Toxic prefibrillar alpha-synuclein amyloid oligomers adopt a distinctive antiparallel beta-sheet structure.

ArticleinBiochemical Journal · January 2012with51 Reads
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Abstract

Parkinson's disease is an age-related movement disorder characterized by the presence in the mid-brain of amyloid deposits of the 140-aa protein alpha-synuclein (AS). AS fibrillation follows a nucleation polymerization pathway involving diverse transient prefibrillar species varying in size and morphology. Like for other neurodegenerative diseases, cytotoxicity is currently attributed to these prefibrillar species rather than to the insoluble aggregates. Nevertheless, the underlying molecular mechanisms responsible for cytotoxicity remain elusive and structural studies may contribute to the understanding of both amyloid aggregation mechanism and oligomer-induced toxicity. It is already recognized that soluble oligomeric AS species adopt beta-sheet structures that differ from those characterizing the fibrillar structure. In the present work we used ATR-FTIR spectroscopy, a technique especially sensitive to beta-sheet structure, to get deeper insight into the beta-sheet organization within oligomers and fibrils. Careful spectral analysis revealed that AS oligomers adopt an antiparallel beta-sheet structure whereas fibrils, a parallel arrangement. The data are discussed in terms of regions of the protein involved in the early beta-sheet interactions and the implications of such conformational arrangement for the pathogenicity associated to AS oligomers.

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Available from: Rabia Sarroukh
Biochem. J. (2012) 443, 719–726 (Printed in Great Britain) doi:10.1042/BJ20111924
719
Toxic prefibrillar α-synuclein amyloid oligomers adopt a distinctive
antiparallel β-sheet structure
Mar
´
ıa Soledad CELEJ*
1
, Rabia SARROUKH, Erik GOORMAGHTIGH, Gerardo D. FIDELIO*, Jean-Marie RUYSSCHAERT and
Vincent RAUSSENS
*Centro de Investigaciones en Qu
´
ımica Biol
´
ogica de C
´
ordoba (CIQUIBIC, UNC-CONICET), Departamento de Qu
´
ımica Biol
´
ogica, Facultad de Ciencias Qu
´
ımicas, Universidad Nacional
de C
´
ordoba, Haya de la Torre y Medina Allende, Ciudad Universitaria, X5000HUA C
´
ordoba, Argentina, and Centre for Structural Biology and Bioinformatics, Laboratory for Structure
and Function of Biological Membranes, Universit
´
e Libre de Bruxelles, CP 206/2, Blvd. du Triomphe, B-1050 Brussels, Belgium
Parkinson’s disease is an age-related movement disorder
characterized by the presence in the mid-brain of amyloid deposits
of the 140-amino-acid protein AS (α-synuclein). AS fibrillation
follows a nucleation polymerization pathway involving diverse
transient prefibrillar species varying in size and morphology.
Similar to other neurodegenerative diseases, cytotoxicity is
currently attributed to these prefibrillar species rather than to
the insoluble aggregates. Nevertheless, the underlying molecular
mechanisms responsible for cytotoxicity remain elusive and
structural studies may contribute to the understanding of both the
amyloid aggregation mechanism and oligomer-induced toxicity.
It is already recognized that soluble oligomeric AS species
adopt β-sheet structures that differ from those characterizing the
fibrillar structure. In the present study we used ATR (attenuated
total reflection)–FTIR (Fourier-transform infrared) spectroscopy,
a technique especially sensitive to β-sheet structure, to
get a deeper insight into the β-sheet organization within
oligomers and fibrils. Careful spectral analysis revealed that
AS oligomers adopt an antiparallel β-sheet structure, whereas
fibrils adopt a parallel arrangement. The results are discussed
in terms of regions of the protein involved in the early β-
sheet interactions and the implications of such conformational
arrangement for the pathogenicity associated with AS oligomers.
Key words: amyloidogenesis, amyloid oligomer, Parkinson’s
disease, secondary structure, structure–toxicity relationship,
α-synuclein.
INTRODUCTION
In the so-called protein deposition diseases, specific proteins
or peptides fail to adopt or remain in their native functional
conformations and subsequently aggregate in amyloid fibrils
with a canonical cross-β structure. These pathological conditions
include well-known debilitating neurodegenerative disorders,
such as Alzheimer’s and Huntington’s diseases and PD
(Parkinson’s disease) [1].
Accumulating evidence suggests that oligomeric intermediates,
rather than final insoluble aggregates, are the primary toxic
species [1]. Although the underlying molecular mechanism
remains elusive, toxicity pathways have been associated with:
(i) the establishment of aberrant protein interactions mediated
by the exposure of flexible hydrophobic surfaces [1,2]; (ii) the
propagation of folding defects by interfering with protein quality
control and clearance mechanisms [2,3]; and (iii) the impairment
of biomembranes [1,4]. These different patterns of phatobiology
are not mutually exclusive and may operate simultaneously.
It appears that oligomer-induced toxicity is related to their
structure rather than to their sequence, since prefibrillar aggregates
comprising non-disease-related proteins showed similar toxicity
in cell culture [5]. Besides, a variety of prefibrillar oligomers
share common structural epitopes [6] and toxicity of such
species seems to also correlate with the accessibility of ANS
(1-anilinonaphthalene 8-sulfonate)-binding hydrophobic patches
[2,7,8].
PD is an age-related movement disorder affecting more
than 2 % of the population over 65. PD is associated
with a dying back of axons projecting from the SNpC
(substantia nigra pars compacta) to the striatum, culminating
in a massive loss of dopaminergic neurons in the SNpC. The
histopathological hallmark of PD is the presence of fibrillar
intraneuronal proteinacious inclusions, termed Lewy bodies, the
main component of which is the 140-amino-acid presynaptic
protein AS (α-synuclein), whose normal function remains unclear.
Growing evidence suggests a link between misfolded AS and the
pathogenesis of PD as well as several other related disorders [9].
Structurally, recombinant human AS is a natively unfolded
monomer [10], with transient long-range domain interactions
stabilizing a closed conformation [11]. In the fibrillar form,
the monomers adopt a parallel in-register structure, with a β-
sheet-rich core region spanning residues 38–95, from which
the two termini are excluded [12–14]. A model for the fold of
AS fibrils has been proposed [15]. Fibrillation of AS follows
a nucleation polymerization pathway involving major structural
rearrangements and the population of diverse transient prefibrillar
species varying in size and morphology [16,17].
Compelling evidence points to AS oligomers being the most
neurotoxic species. It has been demonstrated that AS oligomers
disrupt membranes [18–21] and cause cell death in vitro [22,23]
and in animal models [24,25]. Additionally, certain AS aggregates
may be involved in the prion-like cell-to-cell spreading of the PD
pathology [26].
Despite their relevance in neurodegeneration and disease,
structural information about oligomeric AS is still limited.
Several technical limitations arise from the transient [17]
and heterogeneous [23,27] nature of oligomeric intermediates,
Abbreviations used: Aβ, amyloid-β peptide; AS, α-synuclein; ATR, attenuated total reflection; AVD, avidin; ConA, concanavalin A; FTIR, Fourier-transform
infrared; NAC, non-Aβ component; PD, Parkinson’s disease; SNpC, substantia nigra pars compacta; TBST, TBS/Tween 20; TEM, transmission electron
microscopy; ThioT, thioflavin T; TPI, triose phosphate isomerase.
1
To whom correspondence should be addressed (email mcelej@mail.fcq.unc.edu.ar).
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720 M. S. Celej and others
and the low yield in which they can be produced [18]. CD and IR
spectroscopies indicated that AS oligomeric intermediates contain
substantial β-sheet structure [18,28–30], although some α-helical
contribution has also been observed by Raman spectroscopy [31].
Also,
13
C cross-polarization solid-state NMR studies revealed that
AS oligomers obtained by cold-assisted dissociation of fibrils
have non-fibrillar β-sheet structure [21]. In addition, fluorescence
measurements on engineered tryptophan AS variants showed that
residues in the N-terminal and central part of the protein (towards
at least residue 90) comprise the core of oligomeric AS, the
C-terminal part being the most solvent-exposed region [32].
In the present study, we used ATR (attenuated total reflection)–
FTIR (Fourier-transform infrared) to gain insights into the
structure of AS oligomers. Unlike the above-mentioned structural
studies, we were able to discriminate between the β-sheet
organization within oligomers and fibrils. We demonstrate that
AS oligomers adopt an antiparallel β-sheet structure, as opposed
to the parallel arrangement present in fibrils. We discuss possible
regions that may be involved in such early β-sheet interactions.
As previously shown for oligomeric Aβ (amyloid-β peptide), a
peptide linked to Alzheimer’s disease [33], AS oligomers adopt
an antiparallel β-sheet structure that might represent a distinctive
signature of amyloid oligomers.
EXPERIMENTAL
Protein expression and purification
Recombinant human AS was expressed in Escherichia coli
BL21(D3) cells transformed with a pT7-7 plasmid encoding
for the protein. Bacterial cultures were incubated at 37
Cin
LB (Luria–Bertani) broth containing ampicillin (100 mg/ml). At
D
600
= 0.7, cells were induced with 0.5 mM IPTG (isopropyl β-D-
thiogalactopyranoside), cultured at 37
C for 4 h and centrifuged
at 4000 g for 15 min. The pellet was resuspended in 10 mM
Tris/HCl, pH 8.0, 1 mM EDTA and 1 mM PMSF, and lysed by
three freeze-thaw cycles and sonication (duty cycle 70, output
control 5, 25
C on a Branson Sonifier 250). The cell suspension
was boiled for 30 min and centrifuged at 13 000 g. Streptomycin
sulfate (10 mg/ml) was added to the supernatant, and the mixture
was stirred for 15 min at 4
C and centrifuged as above. Upon
addition of ammonium sulfate (360 mg/ml) to the supernatant,
the solution was stirred for 15 min at 4
C and centrifuged as
above. The pellet was resuspended in 25 mM Tris/HCl, pH 7.7,
loaded on to a POROS HQ (Applied Biosystems) column on an
¨
Akta purifier (GE Healthcare), and eluted with a 0–600 mM NaCl
salt gradient. The pure AS (assessed by SDS/PAGE) was dialysed
overnight against Milli-Q water or 10 mM Hepes, pH 7.4, and
0.02 % sodium azide, freeze-dried when needed, and stored at
20
C.
Preparation of monomeric and aggregated AS
Monomeric AS stock solutions were prepared in 10 mM Hepes,
pH 7.4, and 0.02 % sodium azide and centrifuged (14 100 g,
30 min) before use in order to remove possible aggregates. Protein
concentration was determined by absorbance using an ε
275
of 5600
M
1
· cm
1
.
Oligomerization protocols were adapted from previous studies
[18,30,31]. Monomeric AS stock solutions (250 μM) in 10 mM
Hepes, pH 7.4, were freeze-dried and redissolved using Milli-Q
water at a concentration of 1 mM, and then incubated for 19 h in
a Thermomixer5436 (Eppendorf) at 25
C and 800 rev./min (this
oligomeric preparation will be referred to as condition O-I for
the rest of the paper). The samples were centrifuged (14 100 g,
30 min) and filtered through a 0.22 μm-pore-size filter to remove
possible extremely high-molecular-mass species. Oligomeric AS
was separated from the monomer using an Amicon Ultra-0.5
100 kDa cut-off filter (Millipore). Diafiltration was repeated until
no monomer was detected by native gradient PAGE. Alternatively,
freeze-dried AS from Milli-Q water was dissolved in 10 mM
Hepes, pH 7.4 (condition O-II), and oligomeric species were
purified as described above.
Fibrillation was achieved by incubating 400 μM monomeric
AS stock solutions in glass vials (Zinsser Analytik) at
37
C (Dalvo incubator) with constant stirring at 350 rev./min
(Telesystem 15.20, Variomag) using Teflon magnetic microbars
(Roth) (condition F-I). Alternatively, AS solutions were incubated
at 70
C and 800 rev./min in a Thermomixer5436 (Eppendorf),
conditions that lead to faster aggregation kinetics [34] (condition
F-II). Fibril formation was monitored using the ThioT (thioflavin
T) fluorescence assay. Fibrils were isolated by three consecutive
cycles of centrifugation (14 000 g, 30 min) and resuspended in
10 mM Hepes buffer. Protein concentrations in monomeric units
were determined by the absorbance of aliquots incubated in 6 M
guanidinium chloride at 25
C for 24 h.
Dot blot
Monomeric, oligomeric or fibrillar AS (2 μg) were spotted on
to a nitrocellulose membrane and fixed for 10 min with 10 %
(v/v) acetic acid and 25% (v/v) propan-2-ol. After blocking
for 1 h with 10 % (w/v) non-fat dried skimmed milk powder in
0.01 % TBST (TBS/Tween 20; 10 mM Tris/HCl, pH 8, 150 mM
NaCl and 0.01% Tween 20) and washing, the membranes were
incubated with either a mouse anti-AS antibody (Sigma) for 3 h at
25
C (1:4000) or a rabbit anti-oligomer A11 antibody (Millipore)
overnight at 4
C (1:3000), both in 5 % (w/v) non-fat dried
skimmed milk powder and 0.01 % TBST. After washing three
times with 0.01 % TBST and once with PBS, blots were probed
with either IRDye 800CW anti-mouse IgG or IRDye 800CW anti-
rabbit IgG (both from Li-Cor Biosciences) and scanned using an
Odyssey infrared scanner (Li-Cor Biosciences).
Native gradient PAGE
Native polyacrylamide gels were cast with a linear gradient from
4to15%. HMW Native Marker (GE Healthcare) was loaded as
a molecular mass marker. Electrophoresis was performed under
non-denaturing conditions at a constant 80 V. Gels were stained
with silver nitrate.
ThioT fluorescence assay
Corrected emission spectra were acquired with a Cary Eclipse
spectrofluorimeter (Agilent Technologies), using excitation at
446 nm, spectral bandwidths of 10 nm and a 1 cm path cuvette.
Experiments were performed at 25
C using final concentrations
of 0.25 μM protein and 5 μM dye.
Seeding aggregation assay
The aggregation of AS was carried out in 25 mM Tris/HCl,
pH 7.4, and 150 mM NaCl. The protein was filtered through a
100 kDa cut-off filter (Amicon Ultra-0.5, Millipore) and aliquots
of 350 μl were incubated as described previously for fibrillation
condition F-I. For seeding experiments, oligomers O-I and O-II
and fibrils F-I were prepared as described above. Aggregation
reactions were seeded with purified oligomers O-I, O-II or
sonicated fibrils (5 min in a bath sonicator at 25
C), at a final
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Structural signature of α-synuclein oligomers 721
concentration of 8 μM (oligomer seeds) or 3 μM (fibrillar seeds).
In all cases, the total protein concentration was kept at 108 μM
in terms of monomeric units. The extent of aggregation was
monitored by the ThioT fluorescence assay as described above.
A total of two to four replicates were run independently. The
results were averaged and normalized to the averaged final values
obtained after curve fitting.
TEM (transmission electron microscopy)
Aggregated AS (5 μl) was adsorbed on to Formvar-coated carbon
grids (200 mesh). Grids were washed with Milli-Q water and
stained with 1 % (w/v) uranyl acetate. The samples were imaged
in a JEM-1200 Ex (Jeol) transmission electron microscope
equipped with a GATAN camera, model 785.
ATR–FTIR
IR spectra were recorded on an Equinox 55 IR spectrophotometer
(Bruker Optics) equipped with a single reflection diamond
reflectance accessory (Golden Gate, Specac). The spectrometer
was continuously purged with dried air. A total of 256
accumulations were performed to improve the signal/noise ratio.
Spectra were recorded at 21
C using a resolution of 2 cm
1
.
The sample (1.5–5 μl) was spread on the diamond crystal surface
and excess water was removed under nitrogen flow. The protein
films were rehydrated using nitrogen saturated in
2
H
2
O to obtain
deuterated protein spectra.
Secondary structure analysis
The water vapour and side chain contributions were subtracted and
then the spectra were baseline corrected and normalized for equal
area between 1700 and 1500 cm
1
. All spectra were deconvoluted
using a Lorentzian deconvolution factor with a full width at the
half maximum of 20 cm
1
and a Gaussian apodization factor
with a full width at the half maximum of 13.33 cm
1
to obtain
a resolution enhancement factor K = 1.5. The bands identified
in the deconvolved spectra were used for curve fitting of the
deuterated original (K = 1) spectra using Gaussian/Lorentzian
bands [35].
β-Sheet organizational index
For comparative analysis, FTIR spectra of well-known β-sheet
proteins were extracted from the RASP50 database [36]: AVD
(avidin) from hen egg white, ConA (concanavalin A) from jack
bean and TPI (triose phosphate isomerase) from Saccharomyces
cerevisiae. FTIR spectra of bacterial outer membrane porin
(OmpF), as well as oligomers (oAβ) and fibrils (fAβ) from Aβ(1–
42) were described in a previous study [33]. The Aβ peptide
was incubated at 4
C in 20 mM TBS, pH 7.4, and 100 mM
NaCl over 24 h for oligomerization or at room temperature in
0.5 mM Hepes, pH 7.4, over 36 days for fibrillation [33]. For
the sake of comparison, all baseline corrected, non-deuterated
and self-deconvoluted (using the same deconvolution parameters
as described above) spectra were fitted using the same eight
band components (these bands were centred, before fitting,
respectively at 1695, 1683, 1678, 1664, 1657, 1653, 1640 and
1624 cm
1
). Then, the so-called β-sheet organizational index’,
allowing differentiation between the parallel and antiparallel β-
sheet, was defined as the quotient between the intensity of the
fitted bands assigned to the high and low wavenumber β-sheet
components at their corresponding final (after fitting) positions.
For the different spectra, the final position of the high wavenumber
β-sheet component was located between 1693 and 1697 cm
1
,
and for the low wavenumber component, the final position varied
between 1624 and 1632 cm
1
(with the exception of TPI, which
was at 1641 cm
1
, refer to [35,36] and references therein for
details).
RESULTS AND DISCUSSION
Elucidating the conformational organization of oligomeric AS
is fundamental for understanding the amyloid aggregation
mechanism and oligomer-induced cell toxicity. It is already
recognized that soluble oligomeric AS species contain β-sheet
structural elements [18,30,31] that may differ from the typical
fibrillar structure [21,31]. In the present study, we used ATR–
FTIR, a technique especially sensitive to β-sheet structure, to
unravel the structural signature of AS oligomers.
Oligomer characterization
Several methods for producing stable AS oligomers have
been reported [18,21–23,30,31]. We used freeze-drying and
high protein concentration as a simple method to induce
oligomerization. This approach results in β-sheet oligomers that
exhibit vesicle disruption properties [18,30,31]. Since different
conditions could lead to structurally and functionally different
oligomeric species, we employed five complementary techniques
to characterize the oligomers produced.
The purity of the oligomeric preparations after diafiltration was
assessed by native PAGE electrophoresis (Figure 1A). Since the
migration of a protein in this system depends on its mass, charge
and conformation, the assignment of a correct molecular mass
is rather difficult. However, we could conclude that oligomeric
AS, observed as broad high-molecular-mass bands (Figure 1A,
lines 3 and 4) near the ferritin band ( 440 kDa, Figure 1A,
line 1), did not contain monomeric protein (Figure 1A, line
2). A similar electrophoretic pattern was reported previously
[30]. Moreover, the smeared oligomeric bands indicated that
these samples contain a broad size distribution of protein
aggregates.
The morphology of the aggregates was appraised by electron
microscopy. Fibrillar AS samples were unbranched and several
micrometres long, with widths of 6–10 nm (Figure 1B, bottom
panel) as expected for mature amyloid fibrils [12]. The same
morphological features were observed for fibrils prepared at 70
C
(results not shown), as reported previously [34]. On the other
hand, the purified oligomers appeared as spheroidal, polydisperse
species with sizes in the range 10–60 nm (Figure 1B, top and
middle panels), in agreement with PAGE data (Figure 1A).
Importantly, no fibrillar aggregates were observed in these
preparations.
Furthermore, neither oligomeric nor monomeric AS enhanced
ThioT fluorescence (Figure 1C), a sensitive amyloid dye that
exhibits significant changes in fluorescence upon intercalation
into the fibrillar core. As expected, ThioT became highly
fluorescent in the presence of AS fibrils (Figure 1C). Since there is
no evidence of the presence of fibrillar material in the oligomeric
samples, these results suggest that the produced AS oligomers lack
the stacked in-register β-sheet structure distinctive for amyloid
fibrils. This fact correlates with previous findings that soluble
on-pathway oligomers have a non-fibrillar β-sheet structure [21].
Moreover, the oligomeric species were recognized by the
A11 antibody (Figure 1D), a conformation-specific antibody
targeting oligomeric assemblies of many amyloidogenic proteins
[6]. Conversely, neither the monomer nor the fibrils showed
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722 M. S. Celej and others
Figure 1 Characterization of AS oligomers
(A) Nativegradient PAGEwith a polyacrylamide gradientfrom 4 to 15% stained with silvernitrate.
marker, native molecular marker (ferritin, 440 kDa; catalase, 232 kDa; lactate dehydrogenase,
140 kDa; and BSA, 66 kDa); AS M, monomeric AS; and AS O-I and AS O-II, oligomeric AS
obtained under oligomer-forming conditions O-I and O-II respectively. (B) TEM images of
uranyl-acetate-stained AS oligomers self-assembled under oligomer-forming conditions O-I
(top panel) and O-II (middle panel), and AS fibrils formed under fibril-forming condition F-I
(bottom panel). (C) Fluorescence emission spectra of ThioT in the presence of AS monomer
(-·-.), oligomers (oligomerization condition O-I, - -, and O-II, ···) and fibrils (fibrillation
condition F-I, continuous line). (D) Immunobloting with anti-AS and the A11 anti-oligomer
antibody against the same samples detailed in (C). (E) Kinetics of AS aggregation monitored
by ThioT fluorescence in the absence () and the presence of oligomer seeds (oligomerization
condition O-I, , and O-II, ) and fibrillar seeds ().
significant reactivity against A11 (Figure 1D). This indicates that
the oligomers produced share conformational features with other
toxic prefibrillar intermediates [6].
Finally, we tested whether the oligomeric species were able
to seed amyloid AS formation. Monomeric AS showed the
characteristic sigmoidal ThioT profile (Figure 1E), as expected for
a nucleation-polymerization process [16]. The time point of 50%
apparent conversion (t
1/2
) was approximately 118 h. Consistent
with this model [16], sonicated fibrils bypassed the lag-phase
and caused rapid aggregation (t
1/2
=∼5.5 h, Figure 1E). Addition
of oligomers O-I or O-II shortened the lag-phase for nucleation,
yielding an aggregation half-time of 97 h (Figure 1E). This
observation indicates that the oligomers produced are on-pathway
intermediates.
Overall, we conclude that the isolated AS oligomers are on-
pathway intermediates that share the same structural motif as other
prefibrillar oligomers and that they do not possess the canonical
cross-β-fibril structure.
Figure 2 Secondary structural features of AS oligomers and fibrils assessed
by ATR–FTIR
Normalized FTIR spectra in the amide I region of (A) AS oligomers formed under
oligomer-forming condition O-I, AS oligomers formed under oligomer-forming condition O-II
and oligomeric Aβ (oAβ); (B) the three selected antiparallel β-sheet proteins AVD, ConA
and OmpF; (C) AS fibrils formed under fibril-forming condition F-I, AS fibrils formed under
fibril-forming condition F-II and fibrillar Aβ (fAβ); (D) monomeric AS (AS M) and the parallel
β-sheet protein TPI. All spectrawere deconvoluted with a resolutionenhancement factor
K
= 1.5
and shifted for better visualization.
Secondary structural features of distinct AS species
IR spectroscopy has proven useful to discriminate between
parallel and antiparallel β-sheets, since the former shows a
major band component at 1640–1630 cm
1
, whereas the latter
displays a major band at 1630 cm
1
and an additional
approximately 5-fold weaker band at 1695 cm
1
[35,37,38].
The FTIR absorbance spectra showed distinctive features
depending on the aggregation state of the protein (Figure 2). The
spectrum of the monomeric protein was centred at 1650 cm
1
,
as expected for a substantially unfolded protein (Figure 2D). The
most striking feature of the amide I absorption band of AS in
the two oligomer-forming conditions was the presence of a band
at 1625 cm
1
along with a prominent shoulder at 1695 cm
1
(Figure 2A), the two characteristic components of an antiparallel
β-sheet structure. However, these features were absent in the
FTIR spectra of AS in the two fibril-forming conditions, which
displayed the typical parallel β-sheet features characterized by a
maximum at 1628 cm
1
(Figure 2C). These data clearly indicate
that fibrillar and prefibrillar aggregated amyloid species adopt
different structures, in agreement with recent NMR data reporting
on the non-fibrillar β-sheet nature of AS oligomers [21]. This band
assignment became unambiguously clear when we compared
the FTIR profiles of AS aggregated species with those of well-
known β-sheet-rich proteins, along with oligomeric and fibrilar
Aβ amyloid aggregates. First, AS oligomers were compared with
oligomeric Aβ (Figure 2A) as well as other antiparallel β-sheet
proteins, namely AVD, ConA and OmpF (Figure 2B), which
correspond respectively to the following architecture/topology:
β-barrel/Lipocalin, β-sandwich/Jelly Roll and β-barrel/Porin,
according to the CATH nomenclature. Secondly, AS fibrils were
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Structural signature of α-synuclein oligomers 723
compared with fibrillar Aβ (Figure 2C) and to the parallel β-
sheet protein TPI (Figure 2D), which is classified as an α/β-
barrel/TIM barrel protein according to the CATH nomenclature.
The visual inspection of the FTIR profiles shown in Figure 2
clearly confirms the different β-strand orientation adopted by
AS in the oligomeric and fibrillar forms. A baicalein-stabilized AS
oligomer FTIR spectrum has been published previously [28]. We
could notice that it presented the two characteristic IR components
of antiparallel β-sheets, although this structural conformation was
not suggested by the authors. In addition, similar spectral features
were observed for oligomers formed by Aβ(1–40) [39,40], a
prion-related peptide [41] and β2-microglobulin [42]. On the
basis of our results (the present study and [33]) and the examples
cited above, we postulate that an antiparallel β-sheet organization
might represent a common structural motif in amyloid oligomers.
In order to estimate the secondary structure content of each
species, we performed a quantitative analysis of the amide
I region on deuterated samples by self-Fourier deconvolution
followed by curve-fitting. The β-sheet content was lower than
8 % in AS monomers and increased up to approximately 26 %
in AS oligomers and 51 % in AS fibrils, in good agreement
with previous results obtained by vibrational spectroscopy
[28,31]. The progressive increase in β-sheet content was
accompanied by a concomitant decrease in random coil and/or
helical contribution (observed absorbance 1650–1660 cm
1
) from
60 % in monomers to 52 % in oligomers and 27 % in fibrils. In
both aggregated species, the β-turn content ( 1670 cm
1
)was
approximately 22 %. The estimates of secondary structure in AS
fibrils correlated with EPR and NMR data that revealed a folded
region (residues 34–101) containing extended stretches of
β-strands along with loops or turns, from which the two termini
protrude [13–15], whereas the N-terminus exhibits static disorder
from at least residue 22 onward, the C-terminus, starting from at
least residue 107, is unstructured and mobile [14].
Although the accuracy of the determination of secondary
structure composition is prone to some error, the amide I profiles
of AS oligomers and fibrils unequivocally indicate a change in
the proportion and quality of the β-structures (Figure 2). The
1695/1630 cm
1
intensity ratio can be considered as proportional
to the percentage of antiparallel arrangement of β-strands in
β-sheets [38]. Since the precise positions of the β-sheet band
components depend on the number, length and twisting of the
strands [35] (see, for example, the upshifted spectrum for TPI in
Figure 2D), we defined the β-sheet organizational index’ as the
intensity ratio of the corresponding high and low wavenumber
regions assigned to β-sheets (see the Experimental section). The
calculated values for this index for the samples studied included
inFigure2aredepictedinFigure3.Theβ-sheet organizational
indexes for AS oligomers were high and close to those calculated
for the three selected mainly β proteins (Figure 3), whose
β-strands are clearly arranged in an almost complete antiparallel
fashion. This suggests that 100 % of the β-strands in AS oligomers
also adopt an antiparallel conformation, similar to what was found
for Aβ oligomers [33,40]. In contrast, the corresponding indexes
for both types of AS fibrils were low and similar to the values
determined for TPI and Aβ fibrils, which are folded in parallel
β-sheet structures, in perfect agreement with the EPR results
reporting on the parallel in-register AS fibril conformation [13].
The β-sheet antiparallel oligomer organization in the context
of current knowledge on AS
Three functional domains are distinguished in the AS sequence
(Figure 4A): (i) the amphipatic N-terminus (residues 1–60),
Figure 3 β-Sheet organizational index of AS oligomers and fibrils
Intensity ratio between the high and low wavenumber β-sheet components for AS and Aβ
amyloid aggregates and representative proteins as detailed in Figure 2 (see the Experimental
section for details).
which bears the three genetic mutations linked to early onset of
PD; (ii) the central hydrophobic region (residues 61–95) known
as the NAC (non-Aβ component); and (iii) the highly acidic
C-terminus (residues 96–140) that modulates fibril formation.
In the monomeric state, the C-terminus folds back on to the
central hydrophobic region of the protein, establishing long-
range interactions that prevent aggregation [11]. On the basis
of a number of NMR determinations, five β-strands (namely β1–
β5, encompassing residues 37–43, 52–59, 62–66, 68–77 and 90–
95 respectively, Figure 4B) have been assigned to the AS fibril
core [15]. Intramolecular interactions between the consecutive
β-strands would contribute to the formation of the five-layered β-
sandwich that gives rise through intermolecular interactions to the
parallel in-register β-sheet fibril structure [15] (Figure 4C). The
sequence encompassing β3–β5 corresponds to the amyloidogenic
NAC region (Figure 4B). Indeed, the contiguous β3–β4 stretch
bears the highest predicted β-sheet [43] and aggregation [44]
propensities. In addition, β4 overlaps with the span of 12 residues
(from Val
71
to Val
82
) identified as a key element for the assembly
of the protein into fibrils [45].
Fluorescence determinations on a number of engineered
tryptophan AS variants revealed that the region spanning residues
from at least 4 to 90 comprise the solvent-protected area in
the oligomeric state [32]. However, the exact regions involved
in β-sheet interactions are unknown. Since β3andβ4are
prone to aggregate and, upon fibrillation, they are located in
the centre of the fibril, we could expect them to form stable
parallel β-sheets. In addition, bearing in mind that the release
of long-range interactions constitutes a pre-requisite to trigger
fibrillation [11], we speculate that regions in the protein involved
in nucleation and early stages of oligomerization may differ
from those that constitute the inner core of the fibrils. In order
to prompt aggregation and allow reorganization of the β-sheet
network, these regions should display propensity to establish
inter-molecular β-sheet interactions and, more likely, be located
in the periphery of the AS fibril. Both β1andβ5 would
fulfill such criteria [15,43,44]. Since β5 is highly shielded by
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724 M. S. Celej and others
Figure 4 Schematic representation of AS primary sequence, self-assembly and toxicity
(A) AS functional domains. The N-terminal amphipathic region contains the three point mutations linked to autosomal dominant early-onset PD. The central NAC region encompasses the most
hydrophobic residues and promotes aggregation. The C-terminal portion modulates aggregation. (B) β-Strand localization in the core fibrillar region according to [15]. The NAC region is represented
in bold type. (C) Schematic representation of AS aggregation and oligomer-induced toxicity. A significant rearrangement of the protein structure, involving the population of oligomeric species,
occurs during fibrillogenesis. Whereas amyloid fibrils are folded in a parallel in-register β-sheet structure, β-strands in AS oligomers are arranged in an antiparallel fashion. Oligomeric species
may be trapped by small molecules (green hexagons) that preferentially target regions encompassing residues 3–23 and/or 38–51 and inhibit fibril formation. Oligomers may expose hydrophobic
regions that can mediate aberrant interactions with multifunctional proteins (yellow circles), leading to the collapse of essential cellular functions. As another possible co-existent mechanism, amyloid
oligomers can compromise the integrity of membranes, probably through their antiparallel β-sheet-structured regions.
interactions with the C-terminus [11], we postulate that the region
enclosing β1 is probably involved in early β-sheet interactions.
In this context, we note that several small-molecule inhibitors of
fibrillogenesis preferentially interact with regions encompassing
residues 3–23 and/or 38–51 [46–48], where Tyr
39
plays a
major role in anchoring the anti-amyloid compounds [47,48].
Interestingly, some of these inhibitors trap AS oligomers that do
display β-sheet structure to some extent [28,48]. Thus they seem
to bias the structural ensemble in a fashion that constrains β-sheet
elongation and reorientation. In addition, a Trp
39
AS variant was
sensitive to the build-up of oligomers formed during the lag-phase
of fibrillation [49]. Finally, fluorescence resonance energy transfer
measurements between Tyr
39
and Trp
125
pointed to conformational
differences between oligomers populated at different stages of
aggregation [29]. Taken together, these results speak in favour
of the key role of the β1 region in the early steps of AS amyloid
formation. In addition, to account for the percentage of β-structure
content estimated from fitting analysis, other regions of the protein
must participate in such a structural arrangement. It has been
shown recently that disruption of potential specific salt bridges in
adjacent regions to β1andβ2 led to the formation of oligomers
which were highly toxic in vivo [25]. Thus one could expect β2
to also be involved in these oligomeric β-sheet interactions.
The inherent cytotoxicity of oligomers was postulated to be a
general phenomenon related to their structure [5]. Major efforts
are currently being directed at unravelling the structural basis of
gain-of-function toxicity associated with amyloid oligomers, and
mounting evidence suggests that they share structural similarities
that would underlie their common pathways of pathobiology
[7,22]. For instance, it has been proposed that the exposure of
hydrophobic surfaces [7,8] would endow oligomers with the
ability to engage in aberrant interactions with multifunctional
proteins leading to the collapse of essential cellular functions [2].
In this regard, the higher proportion of random/helical structures
found in AS oligomers as compared with fibrils speaks in favour of
such mechanism and correlates with the higher toxicity associated
with the prefibrillar intermediates.
On the other hand, several reports indicate that AS oligomers
compromise the integrity of membranes both in vitro and
in vivo [18,21,23,25,30]. The molecular bases of such effects are
still a matter of debate and diverse mechanistic models, i.e. pore
formation, membrane thinning and lipid extraction, have been
proposed (recently reviewed in [4]). On the basis of our findings
that AS oligomers adopt an antiparallel β-sheet conformation, a
structural feature also present in other prefibrillar amyloid species
[33,39,41,42], we suggest that this conformational motif is central
in the membrane-disruptive properties displayed by amyloid
oligomers. In this context, it is worth noting that a porin-like
structure was suggested for Aβ oligomers on the basis of striking
spectral similarities between these species and the pore-forming
OmpF porin [33]. Additionally, it has been shown that the A11
antibody recognizes amyloidogenic oligomers, as well as the pore-
forming proteins α-haemolysin and human perforin [50], both of
which self-assemble as β-barrels in biomembranes. Therefore,
on the basis of the A11 reactivity (Figure 1) and our FTIR data
(Figure 3), we hypothesize that the β-sheet-structured motif in
AS oligomers might adopt a porin-like fold. In order to form such
a supramolecular structure, each AS molecule would provide (at
least) two β-strands that self-assemble as an antiparallel β-barrel
which, although it spans the membrane, would place the unordered
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Structural signature of α-synuclein oligomers 725
regions towards one side of the bilayer. A cartoon representation
of such an arrangement is included in Figure 4(C).
The various aspects of AS amyloid self-assembly and
oligomer-induced toxicity discussed above are schematically
depicted in Figure 4(C). Long-range interactions maintain
the protein in its monomeric state. During self-assembly,
antiparallel β-sheet-rich oligomers are formed, probably through
intermolecular interactions mediated by regions enclosing β1and
β2. Oligomeric species are trapped by several small-molecule
inhibitors that target the region near Tyr
39
, preventing the
structural reorganization required for fibril growth. Oligomers
may compromise cell viability via protein–protein and/or protein–
membrane interactions mediated by structurally distinct regions
of AS.
In summary, although our approach using ATR–FTIR cannot
compete with NMR determinations in terms of atomic-scale
resolution, we were able to show for the first time that on-pathway
AS oligomers adopt a distinct antiparallel β-sheet structure, a
signature that might be shared by other amyloid oligomers,
underlying their common membrane-disrupting pathogenic
action. In addition, the unordered structure may be central in
defining the AS oligomer interactome, leading to the impairment
of essential cellular functions. Therefore our findings provide
fundamental knowledge on the structural organization within AS
oligomers important for the understanding of oligomer-induced
toxicity and for the development of therapeutic agents.
AUTHOR CONTRIBUTION
M. Soledad Celej, Jean-Marie Ruysschaert and Vincent Raussens designed the research.
M. Soledad Celej and Rabia Sarroukh performed the research. M. Soledad Celej, Rabia
Sarroukh and Vincent Raussens analysed the results. M. Soledad Celej, Rabia Sarroukh,
Erik Goormaghtigh, Gerardo D. Fidelio, Jean-Marie Ruysschaert and Vincent Raussens
discussed the results and wrote the paper.
ACKNOWLEDGEMENTS
We thank Dr T.M. Jovin (Laboratory of Cellular Dynamics, Max Planck Institute for
Biophysical Chemistry, G
¨
ottingen, Germany) for providing the pT7-7 plasmid encoding
the AS protein, A. Valiente, G. Lamberto, Dr C. Fernandez and Dr C. Bertoncini for insightful
discussions, Dr C. Nome for technical assistance on TEM imaging, G. Heim and Dr D.
Riedel for providing TEM grids and discussion on electron micrographs.
FUNDING
This work was supported by the Secretaria de Ciencia y T
´
ecnica-Universidad Nacional de
C
´
ordoba (SECyT-UNC), Consejo Nacional de Investigaciones Cient
´
ıficas y Tecnol
´
ogicas
(CONICET) [grant number PIP 2011-2013 GI 11220100100012], Fundaci
´
on Florencio
Fiorini (year 2010), Alexander von Humboldt Foundation (equipment grant to M.S.C.),
Fondo para la Investigaci
´
on Cient
´
ıfica y Tecnol
´
ogica (FONCyT) [grant number PICT 34084
(to G.D.F.)] and Ministerio de Ciencia, Tecnolog
´
ıa e Innovaci
´
on Productiva-Fonds de la
Recherche Scientifique (MINCyT-FNRS) [grant number BE0903 (to G.D.F. and E.G.)].
M.S.C. and G.D.F. are Researcher Career members of CONICET. V.R. and E.G. are Senior
Research Associate and Research Director at the National Funds for Scientific Research
(Belgium) respectively.
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Received 28 October 2011/30 January 2012; accepted 8 February 2012
Published as BJ Immediate Publication 8 February 2012, doi:10.1042/BJ20111924
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    • "This would imply reorientation of monomer units as an obligatory step before formation of the fibril. However , at least one report demonstrates that toxic prefibrillar amyloid aggregates adopt an antiparallel orientation [93], and in this sense we cannot draw any analogy to this pseudo-equilibrium dimer population that exists with the fibril accessible monomer ensemble, and how the monomers therein may interact with it. As the monomeric ensemble is shifted towards more fibril prone conditions, previously described conformational changes (Section 2.1), as well as changes in population of oligomeric species occurs. "
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  • [Show abstract] [Hide abstract] ABSTRACT: The toxicity of amyloids is a subject under intense scrutiny. Many studies link this toxicity to the existence of various intermediate structures prior to the fiber formation and/or their specific interaction with membranes. Membranes can also be a catalyst of amyloidogenesis and the composition or the charge of membrane lipids may be of particular importance. Despite intensive research in the field, such intermediates are not yet fully characterized probably because of the lack of adapted methods for their analyses, and the mechanisms of interaction with the membrane are far to be understood. The purpose of this mini-review is to highlight some in vitro characteristics that seem to be convergent to explain the toxicity observed for some amyloids. Based on a comparison between the behavior of a model non-toxic amyloid (the Prion Forming Domain of HET-s) and its toxic mutant (M8), we could establish that short oligomers and/or fibers assembled in antiparallel β-sheets strongly interact with membrane leading to its disruption. Many recent evidences are in favor of the formation of antiparallel toxic oligomers assembled in β-helices able to form pores. We may also propose a new model of amyloid interaction with membranes by a "raft-like" mode of insertion that could explain important destabilization of membranes and thus amyloid toxicity.
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