In contrast, synaptosomal membrane fluidity was decreased by A53T a-synuclein binding with concomitant packing of the lipid headgroups.. On the other hand, the A53T mutant could not be di
Trang 1a-Synuclein–synaptosomal membrane interactions
Implications for fibrillogenesis
Euijung Jo1, Audrey A Darabie1, Kyung Han1, Anurag Tandon1,3, Paul E Fraser1,2and JoAnne McLaurin1,4
1
Centre for Research in Neurodegenerative Diseases; Departments of2Medical Biophysics,3Medicine and4Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada
a-Synuclein 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 has been
sug-gested that these environmental and structural differences
may play a role in aggregation propensity and development
of pathological lesions observed in Parkinson’s disease (PD)
Such effects may be accentuated by mutations observed in
familial PD kindreds In order to test this hypothesis,
wild-type and A53T 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 microscopy demonstrated that wild-type
a-synuclein fibrillogenesis is accelerated in the presence of
synaptosomal membranes whereas the A53T a-synuclein
fibrillogenesis is inhibited under the same conditions These
results suggested that subtle sequence changes in a-synuc-lein could significantly alter interaction with membrane bilayers Fluorescence and absorption spectroscopy using environment sensitive probes demonstrated variations in the inherent lipid properties in the presence and absence of a-synuclein Addition 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 In 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 spectroscopy; membrane
The link between a-synuclein and Parkinson’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 the discovery of
two missense mutations A53T and A30P, which suggested
an etiological significance rather than a secondary
patho-logical event [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 sequence,
expres-sion level or function 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 protein
aggregation might play an active part in these
neurodegen-erative diseases
The physiological function of a-synuclein remains largely unknown, but it has been suggested that it may play a role
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-helices, a central hydrophobic domain followed by an acidic C-terminal, rich in glutamate [15,17] These primary and secondary structure characteristics suggested a role for a-synuclein in protein–membrane interactions Initial in vitro experiments showed that the helical structure of wild-type (WT) a-synuclein was induced and stabilized by binding to synthetic membranes [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 WT 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] More detailed analyses of the A30P mutation demonstrated that 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: j.mclaurin@utoronto.ca
Abbreviations: DPH, 1,6-diphenyl-1,3,5-hexatriene; MC540,
Merocyanine 540; PD, Parkinson’s disease; WT, wild-type.
(Received 10 May 2004, accepted 8 June 2004)
Trang 2WT a-synuclein, the 3D conformation was different
[26] Furthermore, the differences in 3D structure results
in defective membrane binding of the A30P mutant
a-synuclein [27,28] On the other hand, the A53T mutant
could not be distinguished from WT a-synuclein 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 investigate 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 order to determine whether properties
associated with a-synuclein-lipid binding in homogeneous
lipid environments are replicated in environments consisting
of relevant ratios of phospholipids, gangliosides and
sphingomyelin Furthermore, these studies may help to
distinguish subtle differences between mutant and WT
a-synuclein interactions and to further elucidate potential
roles for a-synuclein–lipid interactions in vivo In order to
examine differences and explain pathological findings, we
examined the ability of these membranes to facilitate
a-synuclein assembly by electron microscopy Changes in
the membrane physical characteristics as a result of
a-synuclein interactions were examined by fluorescence
spectroscopy using environment sensitive probes These
parameters define the extent to which a-synuclein penetrates
the lipid bilayer or disrupts lipid headgroup packing
Materials and methods
Expression and purification of recombinant a-synuclein
Human a-synuclein cDNAs, wild-type and A53T mutant
were subcloned into the plasmid pET-28a (Novagen), using
NcoI and HindIII restriction sites a-Synuclein was
over-expressed in Eschericia coli BL21 (DE3) and isolated over a
Q-Sepharose column as described previously [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
recor-ded on a Jasco Circular Dichroism Spectrometer (Tokyo,
Japan) at 25C 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 peptide conformation was determined by
adding an aliquot of stock peptide solution into NaCl/Pi
(pH 7.4) at a final peptide concentration of 10 lM
Synaptosome isolation
Rat grey matter was dissected after cervical dislocation
(according to CACC guidelines) and homogenized in 10
volumes of 320 mMsucrose, 5 mMHepes, pH 7.4
(homo-genizing buffer) using a glass homogenizer The homogenate
was spun at 1050 g for 10 min The supernatant was re-spun
at 13 300 g for 15 min, 4C The pellet 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 4C The synaptosomes were
isolated from the 9–13% layer and diluted into Hepes buffer
Final synaptosome isolation was achieved after
centrifuga-tion at 13 300 g for 5 min, 4C [29,30] Lipids were extracted
from the synaptosomes using Folch partition [chloroform/ methanol/water (v/v/v); 2 : 1 : 0.6] and subsequently con-centrated under a stream of N2 The samples were stored at )20 C until use Phospholipid concentration was deter-mined using the Bartlett assay [31]
Electron microscopy
WT and A53T a-synucleins were incubated in the presence and absence of synaptosomal vesicles at a final peptide concentration of 5.8 lM The a-synuclein to lipid ratio was maintained at 1 : 20 (by mass) For negative stain electron microscopy, carbon-coated pioloform grids 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 fluorescence was measured at 20C using
a Photon Technology International (PTI, London, ON, Canada) QM-1 Fluorescence spectrophotometer equipped with excitation intensity correction and a magnetic 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 studies, 10 lMof WT or A53T a-synuclein
or hen egg-white lysozyme was incubated in the presence or absence of synaptosomal membrane vesicles at 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 equipped with manual polarizers as described previ-ously [34] Excitation and emission wavelengths were set 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)1in NaCl/Pi, incubated for 20–30 min in the 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 (IVV) and horizontal (IVH) positions and anisotropy, r, was calculated using Eqn (1);
r¼ IVV gIVH
IVVþ 2gIVH
ð1Þ Lipid vesicles in the absence of DPH were measured in order
to evaluate the effect of light scattering on our measurements
Trang 3Laurdan generalized polarization
Steady-state excitation and emission 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):
GPem¼I400 I340
I400þ I340
ð2Þ where, I400and I340are the fluorescence 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):
GPex¼I440 I490
I440þ I490
ð3Þ where, I440and I490are the fluorescence intensities at each
excitation wavelength from 320 to 420 nm, measured at
fixed emission wavelengths of 440 nm and 490 nm,
respect-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 probe/lipid ratio of 1 : 500 [36] Final MC540 molar
concentration in the cuvette was 21.3 lM Absorption
spectra were obtained between 400 and 600 nm with 1 nm
steps The lipid alone baseline in the absence of MC540 was
subtracted from all spectra, and then corrected by referring
the absorbances at 600 nm to zero (Eqns 4 and 5)
[monomer]¼A ½e
D C=2
[dimer]¼ðC [monomer]Þ
Where, A is the absorbance at 569 nm, e is the constant for MC540 dimer or monomer at the given wavelength,
em¼ 1.511 · 105 and eD¼ 5400, while C is the final MC540 concentration
After this correction, the absorbance values at 569 nm were used to calculate the dimerization constant (Kdapp) as described by Bernik & Disalvo [37] (Eqn 6)
Kdapp¼ [dimer]
Results
a-Synuclein morphological characteristics Recombinant a-synucleins, both wild-type (WT) and A53T mutant, were over-expressed and purified from Eschericia coliBL21 as described previously [19] After purification, both WT and A53T mutant a-synuclein ran as single bands
on SDS/PAGE (Fig 1A) and displayed typical random secondary structure when diluted into NaCl/Pi, pH 7.3 (Fig 1B) These results confirm both the purity and the random structure of these 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 rat brain
or neuroblastoma cells [22,39] Furthermore, a-synuclein has been located to and associated with the presynaptic terminals and synaptosomal membrane surfaces by immu-nogold localization studies [16] Therefore, bilayers com-posed of lipids isolated from rat 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 WT and A53T mutant a-synuclein Both WT (lane 1) and A53T mutant (lane 2) a-synuclein demonstrated pre-dominantly single bands when electrophoresed by SDS/PAGE (A) The secondary structure of the a-synucleins was determined using circular dichroism spectroscopy (B) Both WT (h) and A53T (s) mutant a-synuclein were randomly structured when diluted into NaCl/P i , as illustrated by
a single minimum below 200 nm.
Trang 4partition 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 lMconcentrations, WT a-synuclein did
not form detectable amyloid fibres after a 3-day incubation
(Fig 2A) 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 2B) 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 demonstrated that membrane-bound WT a-synuclein 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 the 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 fibrils When incubated in the presence of synaptosomal membranes (B) abundant a-synuclein fibrils could be detected with organization along the vesicle surface A53T mutant a-synuclein formed abundant fibrils of varying length and lateral aggregation (C) In the presence of synaptosomal membranes only a few protofibrils of A53T mutant a-synuclein were detected (D) When incubated 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.
Trang 5proven 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 incubation 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 the presence of
synaptosomal lipid vesicles, very few A53T a-synuclein
fibrils could be detected as compared to when incubated 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 white lysozyme was
used as a control amyloid-forming protein that is not found
in the nervous system Under identical conditions, lysozyme
incubated alone for 3 days demonstrated very few fibres of
varying lengths (Fig 2E) and was not distinguishable from
fibres formed in the presence of synaptosomal membranes
(Fig 2F) These 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 amount of soluble protein after incubating in
the presence of synaptosomal membrane vesicles [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 sufficient 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 significantly over time whereas 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 lesser
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 and to
deter-mine the most influential lipid properties 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 us to address
the extent to which a-synuclein inserts into the lipid bilayer
Specifically, the effects of a-synuclein on the mobility of the
fatty acyl chains within the bilayer can be determined using
the steady-state fluorescence anisotropy of the dye, DPH
[34] The relative motion of the DPH 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 membrane 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 bilayer or alternatively that synaptosomal membranes have a high capacity for a-synuclein binding These results appear to be in contrast with the report by Sharon and colleagues, who showed 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-synuclein 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 A53T mutant a-synuclein either inserts directly into the fatty acyl chains or that synaptosomes have a low capacity for A53T mutant binding due to subtle changes in structure The substitution of Ala53fi 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 for 2 (solid bars), 48 (hatched bars) and
96 h (open bars) The extent of aggregation was determined using a ratio of tyrosine fluorescence before and after centrifugation The ratio
of tyrosine fluorescence after immediate mixing was set at 100% and all other conditions were normalized to this value The results are the mean ± SEM for three independent experiments.
Trang 6b-sheet structure of the central hydrophobic domain [15,45].
The decreased membrane fluidity as a result of the A53T
substitution may be explained by the fact that lipid bilayers
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 vesicles in a
concentration dependent manner suggesting that peptide
sequence is 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 further insight into the mechanism of
a-synuclein–bilayer interactions, laurdan fluorescence
spectroscopy was utilized Laurdan’s naphthalene ring is
located at the glycerol backbone and is anchored in the
bilayer by the lauroyl moiety, thereby imparting fluorescent
characteristics that are dependent on 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 fluorescence only reflects the polarity of the probe
associated with the bilayer The spectral properties of
laurdan are described by the general polarization equation
and render information about the lipid phase, polarity and coexistence of multiple lipid phases within a membrane [35,46]
Laurdan excitation and emission spectra in the presence
of synaptosomal membrane bilayers demonstrate the char-acteristic red excitation at 340 nm and blue excitation at
380 nm, whereas the emission spectra indicate a single maximum at 430 nm indicative of the blue emission (Fig 5A) The intensity of the red excitation band correlates with a polar environment or strong hydrogen bonding
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 overall spectral characteristics (A) The decreased 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 dependent on wavelength (B) Generalized polarization values were calculated from excitation and emission scans before (ÆÆÆÆ) and after addition of WT (––) and A53T ( -).
Fig 4 The effect of a-synuclein on membrane fluidity of synaptosomal
lipid bilayers as determined by DPH anisotropy The addition of 5 and
10 lg of WT a-synuclein to synaptosomal membrane vesicles did not
affect membrane fluidity Addition of A53T mutant a-synuclein
significantly decreased the membrane fluidity in a concentration
dependent manner In contrast, lysozyme increased membrane fluidity
in a concentration dependent manner Data represent the mean ± SD
of three independent experiments Student t-test indicates *P < 0.01,
P < 0.001 compared to lipid alone.
Trang 7which occur in gel phase lipid bilayers where little relaxation
occurs The ratio of the blue to red components of the
excitation scan generates data on the polarity of the probe
The addition of both WT and A53T a-synuclein to laurdan
containing synaptosomal membranes generates a blue-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 the excitation scan
represents a less polar environment, suggestive of decreased
H2O molecules and increased packing of lipid molecules
Furthermore, the general polarization emission (GPem) 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 GPem indicating an altered packing of the
interfacial region Finally, the wavelength dependence of the
general polarization lends evidence for the phase behaviour
of bilayers in the presence and absence of proteins The GPex
and GPem of synaptosomal membranes are independent
of wavelength in the presence of both 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 interfacial 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-synuclein 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 dependent on lipid headgroup packing and fluidity
[36] MC540 spectra in the presence of synaptosomal lipid
vesicles are characteristic of a fluid headgroup packing with
characteristic maxima at 530 and 570 nm (Fig 6) The fluid
headgroup space allows for extensive monomeric MC540
insertion as indicated by the predominance of the 570 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 MC540 spectra are consistent
with WT a-synuclein–synaptosomal interactions occurring
predominantly at the 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 formation after lipid binding
of WT a-synuclein Predominant headgroup binding would
position WT a-synuclein in an ideal location to act as a seed
for fibril formation In contrast, addition of A53T mutant
a-synuclein significantly decreased the intensity of the
MC540 spectra indicating increased packing of the lipid
headgroups and decreased accessibility for MC540 binding
(Fig 6) These results are consistent with varying levels of
A53T insertion into the synaptosomal bilayer, which affect
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 and can be used as an indication of lipid headgroup spacing [36] We have calculated the apparent dimerization constant, Kdapp, for MC540 in synaptosomal lipid bilayers in the presence and absence of WT and A53T mutant a-synuclein (Table 1) The Kdapp 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-synuclein increased the Kdapp by 10-fold indicating that dimerization of MC540 within the headgroup space was decreased 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 headgroups (––) Addition of WT a-synuc-lein ( -) resulted in an increase in the intensity of the MC540 spectra indicative of a-synuclein–headgroup interactions In contrast, A53T a-synuclein decreased the intensity of the spectra indicating increased packing of the headgroups (ÆÆÆÆ).
Table 1 Effect of a-synuclein mutations on the apparent dimerization constant (K dapp ) of merocyanine 540 in synaptosomal membranes 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
Trang 8relevant lipid mixtures causing packing defects in the
headgroup space The molecule penetrates into the
interfa-cial lipid space as evidenced by the decrease in H20 content
and increased order of this region, but does not penetrate to
the fatty acyl chains as no change in fluidity was detected
Predominant surface binding of WT to synaptosomal
membranes may 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, subtle changes in the lipid interfacial
space and a decrease in the fluidity of the fatty acyl chains
These results are consistent with insertion of A53T into the
bilayer
Our data suggest that a mutation linked to familial early
onset PD affects not only the self-assembly 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 demonstrated that lipids are distributed
diffusely in homogenous Lewy bodies or are highly localized
to the periphery of concentric Lewy bodies It has been
proposed that lipids may either facilitate the incorporation
of a-synuclein or influence a-synuclein fibril elongation [48]
Our results 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 may 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 membrane-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 Lipid
loading of primary neuronal cells demonstrated the
redis-tribution of WT a-synuclein from cytosol to the surface of
lipid droplets, resulted in the prevention of 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 presdiffer-ence 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 enhanced fibrillogenesis of the
A53T mutant in comparison to WT a-synuclein [25,50–52]
However, these previous in vitro fibrillogenesis studies were
performed in the absence of membranes Our data suggest
that the effects of the A53T mutant a-synuclein may be
elicited by altering the ratio of membrane-bound to
cytosolic a-synuclein If decreased synaptosomal membrane
fluidity resulting from A53T mutant a-synuclein binding
further inhibits a-synuclein–membrane interactions, then
the relative a-synuclein concentration in the cytosol would
be elevated and self-assembly may dominate These results are consistent with the hypothesis that molecular crowding within the cytoplasm may contribute to amyloid-related disorders [53,54] Furthermore, decreased membrane fluidity will also affect normal cellular function and specifically synaptic signalling In conclusion, 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 cause abnormal protein aggregation and Lewy body formation
Acknowledgements
The authors would like to thank the Electron Microscopy Suite at the University of Toronto for use of Hitachi 7000 electron microscope (CIHR Maintenance 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 Council of Canada (J M.), Ontario Mental Health Foundation (P E F.) and the Scottish Rite Charitable Foundation (P E F., J M.) The authors acknowledge support from the Ontario Alzheimer’s Association J M was the Year
2001 Young Investigator Fund Scholarship recipient E J was the recipient of a Postdoctoral Fellowship from the Parkinson’s Founda-tion of Canada.
References
1 Goedert, M., Jakes, R., Crowther, R.A., Hasegawa, M., Smith, M.J & Spillantini, M.G (1998) Intraneuronal filamentous tau protein and alpha-synuclein deposits in neurodegenerative dis-eases Biochem Soc Trans 26, 463–471.
2 Mezey, E., Dehejia, A.M., Harta, G., Tresser, N., Suchy, S.F., Nussbaum, R.L., Brownstein, M.J & Polymeropoulos, M.H (1998) Alpha synuclein is present in Lewy bodies in sporadic Parkinson’s disease Mol Psychiatry 3, 493–499.
3 Spillantini, M.G., Schmidt, M.L., Lee, V.M., Trojanowski, J.Q., Jakes, R & Goedert, M (1997) Alpha-synuclein in Lewy bodies Nature 388, 839–840.
4 Trojanowski, J.Q & Lee, V.M (1998) Aggregation of neurofila-ment and alpha-synuclein proteins in Lewy bodies: implications for the pathogenesis of Parkinson disease and Lewy body dementia Arch Neurol 55, 151–152.
5 Wakabayashi, K., Matsumoto, K., Takayama, K., Yoshimoto,
M & Takahashi, H (1997) NACP, a presynaptic protein, immunoreactivity in Lewy bodies in Parkinson’s disease Neurosci Lett 239, 45–48.
6 Polymeropoulos, M.H., Lavedan, C., Leroy, E., Ide, S.E., Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer, R., et al (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease Science 276, 2045–2047.
7 Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, H., Epplen, J.T., Schols, L & Riess, O (1998) Ala30Pro mutation in the gene encoding alpha-synuclein in Par-kinson’s disease Nat Genet 18, 106–108.
8 Singelton, A.B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J., Hulihan, M., Peuralinna, T., Dutra, A., Nussbaum, R., et al (2003) a-Synuclein locus triplication causes Parkinson’s Disease Science 302, 841.
9 Arai, T., Ueda, K., Ikeda, K., Akiyama, H., Haga, C., Kondo, H., Kuroki, N., Niizato, K., Iritani, S & Tsuchiya, K (1999) Argyrophilic glial inclusions in the midbrain of patients with Parkinson’s disease and diffuse Lewy body disease are immuno-positive for NACP/a-synuclein Neurosci Lett 259, 83–86.
Trang 910 Arima, K., Ueda, K., Sunohara, N., Arakawa, K., Hirai, S.,
Nakamura, M., Tonozuka-Uehara, H & Kawai, M (1998)
NACP/alpha-synuclein immunoreactivity in fibrillary components
of neuronal and oligodendroglial cytoplasmic inclusions in the
pontine nuclei in multiple system atrophy Acta Neuropathol.
(Berl) 96, 439–444.
11 Spillantini, M.G., Crowther, R.A., Jakes, R., Cairns, N.J.,
Lan-tos, P.L & Goedert, M (1998) Filamentous a-synuclein
inclu-sions link multiple system atrophy with Parkinson’s disease
and dementia with Lewy bodies Neurosci Lett 251, 205–
208.
12 Tu, P.H., Galvin, J.E., Baba, M., Giasson, B., Tomita, T., Leight,
S., Nakajo, S., Iwatsubo, T., Trojanowski, J.Q & Lee, V.M.
(1998) Glial cytoplasmic inclusions in white matter
oligoden-drocytes of multiple system atrophy brains contain insoluble
alpha-synuclein Ann Neurol 44, 415–422.
13 Wakabayashi, K., Hayashi, S., Kakita, A., Yamada, M.,
Toyoshima, Y., Yoshimoto, M & Takahashi, H (1998)
Accu-mulation of alpha-synuclein/NACP is a cytopathological feature
common to Lewy body disease and multiple system atrophy Acta
Neuropathol (Berl) 96, 445–452.
14 Wakabayashi, K., Hayashi, S., Yoshimoto, M., Kudo, H &
Takahashi, H (2000) NACP/alpha-synuclein-positive filamentous
inclusions in astrocytes and oligodendrocytes of Parkinson’s
dis-ease brains Acta Neuropathol (Berl) 99, 14–20.
15 Clayton, D.F & George, J.M (1998) The synucleins: a family of
proteins involved in synaptic function, plasticity,
neurodegenera-tion and disease Trends Neurosci 21, 249–254.
16 Clayton, D.F & George, J.M (1999) Synucleins in synaptic
plasticity and neurodegenerative disorders J Neurosci Res 58,
120–129.
17 Goedert, M (1997) Familial Parkinson’s disease The awakening
of alpha-synuclein Nature 388, 232–233.
18 Davidson, W.S., Jonas, A., Clayton, D.F & George, J.M (1998)
Stabilization of a-synuclein secondary structure upon binding to
synthetic membranes J Biol Chem 273, 9443–9449.
19 Jo, E., McLaurin, J., Yip, C.M., St George Hyslop, P & Fraser,
P.E (2000) a-Synuclein membrane interactions and lipid
speci-ficity J Biol Chem 275, 34328–34334.
20 Uversky, V.N., Lee, H.-J., Li, J., Fink, A.L & Lee, S.-J (2001)
Stabilization of partially folded conformation during synuclein
oligomerization in both purified and cytosolic preparations.
J Biol Chem 276, 43495–43498.
21 Cole, N.B., Murphy, D.D., Grider, T., Rueter, S., Brasaemle, D.
& Nussbaum, R.L (2002) Lipid droplet binding and
oligomer-ization properties of the Parkinson’s disease protein a-synuclein.
J Biol Chem 277, 6344–6352.
22 Leng, Y., Chase, T.N & Bennett, M.C (2001) Muscarinic
receptor stimulation induces translocation of an a-synuclein
oligomer from plasma membrane to a light vesicle fraction in
cytoplasm J Biol Chem 276, 28212–28218.
23 Eliezer, D., Kutluay, E., Bussell, R & Browne, G (2001)
Con-formational properties of a-synuclein in its free and
lipid-asso-ciated states J Mol Biol 307, 1061–1073.
24 Perrin, R.J., Woods, W.S., Clayton, D.F & George, J.M (2000)
Interaction of human-synuclein and parkinson’s disease variants
with phospholipids Structural analysis using site-directed
muta-genesis J Biol Chem 275, 34393–34398.
25 Conway, K.A., Lee, S.-J., Rochet, J.-C., Ding, T.T., Williamson,
R.E & Lansbury, P.T Jr (2000) Acceleration of oligomerization,
not fibrillization, is a shared property of both alpha-synuclein
mutations linked to early-onset Parkinson’s disease: implications
for pathogenesis and therapy Proc Natl Acad Sci USA 97,
571–576.
26 McLean, P.J., Kawamata, H., Ribich, S & Hyman, B.T (2000)
Membrane association and protein conformation of a-synuclein in
intact neurons Effect of Parkinson’s disease-linked mutations.
J Biol Chem 275, 8812–8816.
27 Jenson, P.H., Nielsen, M.S., Jakes, R., Dotti, C.G & Goedert, M (1998) Binding of a-synuclein to brain vesicles is abolished by familial Parkinson’s disease mutation J Biol Chem 273, 26292– 26294.
28 Jo, E., Fuller, N., Rand, R.P., St George-Hyslop, P & Fraser, P.E (2002) Defective membrane interactions of familial Parkin-son’s disease mutant A30P alpha-synuclein J Mol Biol 315, 799–807.
29 Tandon, A., Bannykh, S., Kowalchyk, J.A., Banerjee, A., Martin, T.F.J & Balch, W.E (1998) Differential regulation of exocytosis
by calcium and CAPS in semi-intact synaptosomes Neuron 21, 147–154.
30 Tandon, A., Tan, P.K., Bannykh, S., Banerjee, A & Balch, W.E (1998) Neurotransmitter release from semi-intact synaptosomes Methods 16, 198–203.
31 Bartlett, G.R (1959) Phospholipid analyses Biochemistry 342, 466–468.
32 Waschuk, S.A., Elton, E.A., Darabie, A.A., Fraser, P.E & McLaurin, J (2001) Cellular membrane composition defines Ab–lipid interactions J Biol Chem 276, 33561–33356.
33 McLaurin, J., Franklin, T., Chakrabartty, A & Fraser, P.E (1998) Phosphatidylinositol and inositol involvement in Alzhei-mer amyloid-b fibril growth and arrest J Mol Biol 278, 183– 194.
34 Van Blitterswijk, W.J., Hoeven, R.P & Van Der Meer, B.W (1981) Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements Biochim Biophys Acta 644, 323–332.
35 Parasassi, T., DeStasio, G., Ravagnan, G., Rusch, R.M & Gratton, E (1991) Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of laurdan fluorescence Biophys J 60, 179–189.
36 Luzardo, M.D.C., Bernik, D.L., Pazos, I.F., Figueroa, S., Lanio, M.E., Verez, V & Disalvo, E.A (1999) Phase and surface prop-erties of lipid bilayers containing neoglycolipids Arch Biochem Biophys 363, 81–90.
37 Bernik, D.L & Disalvo, E.A (1996) Gel state surface properties of phosphatidylcholine liposomes as measured with merocyanine
540 Chem Phys Lipids 82, 111–123.
38 Narayanan, V & Scarlata, S (2001) Membrane binding and self association of a-synuclein Biochemistry 40, 9927–9934.
39 Lee, H.-J., Choi, C & Lee, S.-J (2002) Membrane-bound a-syn-uclein has a high aggregation propensity and the ability to seed the aggregation of the cytosolic form J Biol Chem 277, 671– 678.
40 Yip, C.M & McLaurin, J (2001) Amyloid-b peptide assembly: a critical step in fibrillogenesis and membrane disruption Biophys.
J 80, 1359–1371.
41 Ji, S.R., Wu, Y & Sui, S.F (2002) Cholesterol is an important factor affecting the membrane insertion of b-amyloid peptide (Ab1–40), which may potentially inhibit the fibril formation.
J Biol Chem 277, 6273–6279.
42 Conway, K.A., Harper, J.D & Lansbury, P.T (1998) Fibrils formed in vitro from a-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid Biochemistry 39, 2552– 2563.
43 Giasson, B.I., Uryu, K., Trojanowski, J.Q & Lee, V.M.-Y (1999) Mutant and wild type human a-synucleins assemble into elonga-ted filaments with distinct morphologies in vitro J Biol Chem.
274, 7619–7622.
44 Sharon, R., Bar-Joseph, I., Mirick, G.E., Serhan, C.N & Selkoe, D.J (2003) Altered fatty acid composition of Dopaminergic neurons expressing a-synuclein and human brains with a-synu-cleinopathies J Biol Chem 278, 49874–49881.
Trang 1045 Bussell, R & Eliezer, D (2001) Residual structure and dynamics
in Parkinson’s disease-associated mutants of a-synuclein J Biol.
Chem 276, 45996–46003.
46 Bagatolli, L.A., Parasassi, T., Fidelio, G.D & Gratton, E (1990)
A Model for the interaction of 6-laurolyl-2-(N,N-dimethylamino)
naphthalene with lipid environments: implications for spectral
properties Photochem Photobiol 70, 557–564.
47 Bussell, R & Eliezer, D (2003) A structural and functional role
for 11-mer repeats in a-synuclein and other exchangeable lipid
binding proteins J Mol Biol 329, 763–778.
48 Gai, W.P., Yuan, H.X., Li, X.Q., Power, J.T.H., Blumbers, P.C &
Jensen, P.H (2000) In situ and in vitro study of colocalization and
segregation of alpha-synuclein, ubiquitin, and lipids in Lewy
bodies Exp Neurol 166, 324–333.
49 Zhu, M & Fink, A.L (2003) Lipid binding inhibits a-synuclein
fibril formation J Biol Chem 278, 16873–16877.
50 Conway, K.A., Harper, J.D & Lansbury, P.T (1998) Accelerated
in vitro fibril formation by a mutant alpha-synuclein linked to
early-onset Parkinson disease Nature Med 4, 1318–1320.
51 Conway, K.A., Harper, J.D & Lansbury, P.T Jr (2000) Fibrils formed in vitro from alpha-synuclein and two mutant forms linked to Parkinson’s disease are typical amyloid Biochemistry 39, 2552–2563.
52 Narhi, L., Wood, S.J., Steavenson, S., Jiang, Y., Wu, G.M., Anafi,
D et al (1999) Both familial Parkinson’s disease mutations accelerate a-synuclein aggregation J Biol Chem 274, 9843– 9846.
53 Yang, D.S., Yip, C.M., Huang, T.H., Chakrabartty, A & Fraser, P.E (1999) Manipulating the amyloid-aggregation pathway with chemical chaperone J Biol Chem 274, 32970–32974.
54 Shtilerman, M.D., Ding, T.T & Lansbury, P.T (2002) Molecular crowding accelerates fibrillization of a-synuclein: could an increase
in the cytoplasmic protein concentration induce Parkinson’s dis-ease? Biochemistry 41, 3855–3860.