NeuroMolecular Medicine 115 Volume 2, 2002

Alpha-Synuclein and Presynaptic Function

Implications for Parkinson’s Disease

Simon Lykkebo and Poul Henning Jensen*

Department of Medical Biochemistry, Aarhus University, DK-8000 Aarhus-C, Denmark

Received April 17, 2002; Accepted May 16, 2002

Abstract

This article focuses on α-synuclein’s role in normal and pathological axonal and presynap-tic functions and its relationship to Parkinson’s disease. It is not possible to mention all the con-tributions to aspects of this area. Readers interested in α-synuclein’s relation to aggregation,Lewy lesions, and pathological modifications are referred to the many reviews (see Goldbergand Lansbury 2000; Galvin 2001a; Goedert 2001).

Index Entries: synuclein; synapse; axon; vesicles; Parkinson’s disease; Lewy body.

NeuroMolecular MedicineCopyright © 2002 Humana Press Inc.All rights of any nature whatsoever reserved.ISSN1535-1084/02/02:115–129/$20.00

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected]

α-Synuclein in Parkinson’s Disease

The small presynaptic protein α-synuclein hasreceived great attention since missense mutations inits gene were associated with heritable autosomaldominant early-onset Parkinson’s disease in 1997-98(Polymeropoulos, 1997; Krüger, 1998). It is a memberof the synuclein gene family, wherein the α-, β-, andγ-synucleins, encoded by genes on chromosomes4q21, 5q35 and 10q23, (Campion, 1995; Spillantini,1995; Lavedan, 1998), are predominant in humans(Fig. 1). Synucleins were first identified based on theirpresynaptic nature by expression cloning using asynaptic-vesicle-specific antibody on a vector libraryof mRNA from the electromotor nucleus of the rayTorpedo California (Maroteaux, 1988). Homologous

mRNAs and proteins have been identified in birdsand mammals, thus demonstrating the highly con-served structure between fish and mammals(Maroteaux, 1988) and birds (George, 1995) (Fig. 1).The initial report coined the term “synuclein” becauseof its localization to the presynaptic terminus and thenuclear envelope. The latter localization was latershown not to be normal for synucleins.

Parkinson’s disease is the second most commonneurodegenerative disease—the first is Alzheimer’sdisease (AD). It is primarily a movement disorder,and is characterized by rigidity, tremor, and bradykinesia, but a wider effect on the nervous system isfrequently noticed, as evidenced by autonomic andcognitive dysfunctions. The definitive diagnosisrelies on the neuropathological demonstration of

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NeuroMolecular Medicine Volume 2, 2002

nerve-cell loss in the pars compacta of the substan-tia nigra in the brainstem, accompanied by the pres-ence of proteinaceous inclusions, Lewy bodies, andLewy neurites. Similar neurodegenerative changesare often found in other parts of the brain—e.g., thenucleus basalis of Meynert, hypothalamus, cingu-lated gyrus, entorhinal cortex, sympathetic ganglia,and peripheral parasympathetic neurons, wherethey may explain the autonomic and cognitivesymptomatology. For a recent review on clinical andpathophysiological aspects of Parkinson’s disease,see (Lang, 1998). The identification of two missense

mutations in the α-synuclein gene on chromo-some 4q21 causing Parkinson’s disease representedthe first demonstration of a single gene as causativefor the disease. Both the mutant Ala53Thr in thelarge Contoursi kindred (Polymeropoulos, 1997)and the Ala30Pro mutations in a German family(Krüger, 1998) cause an early-onset Parkinson’s dis-ease with a dominant heritability of high penetrancethat is indicative of a gain-of-toxic-function by themutant peptide. Mutations in the α-synuclein genehave subsequently turned out to be exceedingly rare. The identification of the mutations in the

Fig. 1. Sequence comparison among synucleins. (A) Alignment of human α-synuclein, β-synuclein and γ-synuclein protein sequences. Gray bars indicate the six KTKEGV consensus repeats in the α-synuclein sequence.Note the high degree of sequence identity within the N-terminal 62 residues of the gene products as compared totheir divergent C-termini. (B) Alignment of α-synuclein protein sequences from human, rat, bird, and fish synu-clein. Note the high degree of conservation of the α-synuclein protein in bird and mammals, and even with theunrelated fish synuclein. Dots indicate amino acids identical with the human α-synuclein sequence. Gaps areindicated with a wavy line (~). Alignments were performed using the MegAlign software from DNAstar Inc. Acces-sion numbers used for the alignments are: human α- (P37840), human β- (Q16143), human γ- (AAC27738), rat α- (P37377), bird α- (AAA93538), and fish synuclein (A60887).

Alpha-Synuclein and Presynaptic Function 117


α-synuclein gene prompted the immunohistochem-ical examination of brain tissue affected by Parkin-son’s disease, and this showed that α-synuclein inthe disease is accumulated in the Lewy bodies(Spillantini, 1997). More neurodegenerative diseasesare associated with the development of Lewy bodiesor similar inclusions in which α-synuclein is a prominent component, dementia with Lewy bodies,Lewy body variant of AD, motor neuron disease,Hallervorden-Spatz disease (for recent reviews seeDickson, 2001; Goedert, 2001) and multiple systematrophy, where the inclusions occur in the astrocytes(Gai, 1998). Accordingly, these diseases are collec-tively designated as α-synucleinopathies. Lewybodies were discovered by Frederic Lewy (Lewy,1912), and were for decades identified histochemi-cally by their affinity for the dye eosin. The sensi-tivity of their detection increased significantly withthe development of immunohistochemistry, forwhich ubiquitin antibodies have represented thestandard for their detection until recently. However,anti-α-synuclein antibodies have increased the sensitivity even further by demonstrating the exis-tence of subsets of ubiquitin-negative Lewy bodies(McKeith, 1999). Lewy bodies and Lewy neuritescontain large amounts of protein filaments (Forno,1987), and thus resemble the neuropathological

lesions in other late-onset neurodegenerative dis-eases, such as neuritic tangles in Alzheimer’s dis-ease and intranuclear inclusions in Huntington’sdisease. The filaments in Lewy bodies and glialcytoplasmic inclusions contain highly insoluble α-synuclein, which is partly modified by proteoly-sis, phosphorylation, and oxidations (Baba, 1998; Gai,1999; Giasson, 2000; Jensen, 2000; Fujiwara, 2002),and they resemble the filaments that can be formedin vitro from recombinant human α-synuclein basedon immuno-electronmicroscopical criteria (Conway,1998; Crowther, 1998; Giasson, 1999). This suggeststhat α-synuclein is the building block for the core fil-aments that may possess an additional coat of asso-ciated auxiliary cytoplasmic proteins. Structuralanalysis of in vitro-formed filaments have demon-strated that they possess a large content of β-foldedstructure and exhibit characteristics of classical amyloid filaments such as the β amyloid peptide(Aβ)-filaments in Alzheimer’s disease (Hashimoto,1998; Conway, 2000a). The accumulation of α-synuclein in the Lewy bodies and neurites repre-sents a dramatic relocalization of the protein from itsnormal presynaptic localization (Fig. 2). A less pro-nounced abnormal accumulation of β-synuclein andγ-synuclein in non-Lewy-body structures has alsobeen demonstrated in Parkinson’s disease, but no fil-

Fig. 2. Topological and structural changes in neuronal α-synuclein in Parkinson’s disease. α-synuclein is nor-mally a presynaptic protein. It is synthesized in the cell body and targeted to the nerve terminal by axonal trans-port. A dramatic relocalization from the nerve terminal to the cell body and neurites occurs in the degeneratingneurons during the course of Parkinson’s disease. A structural change takes place concomittant with this relocal-ization as β-folded amyloid-type α-synuclein filaments accumulates in the Lewy bodies and in the neurites. Colorimage available for viewing at www.humanapress.com



aments have been detected for these synucleins(Galvin, 1999). Concomitant with the α-synucleinrelocalization occurs the above-mentioned structuraltransition of some monomeric unfolded α-synucleininto β-folded filaments. A biochemical correlate tothe filamentous transition is a decreased solubilityofα-synuclein in isolated filaments and in brain tissuecontaining numerous α-synuclein-positive inclu-sions (Dickson 1999; Gai 1999; Jensen 2000). A sim-ilar decreased solubility has been demonstrated intransgenic mice expressing human α-synuclein anddisplaying pathological symptoms (Kahle, 2001).

The aggregation of recombinant human α-synuclein in vitro represents a nucleation-dependent aggregation process in which filamentformation is preceded by a lag phase where fila-mentogene oligomers build up (Conway, 1998;Wood, 1999). The oligomers represents an ill-definedstructural entity whose existence has been demon-strated biochemically and by atomic forcemicroscopy (Conway, 2000a, 2000b; Rochet, 2000;Volles, 2001). However, the oligomers’ ability to seedfilament growth emphasizes the importance of fac-tors that increase their concentration. Such factorsmay be subject to dynamic regulation in vivo andcomprise disease-causing mutations, C-terminaltruncation (Crowther, 1998, Jensen, 2000), oxidativemodifications (Hashimoto, 1999; Paik, 2000; Souza,2000), phosphorylations (Fujiwara, 2002), calciumbinding (Nielsen, 2001), and possibly certain α-synuclein binding proteins such as synphilin(Engelender, 1999).

The direct link between α-synuclein alterationsand the development of Parkinson’s disease—established by the pathogenic mutations in the α-synuclein gene, and the demonstration of anabnormal α-synuclein aggregation in all cases withLewy bodies, including Parkinson’s disease—havehad a tremendous impact by allowing directed molecular investigations of disease mechanisms.

Expression

The synucleins are abundant proteins that rep-resent about 0.1% of the total protein in the brain(Shibayama-Imazu, 1993). They are consideredneuron-specific proteins because their levels in theperipheral and central nervous system (CNS) arefar higher than in other tissues (Jakes, 1994; Ueda,1994; Lavedan, 1998), although significant expres-

sion does exist in other tissues. The synucleins are small acidic and cytosolic proteins of about125–140 amino acids with α-synuclein comprisedof 140 residues. The synuclein signature was imme-diately noticed after the first cloning as the pres-ence of repeats of 11 amino acids with the coreconsensus sequence KTKEGV (Maroteaux, 1988;Fig. 1B). These repeats extend over the N-terminalabout two-thirds of the peptide, and this region ishighly conserved between the different synucleinsand between species. The C-terminal one-third ishighly negatively charged and displays the mostvariable segment between the synucleins and con-trast, therefore, to the positively charged repeatregion (Fig. 1A).

The developmental expression of α-synucleinbegins at an early stage in mammals (Hsu, 1998)and has been detected from gestational wk 15 and16 in the human substantia nigra and cortex (Bayer,1999; Galvin, 2001b). The level slowly rises duringthe gestational period, and increases more rapidlyduring the end of pregnancy and in the early post-partum period (Hsu, 1998; Petersen, 1999). The α-synuclein is initially localized in the cell body andproximal neurites, and the mature presynaptic local-ization in the neuropil is first acquired by about ges-tational wk 18 (Galvin, 2001b). Interestingly, theclose homologous β-synuclein and γ-synucleinare first expressed in the substantia nigra at aboutthe time when α-synuclein has acquired its synaptic localization (Galvin, 2001b). Moreover,they first reach their mature presynaptic localiza-tion after being present for several weeks in the cell body (Galvin, 2001b). This indicates either anasynchronous maturation of their respective trans-port mechanisms or expression in different neu-ronal populations. Coexpression of α-synuclein andβ-synuclein within the same synaptic terminal hasbeen demonstrated in vivo (Fig. 3) and in culturedneurons (Murphy 2000). The synaptic targeting ofα-synuclein displays a pattern very similar to synap-tophysin (Hsu, 1998; Galvin, 2001b), indicating aparallel maturation of the axonal transport mecha-nisms for the two proteins in vivo as compared tothe later maturation for synaptotagmin and synap-tobrevin (Galvin, 2001b). A discrepancy existsbetween the previously mentioned in vivo data andin vitro neuronal cell-culture experiments—thelatter demonstrate an earlier expression of andsynaptic translocation of synaptophysin andsynapsin-1 as compared to α-synuclein (Withers,



1997; Murphy, 2000). This probably reflects the non-physiological differentiation and polarization of cultured neurons.

Catabolism

The α-synucleinopathies are characterized by theaccumulation of α-synuclein, some of which dis-plays a decreased solubility. This suggests thatabnormal catabolism of α-synuclein could play apathogenic role. α-synuclein is a stable protein withT-1/2 > 24 h in transfected mitotic cell lines (Ben-nett, 1999; Okochi, 2000). α-synuclein accumulatesin cell lines treated with proteasome inhibitors,although no ubiquitination of native α-synucleinhas been demonstrated (Bennett, 1999; Ancolio, 2000;Tofaris, 2001). However, unmodified α-synucleincan be directly degraded by the 20S proteasome in vitro (Tofaris, 2001), indicating the existence of anunconventional proteasomal catabolism of thesmall-unfolded α-synuclein peptide. The signifi-cance of this observation awaits corroborative stud-

ies in non-mitotic neurons. The subcellular sitesresponsible for the α-synuclein degradation in neu-rons are unsolved, but axons and nerve terminalsare likely to represent major sites as the anterogradetransport exceeds the retrograde transport (Jensen,1999b). Proteasomal degradation at these sites hasbeen demonstrated to play an important role inaxonal organization (Campbell, 2001). Interactionsbetween the ubiquitin proteasome system and apparently unmodified α-synuclein have beenreported by the coimmunoprecipitation of the Park-inson’s disease-related ubiquitin ligase parkin andα-synuclein (Choi, 2001; Jensen, unpublished obser-vation). A prerequisite for a specific α-synucleinmodification—an O-glycosylation—in supportingthe binding and subsequent α-synuclein ubiquitina-tion, has been reported in a study of human braintissue (Shimura, 2001). This α-synuclein modifica-tion has not yet been confirmed by other groups, but O-glycosylated β-synuclein was demonstrated in rat nerve terminals (Cole, 2001). The regulation of α-synuclein catabolism is not a trivial matter, considering that the accumulation of misfolded

Fig. 3. α-synuclein and β-synuclein colocalize in some nerve terminals. Rat hippocampal sections were labeledfor α-synuclein with a monoclonal antibody (MAb) (right panel) and for β-synuclein with a polyclonal antibody (leftpanel), and analyzed by confocal laser scanning fluorescence microscopy. Evidently, the two synucleins display adifferential distribution, but they colocalize in nerve terminals of many regions, including the hippocampus (demon-strated in the inset) and the cortex and striatum (data not shown). The images were kindly provided by Dr. J.-Y. Li,Wallenberg Neuroscience Center, Lund, Sweden. Color image available for viewing at www.humanapress.com



α-synuclein is a key feature of more neurodegener-ative diseases. However, clarification of these issuesneeds much more work.

Axonal Transport

α-synuclein is almost completely confined topresynaptic spaces in the mature nervous system(Shibayama-Imazu, 1993; Jakes, 1994; George, 1995;Iwai, 1995; Kahle, 2000), thus demonstrating the existence of an efficient axonal transport mechanismsthat assures its clearance from its site of synthesis in the somato-dendritic compartment. Disturbancesherein may contribute to the development of thediverse group of α-synucleinopathies characterizedby ectopic accumulation of α-synuclein.

Axonal transport includes the anterograde move-ment of axonal and presynaptic constituents fromtheir site of synthesis in the cell body and the ret-rograde transport of vesicles and proteins, such asendocytosed growth factors. This transport has tra-ditionally been divided into rate components thatcharacterize groups of proteins and vesicular struc-tures based on their similar rate of transport. Theanterograde movement is categorized into threegroups; 1) fast transport, 2) slow component b, and3) slow component a, moving at rates about 100–400,2–4, and 0.2–1 mm/d, respectively (Hirokawa, 1994).By contrast, only one retrograde mode exists: a fastmovement similar in rate to the fast anterogradetransport. The fast axonal transport (FAT) is cat-alyzed by motor proteins that convert ATP hydrol-ysis into mechanical force (Hirokawa, 1998). Themotor proteins, kinesins as principal anterogrademotors and dynein as the prime retrograde motor—use the unidirectional axonal microtubules as tracksfor pulling their cargo, e.g. mitochondria and synap-tic-vesicle precursors. The Alzheimer’s disease-associated amyloid precursor protein (APP) wasrecently demonstrated to act as a linker that tethersa subset of axonal vesicles to kinesin-I (Kamal, 2000).The slow component b transports a heterogenousgroup of soluble proteins, whereas the insolubleproteins, considered to include axoskeletal compo-nents such as neurofilaments, migrate by the slowcomponent a (Hirokawa, 1994). The forces respon-sible for moving the slow components haveremained elusive for decades, although the idea ofa single transport system responsible for all axonaltransport was put forward as early as 1975 (Ochs,1975). Recent experiments supports this hypothe-

sis after video microscopical studies have demon-strated that cargos traditionally considered as“slow,” like neurofilament subunits, move rapidlyfor short distances followed by prolonged pauses(Shea, 2001). This indicates that the slow compo-nent transports constituents only temporarily asso-ciating with the motorproteins and their cargo, andwhose net velocity will depend of the affinitybetween the reactants—an affinity that may be sub-ject to dynamic regulation (e.g., by phosphoryla-tion) (Shea, 2001).

The axonal transport of synuclein in the CNS hasbeen analyzed directly using the rat optic nerve asa model (Jensen, 1998, 2000). This study, whichexamined both α- and β-synuclein, demonstratedthat each was transported by all the anterogradeand retrograde rate components (see Fig. 4). Theslow component b transported about 60% of themetabolically labeled synuclein, and the remainingportion was divided approximately evenly betweenthe fast component and the slow component a. Theproportion being transported retrograde was notquantified, but was demonstrated qualitatively bymicroscopic nerve-crush experiments. Proteins trans-ported by more than one rate component are notunprecedented—e.g., dynein and dynactin displaya pattern similar to synuclein with slow componentb dominating (Dillman, 1996). The involvement ofall the rate components in the axonal synuclein trans-port gives the synucleins ample opportunity to inter-act with many axonal constituents en route to thenerve terminals, such as motor proteins, vesicles(Jensen 1998, 2000), and axoskeletal regulators suchas soluble tau (Jensen, 1999a) and tubulin (Payton,2001; Alim, 2002). Apparently, synucleins can evenbecome incorporated into the axoskeleton, as radio-labeled synuclein was recovered from the axons 21 d after labeling of the nerve cells (Jensen, 2000).

There may be an association between disturbedaxonal α-synuclein transport and its accumulationin Lewy lesions, although this has not been proven.Partial transport defects will lead to an increasedtransit time for α-synuclein, and pending a contin-ued expression, a buildup in the cell body with subsequent increase in the local α-synucleinconcentration within the axon. An increased α-synuclein concentration in the stationary “pause”mode will favor interactions with axonal proteinssuch as the tubulins (Payton 2000; Alim 2002) andthe microtubule-stabilizing proteins MAP-1A,MAP-1B, MAP-4, and tau (Jensen, 1999a, 2000). The



binding to the microtubule-binding proteins mayinhibit their function by more than one mecha-nism—by sequestering them in Lewy lesions(Jensen, 2000), and by inhibiting tau’s ability to bind to microtubules by directly occupying themicrotubule-binding domain in tau and by facili-tating its phosphorylation at Ser262 (Jensen, 1999a).Phosphorylation at this site inhibits tau’s bindingto microtubules (Ackmann, 2000). A direct bindingof α-synuclein to soluble tubulin has been demon-strated in brain extracts (Payton 2000; Alim 2002),but a direct interaction with microtubules is unclearbecause it has not been confirmed that α-synucleincan bind to the polymerized microtubules (Jensen1999; Payton, 2000; Alim, 2002). Still, interactionsbetween soluble tubulin and α-synuclein may have profound effects by facilitating aggregation ofα-synuclein (Alim, 2002). The buildup of filamen-tous α-synuclein in Lewy neurites may disruptaxonal transport, as hypothesized for neurofilamentinclusions in motor neuron disease (Julien, 2001). A toxic role for α-synuclein accumulated in axonsis corroborated by the transgenic models of miceand Drosphila, in which increased α-synucleinexpression leads to neuronal dysfunction (Feany,

2000; Masliah, 2000; Kirik, 2002), particularlypreterminal degeneration (van der Putten, 2000).

Synaptic FunctionHigh concentrations of α-synuclein are localized,

and apparently tolerated, at presynaptic sites innormal neurons, and its function has accordinglybeen investigated by reducing its expression in miceand nerve-cell-culture models (Abeliovich, 2000;Murphy, 2000). Gene-knockout mice represent com-plex models, in which the functional loss of a genein the phenotypes may be hidden by redundancyor other compensatory mechanisms. α-synuclein–/– mice develop normally, and display an intactbrain architecture and morphology of the nigros-triatal dopaminergic system, and especially anormal presynaptic morphology (Abeliovich, 2000).However, the animals display abnormalities in thedopaminergic system—their dopamine-dependentlocomotor response to methamphetamine is atten-uated and their striatal dopamine content isdecreased (Abeliovich, 2000). At the presynapticlevel an increased dopamine release followingpaired stimuli of isolated terminals in striatal slices

Fig. 4. Axonal synuclein transport. Axonal α-synuclein transport was analyzed using the optic system of the rat,where newly synthesised α-synuclein in the ganglion cells of the retina was metabolically labeled by bilateral injec-tion of 35S-methionine into the eye. The transport of the labeled synuclein was monitored by immunoprecipitationfrom different segments of the axons that extend from the ganglion cells to the lateral geniculate body. This enabledthe distinction of the synuclein moving with the fast component (indicated in lat. gen. body), the slow component b(indicated in chiasma), and the slow component a (indicated in initial part of the optic nerve). The existence of bothanterograde and retrograde synuclein transport was demonstrated qualitatively, and the proportions moving in thedifferent anterograde-rate components were quantified to 25% fast transport, 60% slow component b, and 15% slowcomponent a (Jensen, 1999b). Color image available for viewing at www.humanapress.com



is observed (Abeliovich, 2000). This rather weakphenotype of the α-synuclein –/– mice does notallow a definition of α-synuclein’s functional role,although it indicates a link between α-synuclein anddopamine metabolism. This is corroborated by trans-fection studies, where increased α-synuclein alsoaffects the dopamine system in mice and rats, asdemonstrated by a loss of tyrosine hydroxylase-positive terminals and activity in striatum (Masliah,2000; Kirik, 2002). Dopamine is transported from the extracellular space into the cytosol by the dopa-mine transporter that is expressed specifically by thedopaminergic neurons. α-synuclein interacts directlywith the C-terminal part of the human dopaminetransporter and facilitates its translocation to theplasma membrane, thereby increasing dopamineuptake (Lee, 2001). In the cytosol, dopamine is con-sidered toxic because of its pro-oxidative properties(Adams, 1991), and it is efficiently removed from thecytosol into vesicles by the vesicular monoaminetransporter. The dopamine hypothesis states thatcytosolic dopamine, through its oxidative proper-ties, contributes to neurodegeneration, and it has theadvantage that it answers the question about theselective cellular vulnerability of the dopaminergicneurons in Parkinson’s disease. Candidates that favoran increased cytosolic dopamine concentrationsinclude increased activity of the plasma-membranedopamine transporter, where α-synuclein may con-tribute as stated here, or decreased function of thevesicular monoamine transporter. Detrimentaleffects of α-synuclein on the vesicular monoaminetransporter have not been reported, but a studywould be highly justified. Anti-sense oligonu-cleotide treatment represents another model forinvestigating the effect of the loss of a specific pro-tein. Arapid reduction in the gene product can oftenbe achieved, thereby possibly avoiding compen-satory mechanisms. Anti-sense oligonucleotidetreatment of cultured rat hippocampal neurons for 3 d resulted in a reduction of about 50% in α-synuclein protein without affecting the β-synuclein level, and this was accompanied by asignificant reduction of the distal pool of synapticvesicles and the synaptic-vesicle markers synapsin Iand synaptophysin (Murphy, 2000). Accordingly, α-synuclein may play a general role in the vesiclehomeostasis of nerve terminals that are not restrictedto dopaminergic neurons.

α-Synuclein associations with vesicles have beeninvestigated by methods such as immuno-

electronmicroscopy (Maroteaux, 1988; Iwai, 1995;Irizarry, 1996; Withers, 1997), biochemistry(Maroteaux, 1991; George, 1995; Kahle, 2000; Cole,2002; Lee, 2002) and fluorescence energy transfer(McLean, 2000), and these have led to the conclu-sion that α-synuclein is close to synaptic vesicles innerve terminals (Iwai, 1995), directly interactingwith brain vesicles (Jensen, 1998), and close to vesi-cles in cultured neurons (McLean, 2000). Moreover,the FAT of some α-synuclein in intact animals cor-roborates an interaction with vesicles (Jensen, 1998,1999b). Still, the fraction of α-synuclein being boundto vesicles, the characteristics of the interaction, andthe identity of the α-synuclein-containing vesiclesare a matter of debate, probably because thereversible nature of the binding leads to the disso-ciation of some α-synuclein during subcellular frac-tionation procedures. Synthetic liposomes havebeen widely used as models for investigating α-synuclein interaction with phospholipid bilayers(Davidson, 1998; Jo, 2000; Perrin, 2000; Eliezer, 2001;Narayanan, 2001; Volles, 2001), and this has allowedthe demonstration of the impact of membrane bind-ing on α-synucleins structure by circular dichroismspectroscopy. Through these means, a dramaticstructural transition was demonstrated from a pre-dominantly random coiled peptide in solution to astructure with a large α-helical content when boundto the membrane (Davidson, 1998; Jo, 2000; Perrin,2000; Eliezer, 2001; Narayanan, 2001; Volles, 2001).The interaction requires acidic phospholipids, andsome lipids facilitate the aggregation of α-synucleininto putative dimers and higher oligomers (David-son, 1998; Jo, 2000, Sharon, 2001). A pitfall in the interpretation of liposome studies has been uncov-ered as different phospholipid species support the binding of different segments of α-synuclein. Onestudy demonstrated a broad distribution of mem-brane-interacting elements over the N-terminal 103 amino-acid residues when using a certain lipidcomposition (Perrin, 2000) that contrasts with thecritical role of the N-terminal 30 in the interactionwith brain vesicles (Jensen, 1998). Liposomes pre-pared from a specific brain lipid extract, the Folchfraction I, require the same α-synuclein domain forbinding as brain vesicles and induce α-helical struc-ture (Lykkebo & Jensen, unpublished results). Thisfinding suggests that these liposomes may be ben-eficial for further studies on the functional effectsof α-synuclein binding to lipid bilayers and its inter-play with specific reconstituted vesicular proteins



(see Fig. 5). An elegant study has addressed the binding of α-synuclein to phospholipid membranesin vivo using cell lines and hippocampal neurons(Cole, 2002). The cells were fed excess lipids thattriggered the formation of large lipid droplets—inclusions of neutral lipids surrounded by a singlephospholipid layer. Thus, the inclusions do notresemble true cellular vesicles surrounded by abilayer membrane, but rather include a cell-compatible phospholipid composition and certainmembrane proteins. The lipid droplets bound bothwild-type α-synuclein and the disease-causingmutant A53T, whereas the A30P mutant wasseverely impaired in its binding, thus corroborat-ing the inhibitory effect of the A30P mutation onbinding to brain vesicles (Jensen, 1998). However,both the A53T and the A30P mutant peptides weredefective in their protection of the lipid dropletsfrom lipase activity as compared to wild-type

α-synuclein. Furthermore, the use of chemicalcrosslinkers demonstrated that α-synuclein was present in dimeric and higher oligomeric species on vesicles and lipid droplets. The crosslinking sta-bilized the surface binding, along with theoligomeric structures, thus indicating that oligomersrepresents a favorable quaternary structure on mem-branes, although artefactual crosslinking to amine-containing phospholipids cannot be excluded.Formation of correctly folded oligomers on the sur-face may be a prerequisite for the full—yetunknown—activity of α-synuclein. Inhibition ofmembrane phospholipases has previously beenreported as one candidate function (Jenco, 1998;Cole 2002), and this activity may be affected by disturbances in the membrane phospholipid composition or modifications of α-synuclein—e.g.,by phosphorylations by membrane associated G-protein-coupled receptor kinases (Pronin, 2000)

Fig. 5. α-synuclein can tether ligands to lipid membranes. α-synuclein binds to liposomes through a domain inthe N-terminal 55 amino acid residues. The isolated domain posses full binding activity, and acquires a α-helicalconformation upon binding. A segment designated as the spacer domain is placed between the membrane-binding domain and the acidic C-terminal domain. This domain facilitates α-synucleins liposome binding byremoving the negatively charged C-terminus from the acidic phospholipids in the lipid membrane. The modularorganization of α-synuclein allows it to tether ligands for the C-terminal ligand-binding domain to liposomes asdemonstrated using a C-terminal-directed antibody as a model ligand. Such tethering may have functional signif-icance, and allows the differential expression of α- and β-synuclein to mediate the association of different pro-teins to membrane surfaces (Lykkeboe and Jensen, unpublished results). Color image availabe for viewing atwww.humanapress.com



or oxidations (Paxinou, 2001). α-synuclein can tetherC-terminally-bound proteins to liposomes (Fig. 5) and this ability may represent a physiological func-tion of synuclein bound to brain vesicles throughtheir N-terminal membrane-binding domain. α- andβ-synuclein possess very homologous vesicle-binding domains but differ in their C-termini. Thetethering hypothesis would imply that these synu-cleins can target specific subsets of soluble proteinsto vesicles for the facilitation of their axonal trans-port or the modulation of membrane-associatedfunctions. This would be analogous to the recentlyreported function of the membrane spanningAlzheimer’s disease-associated APP, which maytether certain axonal transport vesicles to theKinesin-I motor protein, thereby mediating theiraxonal transport (Kamal, 2000).

α-Synuclein-Mediated Dysregulationof “Presynaptic” Functions in Neurodegenerative Disorders

Autosomal dominant missense mutations in theα-synuclein gene are pathogenic in Parkinson’s disease, and abnormally folded α-synuclein accu-mulates in all Lewy bodies in a range of neuro-degenerative diseases. This forms the basis for the widely accepted hypothesis that a gain-of-toxic α-synuclein function is operating in these α-synucleinopathies. The mechanistic basis is lessclear, but likely involves novel functions of theabnormally filamentous α-synuclein and its proforms (Rochet, 2000b).

It is assumed that α-synuclein possesses physi-ological functions in the presynaptic compartment,such as regulating the activity of the dopamine trans-porter or fine-tuning the neurotransmitter release.The disease-associated relocalization to the cell bodyand neurites will then have a negative impact onthese functions. A putative decreased disease-associated synaptic α-synuclein content may becaused by dysfunctional axonal transport, or bytoxic effects of the accumulated α-synuclein in axonsthat might lead to full-blown axonal strangulation,or more specific cellular lesions caused by theabnormally localized and folded α-synuclein char-acterizing these diseases.

The ultimate cause(s) for the development of theα-synuclein containing Lewy body-lesions are

unknown. They may represent a specific neuronalresponse to a multifactorial yet selective set ofinsults, given that not all degenerating neuronsdevelop Lewy bodies. Still, the direct disease-causing effect of the A53T and the A30P mutationsdemonstrates that α-synuclein dysmetabolismeither activates or is part of a final common pathway in neurodegeneration. The developmentof compact-filament-containing Lewy bodies is preceded by a more diffuse accumulation of α-synuclein of unknown structural forms in thesomato-dendritic compartment (Wakabay-ashi, 1998; Gomez-Tortosa, 2000). This “ectopic” α-synuclein may have a special impact on thedopaminergic substantia nigra neurons at risk inParkinson’s disease, as their plasma membrane andintracellular vesicles at the somato-dendritic com-partment in certain aspects resemble presynapticsites, namely by expressing functional transportersof dopamine (Nirenberg 1996a, 1996b). These transporters may cause an abnormal α-synuclein-stimulated dopamine uptake into their cell bodythat could prime them for oxidative stress, andthereby contribute to their selective vulnerability inParkinson’s disease.

The majority of the current α-synuclein researchfocuses on the abnormal contributions of α-synuclein’s putative gain-of-toxic-function, andthe heritable effects of α-synuclein gene mutationsjustify this. Still, a better understanding of all aspectsof α-synucleins normal biology may reveal mecha-nisms that are dysfunctional in the diseased state,and are therefore worthy of evaluation as points oftherapeutic intervention.

Acknowledgment

Dr. Jia-Yi Li is thanked for generously providingthe image in Fig. 3. This manuscript is based onstudies supported by the Danish Medical ResearchCouncil (9902995), the Lundbeck Foundation, theDanish Parkinson Foundation, Wacherhausenslegat, Aarhus University’s Research Foundation,and Direktør Ib Henriksens Fond. SLwas supportedby a Novo Nordisk scholarship.

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