Báo cáo khoa học: a-Synuclein–synaptosomal membrane interactions Implications for fibrillogenesis pot

a-Synuclein–synaptosomal membrane interactions
Implications for fibrillogenesis
Euijung Jo
1
, Audrey A. Darabie
1
, Kyung Han
1
, Anurag Tandon
1,3
, Paul E. Fraser
1,2
and JoAnne McLaurin
1,4
1
Centre for Research in Neurodegenerative Diseases; Departments of
2
Medical Biophysics,
3
Medicine and
4
Laboratory Medicine
and Pathobiology, University of Toronto, Ontario, Canada
a-Synucle in exists in two different compartments in vivo –
correspondingly existing as two different forms: a mem-
brane-bound form that is predominantly a-helical and a
cytosolic form that is randomly structured. It h as been sug-
gested that these environmental and structural differences
may play a role in aggregation propensity and d evelopment
of pathological lesions observed in P arkinson’s disease (PD).
Such effects may be accentuated by mutations observed in

familial PD k indreds. In order to test this hypothesis, wild-
type and A53 T mutant a-synuclein interactions with rat
brain synaptosomal membranes were examined. Previous
data has demonstrated that the A30P mutant has defective
lipid binding and therefore was not examined in this
study. Electron m icroscopy demonstrated that wild-type
a-synuclein fibrillogenesis is accelerated in the presence of
synaptosomal membranes whereas the A53T a-synuclein
fibrillogenesis i s inhibited under the same conditions. These
results suggested that subtle sequence changes in a-synuc-
lein could s ignificantly a lter interaction with membrane
bilayers. Fluorescence and absorption spectroscopy using
environment s ensitive probes demonstrated variations in the
inherent lipid properties in the presence and absence of
a-synuclein . A ddition of wild-type a-synuclein to synapto-
somes did not significantly alter the membrane fluidity at
either the fatty acyl chains or headgroup space, suggesting
that synaptosomes have a high capacity for a-synuclein
binding. I n contrast, synaptosomal membrane fluidity was
decreased by A53T a-synuclein binding with concomitant
packing of the lipid headgroups. These results suggest that
alterations in a-synuclein–lipid interactions may contribute
to physiological changes detected in early onset PD.
Keywords: anisotropy; electron microscopy; fibrillogenesis;
fluorescence s pectroscopy; m embrane.
The link between a-synuclein and P arkinson’s disease (PD)
is unclear; yet a-synuclein is the major component of the
primary neuropathological feature, Lewy bodies [1–5]. The
association of a-synuclein with familial Parkinson’s disease
was established in several PD kindreds with t he disc overy of

two missense mutations A53T and A30P, which suggested
an etiological significance rather than a secondary patho-
logical e vent [6,7]. More recently, a-synuclein gene triplica-
tion has been identified in a large family of early onset
autosomal dominant PD [8]. These studies suggest that
multiple alterations in a-synuclein protein s equence, expres-
sion level or f unction may lead to the downstream clinical
manifestation of PD. Immunocytochemistry has revealed
a-synuclein positive inclusions within astrocytes and oligo-
dendrocytes of PD-patients and glial and neuronal inclu-
sions of multiple system atrophy patients [9–14]. These
studies suggested that a-synuclein and its abnormal p rotein
aggregation might play an active part in these n eurodegen-
erative diseases.
The physiological function of a-synuclein remains largely
unknown, but it has been suggested that it may play a r ole
in synaptogenesis and lipid trafficking [15,16]. a-Synuclein
protein structure contains seven imperfect repeats of 11
amino acids which form the amino terminal amphipathic
a-h elices, a central hydrophobic domain followed by an
acidic C-terminal, r ich in g lutamate [15,17]. These primary
and secondary structure characteristics su ggested a role fo r
a-synuclein in protein–membrane interactions. Initial in vitro
experiments showed that the helical structure of wild-type
(WT) a-synuclein was in duced and s tabilized by binding to
synthetic m embranes [18]. Subsequently, it was found that
a-synuclein forms a dimer or trimer when bound to lipid
vesicles [19–21]. Furthermore, WT a-synuclein isolated from
human SH-SY5Y cells is monomeric in soluble or cytosolic
form and oligomeric when associated with lipids [ 22].

Subsequent studies demonstrated that the N-terminal region
of a-synuclein bound to lipids while the C-terminus remained
soluble and randomly structured [23]. As both familial PD
mutations are located in the N -terminal lipid-binding region,
it is possible that these mutations may alter the normal
equilibrium between a membrane-bound dimeric/oligomeric
form and a free cytosolic form of the W T a-synuclein.
Previous investigations have shown that A30P and A53T
mutations had no effect on the lipid-induced a-helical
structure of a-synuclein [24], yet these mutations increase
a-synuclein oligomerization, in vitro [25]. M ore d etailed
analyses of the A30P mutation demonstrated t hat although
the secondary structure could not be distinguished from
Correspondence to J. McLaurin, Centre for Research in Neuro-
degenerative Diseases, Tanz Neuroscience Building 6 Queen’s
Park Crescent West, Toronto, Ontario, M5S 3H2, Canada.
Fax: +1 416 978 1878; Tel.: +1 416 978 1035;
E-mail:
Abbreviations: DPH, 1,6-diphenyl-1,3,5-hexatriene; MC540,
Merocyanine 540; PD, Parkinson’s disease; WT, wild-type.
(Received 1 0 May 20 04, accepted 8 June 2004)
Eur. J. Biochem. 271, 3180–3189 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04250.x
WT a-synuclein, the 3D conformation was d ifferent
[26]. F urthermore, t he differences in 3D structure results
in defective membrane b inding of the A30P m utant
a-synuclein [27,28]. On the other hand, the A53T mutant
could not be distinguished from WT a-synu clein with
respect to lipid binding or protein structure, yet A53T
mutant a-synuclein was less effective than WT at destabili-
zing membrane bilayers [19].

In order to i nvestigate this apparent discrepancy, we
undertook the examination of A53T mutant a-synuclein in
the presence of lipid bilayers formed from rat brain
synaptosomes. We chose to evaluate physiologically rele-
vant membranes in o rder to determine whether properties
associated with a-synuclein-lipid binding in homogeneous
lipid environments are replicated in environments consisting
of relevant ratios of phospholipids, ganglios ides and
sphingomyelin. Furthermore, these studies may h elp to
distinguish subtle differences between m utant a nd WT
a-synuclein interactions and to further elucidate potential
roles for a-synuclein–lipid interactions in vivo.Inorderto
examine differences and explain pathological findings, we
examined the ability of these membranes to facilitate
a-synuclein assembly by electron microscopy. Changes in
the m embrane physical characteristics as a result of
a-synuclein interactions were examined by fluorescence
spectroscopy using environment s ensitive probes. These
parameters define the extent to which a-synuclein penetrates
the lipid bilayer or disrupts lipid headgroup p acking.
Materials and methods
Expression and purification of recombinant a-synuclein
Human a-synuclein cDNAs, wild-type and A53T mutant
were subcloned i nto the plasmid pET-28a (Novagen), u sing
NcoIandHindIII restriction s ites. a-Synuclein was over-
expressed in Eschericia coli BL21 ( DE3) and i solated over a
Q-Sepharose column as described p reviously [19]. Aliquots
from all purification steps were analyzed by SDS/PAGE to
confirm purity. Protein concentration was determined by
Lowry assay. Circular dichroism (CD) spectra were re cor-

ded on a Jasco Circular Dichroism Spectrometer (Tokyo,
Japan) at 25 °C. Spectra were obtained from 195 to 250 nm,
with a 0.5 nm step, 1 nm bandwidth and 10 s collection
time per step. The p eptide conformation was determined by
adding an aliquot of stock peptide solution into NaCl/P
i
(pH 7 .4) at a final peptide concentration of 10 l
M
.
Synaptosome isolation
Rat g rey matter was dissected a fter cervical dislocation
(according to CACC guidelines) a nd homogenize d in 10
volumes of 320 m
M
sucrose, 5 m
M
Hepes, pH 7.4 (homo-
genizing buffer) using a glass homogenizer. The homogenate
was s pun at 1050 g for 10 min. The supernatant was re-spun
at 13 30 0 g for 15 min, 4 °C. The p ellet was resuspended in
homogenizing buffer and loaded onto a discontinuous ficoll
gradient, consisting of 13, 9 and 5% ficoll. The gradient was
spun for 35 min at 60 000 g at 4 °C. The synaptosomes were
isolated from the 9–13% layer and diluted into H epes buffer.
Final synaptosome isolation w as achieved after centrif uga-
tion at 13 300 gfor5 min, 4 °C [29,30]. Lipids were extracted
from the s ynaptosomes using Folch partition [chloroform/
methanol/water (v/v/v); 2 : 1 : 0.6] and subsequently con-
centrated under a stream of N
2

.Thesampleswerestoredat
)20 °C until use. Phospholipid concentration was deter-
mined using the Bartlett assay [31].
Electron microscopy
WT and A53T a-synucleins were i ncubated i n the presence
and absence of synaptosomal vesicles at a final peptide
concentration of 5.8 l
M
.Thea-synuclein to lipid ratio was
maintained at 1 : 20 (by mass). For negative stain electron
microscopy, c arbon-coated pioloform g rids were floated on
aqueous solutions of peptides. After the grids were blotted
and air-dried, the samples were stained with 1% ( w/v)
phosphotungstic acid and examined on a Hitachi 7000
electron microscope operated at 75 kV [32].
Tyrosine fluorescence spectroscopy
Steady-state fluo rescence was measured at 20 °Cusing
a Photon Technology International (PTI, London, ON,
Canada) QM-1 Fluorescence spectrophotometer equipped
with excitation intensity correction a nd a m agnetic stirrer.
Tyrosine emission spectra from 290 to 340 nm were
collected (excitation wavelength 281 nm, 0.5 sÆnm
)1
,band
pass 4 nm). A cuvette with a 1 cm path-length was used.
For aggregation s tudies, 10 l
M
of WT or A53T a-synuclein
or hen e gg-white lysozyme was incubated in the presence or
absence of synaptosomal membrane vesicles a t a 1 : 20

molar ratio for up to 96 h with stirring [33]. The samples
were measured for fluorescence as a measure of total
tyrosine fluorescence and then centrifuged for 30 min at
15 600 g in order to sediment fibres. The relative amount of
tyrosine fluorescence in the supernatant was then deter-
mined as a measure of soluble protein fraction.
Steady-state fluorescence anisotropy
Anisotropy experiments were performed on a PTI fluori-
meter e quipped with manual polarizers as described previ-
ously [34]. Excitation a nd emission wavelengths w ere s et at
360 nm and 425 nm with a slit width of 1 and 4 nm,
respectively. Our system was calibrated initially using 1,6-
diphenyl-1,3,5-hexatriene (DPH; Molecular Probes) in
mineral oil, which should give an anisotropy equal to 1.
The g-factor was calculated using horizontally polarized
excitation and subsequent comparison of the horizontal and
vertical emissions, which for our instrument is 0.883. Lipid
vesicles were diluted to 250 lgÆmL
)1
in NaCl/P
i
, incubated
for 20–30 min in t he presence and absence of a-synuclein or
lysozyme, and then incubated subsequently for a further
30 min with DPH at a 1 : 500 probe/lipid ratio. Fluores-
cence intensity was measured with the excitation polarizer in
the vertical position and the analyzing emission polarizer in
the vertical ( I
VV
) and horizontal (I

VH
) positions and
anisotropy, r, was calculated using Eqn (1);
r ¼
I
VV
À gI
VH
I
VV
þ 2gI
VH
ð1Þ
Lipid vesicles in the absence of DPH were measured in order
to evaluate the effect of light scattering on our measurements.
Ó FEBS 2004 Lipid-induced a-synuclein fibrillogenesis (Eur. J. Biochem. 271) 3181
Laurdan generalized polarization
Steady-state excitation and e mission spectra were collected
on the PTI fluorimeter. Laurdan (Molecular Probes) was
added to preformed lipid vesicles in the presence and
absence of a-synuclein at a 500 : 1 lipid/probe ratio. The
laurdan generalized polarization (GP) parameter as devel-
oped by Parasassi and colleagues [35] was calculated as
follows. The emission GP parameter is given by Eqn (2):
GP
em
¼
I
400
À I

340
I
400
þ I
340
ð2Þ
where, I
400
and I
340
are the flu orescence intensities measured
at all emission wavelengths between 420 and 520 nm. Using
the fixed excitation wavelength of 400 and 340 nm,
respectively. The excitation GP is given by Eqn (3):
GP
ex
¼
I
440
À I
490
I
440
þ I
490
ð3Þ
where, I
440
and I
490

are t he fluorescence intensities at each
excitation wavelength from 320 to 420 nm, measured at
fixed emission wavelengths o f 440 nm and 4 90 nm, r espect-
ively.
Merocyanine 540 absorption spectroscopy
Merocyanine 540 (MC540, Molecular Probes) absorption
spectra were obtained at room temperature on a Beckman
spectra DU530. The dye was added to preformed vesicles at
a p robe/lipid ratio o f 1 : 500 [36]. Final MC540 molar
concentration in the cuvette was 21.3 l
M
. Absorption
spectra were obtained between 400 and 600 nm with 1 n m
steps. The lipid alone baseline in t he absence of MC540 was
subtracted from all spectra, a nd then corrected by referring
the absorbances at 600 nm to zer o (Eqns 4 and 5).
[monomer] ¼
A À½e
D
 C=2
e
m
À e
D
=2
ð4Þ
[dimer] ¼
ðC À [monomer]Þ
2
ð5Þ

Where, A is the absorbance at 569 nm, e is the constant
for MC540 dimer or monomer a t t he given wavelength,
e
m
¼ 1.511 · 10
5
and e
D
¼ 5400, while C is the final
MC540 concentration.
After this correction, the absorbance values at 569 nm
were used to calculate the dimerization constant (K
dapp
)as
described by Bernik & Disalvo [37] (Eqn 6).
K
dapp
¼
[dimer]
[monomer]
2
ð6Þ
Results
a-Synuclein morphological characteristics
Recombinant a-synucleins, both wild-type (WT) and A53T
mutant, were over-expressed and purified from Eschericia
coli BL21 as described previously [19]. After purification,
both WT a nd A53T mutant a-synuclein ran as s ingle bands
on SDS/PAGE (Fig. 1A) and displayed typical random
secondary structure when diluted into N aCl/P

i
,pH7.3
(Fig. 1 B). These results confirm both the purity and the
random structure o f t hese monomeric a-synuclein prepara-
tions. Previous studies have suggested that WT a-synuclein
becomes oligomeric upon binding to acidic phospholipids
[28,38] as well as when bound to membranes from r at brain
or neuroblastoma cells [22,39]. Furthermore, a-synuclein
has been located to and associated with the presynaptic
terminals and synaptosomal membrane surfaces b y immu-
nogold localization s tudies [16]. T herefore, bilayers com-
posed of lipids isolated f rom r at brain synaptosomes
represent a physiologically relevant system in which to
examine a-synuclein–lipid interactions. In order to distin-
guish a-synuclein–lipid interactions from a-synuclein–pro-
tein interactions, synaptosomal lipids were isolated by Folch
Fig. 1. Physical properties of W T and A53T mutant a-synuclein. Both WT (lan e 1) and A53T mutant (lane 2) a-synuclein demonstrated pre-
dominantly single band s w hen elec trophores ed b y SDS /PAGE (A). Th e second ary structu re of th e a-synucleins was determined using c ircular
dichroism spectroscopy (B). Both WT (h)andA53T(s)mutanta-synuclein were randomly structured when diluted into NaCl/P
i
, as illustrated by
a single m inimum below 200 nm.
3182 E. Jo et al.(Eur. J. Biochem. 271) Ó FEBS 2004
partition and subsequently sonicated to form small uni-
lamellar vesicles.
To determine if synaptosomal membranes promote
assembly of WT and A53T mutant a-synuclein into amyloid
fibres, we examined a-synuclein structural characteristics by
negative stain electron microscopy (Fig. 2). In the absence
of lipid and at low l

M
concentrations, WT a-synuclein did
not form detectable amyloid fibres after a 3-day incubation
(Fig. 2 A) but upon extensive incubation formed fibres (data
not shown). In contrast, abundant a-synuclein fibres were
detected in the presence of synaptosomal lipid vesicles
(Fig. 2 B). The fibres appeared to be associated with both
the surface and the edge of vesicles suggesting a direct
interaction between fibre and the vesicle bilayer. These
results are consistent with our previous studies in which WT
a-synuclein assembled into aggregates and protofibrils in
the presence of phosphatidylcholine/phosphatidylserine
bilayers [19]. Similarly, Lee and coworkers d emonstrated
that membrane-bound WT a-syn uclein could seed the
aggregation of cytosolic WT a-synuclein as determined
using SDS/PAGE analyses [39]. These data are consistent
with partial insertion of a-synuclein into th e bilayer, which
acts as an anchor for site-directed fibril assembly. Preced-
ence for this mechanism of amyloid formation has been
Fig. 2. Negative stain electron microscopy of a-synuclein in the presence of synaptosomal membranes. WT a-synuclein incubated in buffer alone (A)
did not form fib rils. When incubated in the presenc e of synaptosomal membranes (B) abundant a-synuclein fibrils could be detected with
organization along the v esicle surface. A53T mu tant a-synuclein formed abundant fib rils of varyin g length and l ateral aggregation (C). In the
presence o f syn aptosomal membranes only a few protofibrils of A53T mutant a-synuclein were detected (D). W hen in cubated alone, lysozyme
formed a few fibres of varying lengths (E) and were indistinguishable from fibres found in the presence of synaptosomal membranes (F). Scale bar is
50 nm for all.
Ó FEBS 2004 Lipid-induced a-synuclein fibrillogenesis (Eur. J. Biochem. 271) 3183
proven for Alzheimer’s amyloid-b peptide fibrillogenesis at
the surface of bilayers [40,41].
Under identical conditions and in the absence of syna-
ptosomal lipid vesicles, A53T mutant a-synuclein formed

abundant fibres after a 3-day i ncubation period (Fig. 2C).
The fibrils were of varying lengths with a characteristic
10–12 nm diameter [42,43]. These short fibres demonstra-
ted varying degrees of lateral aggregation into larger
bundles up to 130 nm in diameter. In t he presence of
synaptosomal lipid vesicles, very few A53T a-synuclein
fibrils could be d etected as c ompared to when i ncubated in
the absence of lipid (Fig. 2D). The few fibrils that were
detected had the morphological characteristics of protofi-
brils but were always intimately associated with synapto-
somal vesicle edges. In contrast to WT a-synuclein,
synaptosomal membranes inhibit the formation of A53T
mutant a-synuclein amyloid fibres, suggesting that the
A53T mutation affects the mode of interaction with lipid
bilayers.
To demonstrate the specificity of a-synuclein–synapto-
somal membrane interactions, hen egg w hite lysozyme was
used as a c ontrol amyloid-forming protein that is n ot found
in the nervous system. Under identical conditions, lysozyme
incubated alone for 3 days demonstrated ver y few fibr es of
varying lengths (Fig. 2E) and was not d istinguishable from
fibres formed in the presence of synaptosomal membranes
(Fig. 2 F). Th ese results suggest that synaptosomal lipid
vesicles do not alter lysozyme fibril formation.
In order to correlate the morphological studies of
a-synuclein fibre formation with quantitative fibril growth,
the intrinsic tyrosine fluorescence of a-synuclein was used to
monitor the amou nt of soluble protein after incubating in
the p resence of synaptosomal membrane v esicles [33]. After
2, 48 and 96 h of incubation , soluble a-synuclein or

lysozyme was separated from aggregated and fibrillar
protein by low-speed centrifugation (Fig. 3). These condi-
tions are s ufficient to pellet protein aggregates and fibrils but
not unilamellar lipid vesicles. In agreement with the electron
microscopy studies, the amount of soluble WT a-synuclein
decreased s ignificantly over t ime w hereas A53T a-synuclein
remained soluble. An aliquot from each sample was
examined by electron microscopy; WT but not A53T
a-synuclein fibres were detected as described above.
Furthermore, lysozyme in the presence of synaptosomal
membrane vesicles aggregated over time but to a l esser
extent than WT a-synuclein (Fig. 3).
Fatty acyl chain mobility
To further characterize the differences in WT and A53T
mutant a-synuclein binding to lipid bilayers an d to deter-
mine the most influential lipid prop erties that govern
a-synuclein fibrillogenesis, we examined the effect of
a-synuclein on synaptosomal membrane fluidity. The
availability of fluorescent dyes that penetrate to varying
levels within the bilayer and exhibit fluorescent properties
characteristic of their local environment allow u s to address
the extent t o which a-synuclein inserts into t he lipid bilayer.
Specifically, the effects of a-synuclein on the mobility of t he
fatty a cyl chains within the bilayer can be determined using
the steady-state fluorescence anisotropy of the dye, DPH
[34]. The relative motion of the D PH molecule within the
bilayer is determined by polarized fluorescence and
expressed as r, the anisotropy constant, that is inversely
proportional to the degree of membrane fluidity.
The relative fluidity of synaptosomal membranes was

considered gel-like as indicated by an r-value close to 0.2
(Fig. 4 ). Addition of WT a-synuclein to the synaptosomal
membranes had little effect on the m embrane fluidity and
was not dependent on the a-synuclein/lipid ratio. These
results suggest that although WT a-synuclein binds lipid
vesicles, it may not insert into the bilaye r or alternatively
that synaptosomal membranes have a high capacity for
a-synuclein binding. These results appear to be in contrast
with the r eport by Sharon a nd colleagues, who s howed that
WT a-synuclein decreased the fluidity of whole cell mem-
brane preparations [44]. The differences between the two
studies may be accounted for by either the presence of
proteins or the combination of plasma, nuclear, endosomal,
lysosomal and Golgi membranes in this preparation. Our
experiments address only synaptosomal membrane inter-
actions and therefore represent a small population within
the whole cell membrane preparation.
In contrast, A53T mutant a-synucle in significantly
decreased internal bilayer fluidity of synaptosomal vesicles
as demonstrated by the elevated anisotropy constant
(Fig. 4 ). Increasing A53T concentration further decreased
synaptosomal membrane fluidity. These results suggest that
the A 53T muta nt a-synuclein either inserts directly into the
fatty acyl chains or that synaptosomes h ave a low capacity
for A53T mutant binding due to subtle changes in structure.
The substitution of Ala53 fi Thr of a-synuclein is predicted
to partially disrupt the N-terminal a-helix and extend the
Fig. 3. Tyrosine fluorescence was used to determine the extent of
a-synuclein aggregation in the presence of synaptosomal membranes.
WT and A53T a-synuclein or lysozyme were incubated in the presence

of synaptosomal membranes f or 2 (solid bars), 48 (hatched bars) and
96 h ( open ba rs). The e xtent o f aggr egat ion was det er mined using a
ratio of tyrosine fl uorescence before and after centrifugation. The ratio
of tyrosine fluorescence after immediate mixing was set at 100% an d
all other conditions were normalized to this value. The re su lts are the
mean ± SEM for t hree independent e xperiments.
3184 E. Jo et al.(Eur. J. Biochem. 271) Ó FEBS 2004
b-sheet structure of the central hydrophobic domain [15,45].
The decreased membrane fluidity as a result of the A53T
substitution m ay be explained by the fact that lipid bilaye rs
can readily accommodate a-helices but are disrupted by
b-structured transmembrane features. The specificity of
a-synuclein-synaptosomal membrane preparation was
further probed by examining the effects of lysozyme on
synaptosomal membrane fluidity (Fig. 4). This amyloid-
forming peptide increases the fluidity of these v esicles i n a
concentration dependent manner suggesting that peptide
sequence i s important for specific membrane perturbations.
Dynamics of lipid headgroups and interface
In order to further characterize the differences in WT and
A53T-synpatosomal membrane binding, the dynamics of
the polar headgroups and the polarity of the lipid interface
were analyzed as a measure of protein–membrane inter-
actions. To obtain f urther insight into t he mechanism of
a-synuclein–bilayer interactions, laurdan fluorescence
spectroscopy was utilized. L aurdan’s naphthalene ring is
located at the glycerol backbone and is a nchored in the
bilayer by t he lauroyl moiety, thereby imparting fluorescent
characteristics that are dependent o n the polarity of its
environment [35,46]. The advantage of laurdan over other

fluorescent probes is that it is nonfluorescent in aqueous
media and is independent of pH and lipid headgroups;
therefore fl uorescence only r eflects the polarity of the probe
associated with the bilayer. The spectral p roperties of
laurdan are described by the general polarization equation
and render information about th e lipid phase, polarity and
coexistence of multiple lipid phases within a membrane
[35,46].
Laurdan excitation a nd emission spectra in the presence
of synaptosomal membrane bilayers demonstrate t he char-
acteristic red excitation at 340 nm and blue excitation at
380 n m, whereas the emission spectra indicate a single
maximum at 430 nm indicative of the blue emission
(Fig. 5 A). The intensity of the red excitation b and correlates
with a polar environment o r strong hydrogen bon ding
Fig. 5. Laurdan emission and excitation spectra of synaptosomal
membranes in the presence of a-synuclein. Vesicles alone (ÆÆÆÆ) and after
addition of WT (––) and A53T ( ) a-synuclein show similar o verall
spectral characteristics ( A). T he dec reased intensity as a result of the
presence of a-synuclein demonstrates the decrease in polarity of the
headgroup–fatty acyl chain interface. The excitation and emission
generalized polarization of laurdan in the presence of both WT and
A53T a-synuclein are not depe ndent on wavelength (B). Generalized
polarization values were calculated from excitation and emission scans
before (ÆÆÆÆ) a nd after addition o f WT (––) a nd A53T ( ).
Fig. 4. The effect of a-synuclein on membrane fluidity o f synaptosomal
lipid bilayers as determined by DPH anisotropy. The addition of 5 and
10 lgofWTa-synuclein to synaptosomal membrane vesicles did n o t
affect membrane flu idity. Addition o f A53T m utant a-synuclein
significantly decreased t he membrane fluidity in a concentration

dependent manner. In contrast, lysozym e increased membrane fluidity
in a concentratio n dependent manner. D ata represent the mea n ± SD
of three independent experiments. Student t-test indicates *P <0.01,
P < 0.001 compared to lipid alone.
Ó FEBS 2004 Lipid-induced a-synuclein fibrillogenesis (Eur. J. Biochem. 271) 3185
which occur in gel phase lipid bilayers where little relaxation
occurs. The ratio of the blue to red components of the
excitation scan generates d ata o n the polarity of the probe.
The addition of both WT a nd A53T a-synuclein to laurdan
containing synaptosomal membranes g enerates a b lue-shift
in the excitation curve and decreases the intensity of laurdan
fluorescence (Fig. 5A). Furthermore, the blue : red excita-
tion ratio changes from 1.00 for synaptosomal membranes
to 1 .02 and 1.04 after addition of A53T and WT a-synuc-
lein, respectively. The blue-shift in t he excitation scan
represents a less polar environment, suggestive of d ecreased
H
2
O molecules and increased packing of lipid molecules.
Furthermore, the general polarization emission (GP
em
)is
unaffected by addition of A53T a-synuclein confirming that
the micropolarity or hydration of the interfacial region of
the lipid bilayer is unchanged, whereas WT binding
increases the GP
em
indicating an altered packing of the
interfacial region. Finally, the wavelength dependence of the
general polarization lends eviden ce for the phase behaviour

of bilayers in the presence and absence of proteins. The GP
ex
and GP
em
of synaptosomal membranes are independent
of wavelength in the p resence of b oth WT and A53T
a-synuclein indicating that the lipids do not undergo
protein-induced phase change (Fig. 5B). These results
suggest that binding of WT and A53T a-synuclein to
synaptosomal membranes disrupts the inter facial region of
the lipid molecule to varying extents but both can be easily
accommodated within the lipid structure.
Lipid headgroup packing and surface properties
To distinguish a-synuc lein surface binding from insertion
into the bilayer, we examined the lipid headgroup spacing
using merocyanine 540 (MC540) absorption spectral prop-
erties. The unique spectral properties result from binding of
monomeric MC540 and subsequent dimerization, which are
both dependen t on lipid headgroup packing and fluidity
[36]. MC540 spectra in the presence of synaptosomal lipid
vesicles are characteristic of a fluid headgroup packing w ith
characteristic maxima at 530 and 570 nm (Fig. 6). The fluid
headgroup space allows for extensive monomeric MC540
insertion as i ndicated by the predominance of the 5 70 nm
maxima. Addition of WT a-synuclein increased the intensity
of both maxima, indicating a slightly more fluid environ-
ment and an increased headgroup space or the presence of
packing defects (Fig. 6). The M C540 spectra are consistent
with WT a-synuclein–synaptosomal interactions occurring
predominantly at t he headgroup space. These data correlate

well with our anisotropy studies, which demonstrate no
change in the fatty acyl chain mobility as a result of WT
a-synuclein binding and our electron microscopy data,
which showed enhanced fibre f ormation after lipid binding
of WT a-synuclein. Predominant headgroup b inding would
position WT a-synuclein in an ideal location to act as a seed
for fibril formation. In contrast, a ddition of A53T mutant
a-synuclein significantly decreased the intensity o f the
MC540 spectra indicating increased packing of the lipid
headgroups and d ecreased accessibility f or MC540 binding
(Fig. 6). These results are consistent with varying levels of
A53T insertion into the synapt osomal bilayer, which a ffect
both the headgroup and fatty acyl chain mobility.
One interpretation of these results is that A53T mutant
a-synuclein may insert into the bilayer to a greater degree
than WT a-synuclein such that the hydrophobic domain is
buried within the bilayer. Masking of the hydrophobic, b-
sheet promoting region would effectively inhibit a-synuclein
self-assembly into fibrils.
The MC540 monomer-dimer equilibrium is relevant to
the packing properties of the bilayer a nd can be used as a n
indication of lipid headgroup spacing [36]. W e have
calculated the apparent dimerization constant, K
dapp
,for
MC540 in synaptosomal lipid bilayers in the presence and
absence o f W T and A53T mutant a-synuclein (Table 1).
The K
dapp
of synaptosomal membranes was not altered

significantly after binding WT a-synuclein suggesting that
the membrane can easily accommodate WT a-synuclein.
Addition of A53T a-syn uclein inc reased the Kdapp by
10-fold indicating that dimerization of MC540 within the
headgroup space was d ecreased. These results support the
notion that A53T mutant a-synuclein interactions with
synaptosomal membranes organize the headgroup packing
and ultimately the bilayer fluidity, thereby increasing
membrane rigidity.
Discussion
Overall, our data suggest that WT a-synuclein binds
predominantly to the headgroups of physiologically
Fig. 6. The interaction of a-synuclein with the lipid headgroups of syn-
aptosomal membranes was examined using MC540 absorption spectro-
scopy. MC540 spectra demonstrate the fluid packing of the
synaptosomal membrane h eadgroups (––). Addition o f W T a-synuc-
lein ( ) resulted i n an increase i n the intensity of t h e MC540 spectra
indicative of a-synuclein–headgroup interactions. In c ontrast, A53T
a-synucle in decreased the in tensity of the sp ectra indicating increased
packing o f the headgroups (ÆÆÆÆ).
Table 1. Effect of a-synuclein mutations on the apparent dimerization
constant (K
dapp
) o f merocyanine 540 in synaptosomal m embranes.
Peptides were added to lipid vesicles at a 1 : 20 ratio with a final
peptide concentration of 6.9 lm.
Sample Apparent dimerization constant
Synaptosomes 1.67 · 10
5
WT a-synuclein 2.17 · 10

5
A53T mutant 1.54 · 10
6
3186 E. Jo et al.(Eur. J. Biochem. 271) Ó FEBS 2004
relevant lipid mixtures causing p acking defects in the
headgroup space. The molecule penetrates into the interfa-
cial lipid space as evidenced by the decrease in H
2
0 content
and increased order of this region, but does not penetrate to
the fatty acyl chains as no change in fluidity was detected.
Predominant s urface binding of WT to synaptosomal
membranes m ay help to explain the reversible lipid binding
function of a-synuclein as this would create the least amount
of disturbance in overall membrane structure [19,47]. In
contrast, A53T binding causes increased lipid head-
group packing, sub tle changes in the lipid interfacial
space and a d ecrease in t he fluidity o f the fatty a cyl chains.
These results are consistent with insertion of A53T into the
bilayer.
Our data suggest that a mutation linked t o familial early
onset PD af fects no t on ly the s elf-asse mbly of a-synuclein
but also the interaction with lipid bilayers. These results
have implications for development of PD pathology, such as
Lewy bodies, and extend our understanding of the effect of
mutations in a-synuclein that may result in early onset
forms of the disease. Lewy bodies are composed of
a-synuclein, lipids and ubiquitin [48]. Immunohistochemical
analyses have demon strated that lipids are distributed
diffusely in homogenous Lewy bodies or are highly localized

to the periphery of concentric Lewy bodies. It h as been
proposed that lipids m ay either facilitate the incorporation
of a-synuclein or influence a-synuclein fibril elo ngation [48].
Our r esults demonstrate that WT a-synuclein binding to
synaptosomal membranes not only enhances fibril forma-
tion but also propagates fibril growth along the bilayer
surface. These results suggest that heterogenous seeding of
a-synuclein fibrillogenesis m ay be one mechanism by which
Lewy body formation progresses in PD. These results are
consistent with previous reports that have shown seeding of
WT cytosolic a-synuclein with me mbrane-bound a-synuc-
lein [39]. Furthermore, synaptosomal membranes have a
high capacity to bind WT a-synuclein, raising the possibility
that control of membrane to cytosol distribution of
a-synuclein may be important in nerve terminals. L ipid
loading of primary neuronal cells demonstrated the redis-
tribution of WT a-synuclein from cytosol t o the surface of
lipid droplets, resulted in the prevention o f triglyercides
hydrolysis [21]. Our results are in contrast to single lipid
environments of acidic phospholipids, which have demon-
strated decreased fibre formation in the presence of
phosphatic acid and phosphatidylglycerol [49]. The differ-
ence may be explained by the presence of a full repertoire of
phospholipids, gangliosides, cholesterol and sphingomyelin,
affecting not only overall membrane fluidity but also the
surface charge of the lipid bilayer.
In contrast to WT a-synuclein, A53T mutant a-synuclein
binding to synaptosomal membranes decreases fibril for-
mation, which seems to contradict in vitro self assembly
models that have shown e nhanced fibrillogenesis of t he

A53T mutant in comparison to WT a-synuclein [25,50–52].
However, these p revious in v itro fibrillogenesis studies were
performed in t he absence of membranes. Our data suggest
that the effects of the A53T mut ant a-synuclein may be
elicited by altering the ratio of membrane-bound to
cytosolic a-synuc lein. If decreased synaptosomal membrane
fluidity resulting from A53T mutant a-synuclein binding
further i nhibits a-synuclein–membrane interactions, t hen
the relative a-synuclein concentration in the cytosol would
be elevated and self-assembly may dominate. These results
are consistent with the hypothesis t hat molecular crowding
within the cytoplasm may contribute to amyloid-related
disorders [53,54]. Furthermore, d ecreased m embrane
fluidity will also affect normal cellular function and
specifically synaptic signalling. In conclusio n, the equilib-
rium between membrane-bound and cytosolic a-synuclein
may be crucial for physiological function such that any
significant shift in the equilibrium due to missense mutations
or changes in membrane fluidity may c ause abnormal
protein aggregation and Lewy body formation.
Acknowledgements
The authors would l ike to thank the Electron M icroscopy Suite at the
University of Toronto for use of Hitachi 7000 electron microscope
(CIHR M aintenance Grant). This work was supported by the
Canadian Institutes of Health Research (J. M., P. E. F., P. H.), the
Natural Sciences and Engineering Research C ouncil of Canada (J. M.),
Ontario M ental Health Foundation (P.E.F.)andtheScottishRite
Charitable Foundation (P. E. F., J. M.). The authors acknowledge
support from the Ontario Alzheimer’s Asso ciation. J. M. was the Year
2001 Young Investigator Fund Scholarship recipient. E. J. was the

recipient of a Postdoctoral Fellowship from the P arkin son’s Founda-
tion of Canada.
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