Stabilization of neurotoxic Alzheimer amyloid-beta oligomers by protein engineering.
ABSTRACT Soluble oligomeric aggregates of the amyloid-beta peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the conformation adopted by Abeta within these aggregates is not known, a beta-hairpin conformation is known to be accessible to monomeric Abeta. Here we show that this beta-hairpin is a building block of toxic Abeta oligomers by engineering a double-cysteine mutant (called Abetacc) in which the beta-hairpin is stabilized by an intramolecular disulfide bond. Abeta(40)cc and Abeta(42)cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Abeta aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Abetacc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that beta-sheet-containing Abeta(42)cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Abeta(42), in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Abeta oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Abeta(42) is more toxic than the shorter Abeta(40), and why certain inherited mutations are linked to protofibril formation and early-onset AD.
- SourceAvailable from: M. Mahafuzur Rahman[Show abstract] [Hide abstract]
ABSTRACT: The progressive neurodegeneration in Alzheimer's disease is believed to be linked to the presence of prefibrillar aggregates of the amyloid-β (Aβ) peptide in the brain. The exact role of these aggregates in the disease pathology is, however, still an open question. Any mechanism by which oligomeric Aβ may cause damage to neuronal cells must, in one way or another, involve interactions with other molecules. Here, we identify proteins in human serum and cerebrospinal fluid that bind to stable protofibrils formed by an engineered variant of Aβ42 (Aβ42CC). We find that the protofibrils attract a substantial number of protein binding partners. Many of the 101 identified proteins are involved in lipid transport and metabolism, the complement system or in hemostasis. Binding of representative proteins from all these groups with micromolar affinity was confirmed using surface plasmon resonance. In addition, binding of apolipoprotein E to the protofibrils with nanomolar affinity was demonstrated. We also find that aggregation of Aβ enhances protein binding, as lower amounts of proteins bind monomeric Aβ. Proteins that bind to Aβ protofibrils might contribute to biological effects that these aggregates are involved in. Our results therefore suggest that improved understanding of the mechanisms by which Aβ causes cytotoxicity and neurodegeneration might be gained from studies carried out in biologically relevant matrices in which Aβ-binding proteins are present.ACS Chemical Biology 12/2014; · 5.36 Impact Factor
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ABSTRACT: Many factors are known to influence the oligomerisation, fibrillation and amyloid formation of the Aβ peptide that is associated with Alzheimer's Disease. Other proteins that are present when Aβ peptides deposit in vivo, are likely to have an effect on these aggregation processes. In order to separate specific vs. broad-spectrum effects of proteins on Aβ aggregation, we tested a series of proteins not reported to have chaperone activity: catalase, pyruvate kinase, albumin, lysozyme, α-lactalbumin, and β-lactoglobulin. All tested proteins suppressed the fibrillation of Alzheimer's Aβ(1-40) peptide at sub-stoichiometric ratios, albeit some more effectively than others. All proteins bound non-specifically to Aβ, stabilised its random coils and reduced its cytotoxicity. Surprisingly, pyruvate kinase and catalase were at least as effective as known chaperones in inhibiting Aβ aggregation. We propose general mechanisms for the broad-spectrum inhibition Aβ fibrillation by proteins. The mechanisms we discuss are significant for prognostics and perhaps even for prevention and treatment of Alzheimer's Disease.Journal of Biological Chemistry 08/2014; · 4.60 Impact Factor
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ABSTRACT: A critical step of β-amyloid fibril formation is fibril elongation in which amyloid-β monomers undergo structural transitions to fibrillar structures upon their binding to fibril tips. The atomic detail of the structural transitions remains poorly understood. Computational characterization of the structural transitions is limited so far to short Aβ segments (5-10 aa) owing to the long timescale of Aβ fibril elongation. To overcome the computational timescale limit, we combined a hybrid-resolution model with umbrella sampling and replica exchange molecular dynamics and performed altogether ∼1.3 ms of molecular dynamics simulations of fibril elongation for Aβ17-42. Kinetic network analysis of biased simulations resulted in a kinetic model which encompasses all Aβ segments essential for fibril formation. The model not only reproduces key properties of fibril elongation measured in experiments, including Aβ binding affinity, activation enthalpy of Aβ structural transitions and a large timescale gap (τlock/τdock = 103-104) between Aβ binding and its structural transitions, but also reveals detailed pathways involving structural transitions not seen before, namely fibril formation both in hydrophobic regions L17-A21 and G37-A42 preceding fibril formation in hydrophilic region E22-A30. Moreover, the model identifies as important kinetic intermediates strand-loop-strand (SLS) structures of Aβ monomers, long suspected to be related to fibril elongation. The kinetic model suggests further that fibril elongation arises faster at the fibril tip with exposed L17-A21, rather than at the other tip, explaining thereby unidirectional fibril growth observed previously in experiments.Journal of the American Chemical Society 08/2014; · 11.44 Impact Factor
Stabilization of neurotoxic Alzheimer amyloid-β
oligomers by protein engineering
Anders Sandberga, Leila M. Luheshib, Sofia Söllvanderc, Teresa Pereira de Barrosb, Bertil Macaoa, Tuomas P. J. Knowlesb,
Henrik Biverståld, Christofer Lendeld, Frida Ekholm-Pettersonc, Anatoly Dubnovitskyd, Lars Lannfeltc,
Christopher M. Dobsonb, and Torleif Härdd,1
aInstitute of Biomedicine, University of Gothenburg, SE-405 30 Gothenburg, Sweden;bDepartment of Chemistry, University of Cambridge, Cambridge CB2
1EW, United Kingdom;cDepartment of Public Health and Caring Sciences/Geriatrics, Uppsala University, SE-751 25 Uppsala, Sweden; anddDepartment of
Molecular Biology, Swedish University of Agricultural Sciences (SLU), Uppsala Biomedical Center, SE-751 24 Uppsala, Sweden
Edited by Alan R. Fersht, Medical Research Council Centre for Protein Engineering, Cambridge, United Kingdom, and approved July 13, 2010 (received for
review February 12, 2010)
Soluble oligomeric aggregates of the amyloid-β peptide (Aβ)
have been implicated in the pathogenesis of Alzheimer’s disease
(AD). Although the conformation adopted by Aβ within these
aggregates is not known, a β-hairpin conformation is known to
be accessible to monomeric Aβ. Here we show that this β-hairpin
is a building block of toxic Aβ oligomers by engineering a double-
cysteine mutant (called AβCC) in which the β-hairpin is stabilized by
an intramolecular disulfide bond. Aβ40CC and Aβ42CC both sponta-
neously form stable oligomeric species with distinct molecular
weights and secondary-structure content, but both are unable to
convert into amyloid fibrils. Biochemical and biophysical experi-
ments and assays with conformation-specific antibodies used to
detect Aβ aggregates in vivo indicate that the wild-type oligomer
structure is preserved and stabilized in AβCC oligomers. Stable
oligomers are expected to become highly toxic and, accordingly,
we find that β-sheet-containing Aβ42CC oligomers or protofibrillar
species formed by these oligomers are 50 times more potent in-
ducers of neuronal apoptosis than amyloid fibrils or samples of
monomeric wild-typeAβ42, in which toxicaggregates areonly tran-
siently formed. The possibility of obtaining completely stable and
physiologically relevant neurotoxic Aβ oligomer preparations will
facilitate studies of their structure and role in the pathogenesis of
AD. For example, here we show how kinetic partitioning into dif-
ferent aggregation pathways can explain why Aβ42is more toxic
than the shorter Aβ40, and why certain inherited mutations are
linked to protofibril formation and early-onset AD.
amyloid-β peptide|protein aggregation|protein structure|protofibril|
the brain and several such aggregates have been described (1, 2).
tissue of humans as 70 kDa or larger aggregates containing Aβ
dimers (3), or from transgenic AD mice as smaller aggregates
called Aβ*56 (4). Toxic Aβ oligomers and protofibrils have also
been made in vitro, such as oligomeric ADDLs prepared by di-
lution from organic solvents (5, 6), smaller globulomers formed in
SDS-containing solvents(7), oligomersformed in water at lowpH
(8, 9), and larger nonfibrillar aggregates known as protofibrils (10,
11). However, it is not clear if and how these different aggregates
are related to the pathogenesis of AD. Indeed, although there is
a large body of data on the conformation and “cross-β” packing of
Aβ in amyloid fibrils (12, 13), which are the end-products of ag-
gregation, little is known about the basic building blocks of the
engineering to address these issues and to provide a method to
stabilize toxic Aβ oligomers for structural and functional studies.
Monomeric Aβ does not adopt a unique conformation in water
solution. Nevertheless, NMR experiments (14) and molecular
modeling (15) suggest that the central and C-terminal hydrophobic
regions of Abeta have a propensity to form extended beta-strand
lzheimer’s disease (AD) is linked to the formation of neuro-
toxic oligomeric aggregates of the amyloid-β peptide (Aβ) in
conformations with a connecting turn between them. Such a
“hairpin” conformation (Fig. 1A) is in fact also induced when Aβ
forms a complex with a phage-display selected Affibody-binding
protein (16, 17). The hairpin is topologically similar to the con-
formation of Aβ in fibrils. However, there is a distinct difference
in that the hydrogen bonds are intramolecular, resulting in an-
tiparallel β-strands, whereas they are intermolecular in fibrils,
resulting instead in parallel β-sheets.
We have previously proposed that intermediate oligomeric
a conformational change results in the formation of Aβ subunits
polymerization into amyloid fibrils (17). There is also experi-
mental evidence for the hairpin in oligomers of the globulomer
kind (18), as well as for antiparallel β-sheet secondary structure in
We therefore set out to test if stabilizing Aβ in the hairpin con-
formation observed in the Affibody complex would promote the
formation of oligomeric aggregates but not fibrils, and whether
such stabilized oligomers would possess antigenic and neurotoxic
characteristics similar to those of wild-type Aβ oligomers found
Protein Engineering. The structure of Aβ40in complex with the
suitable for disulfide engineering that would constrain it in its
hairpin conformation. Ala21 and Ala30 are ideally suited for this
purpose, as their β-carbons are located on opposite β-strands at
showed that a Cys21/Cys30 disulfide can be accommodated with
favorable conformational energy and without perturbing the
hairpin structure in which 12 backbone hydrogen bonds are pre-
dicted to form in Aβ40CC.
We produced AβCC in Escherichia coli bacteria by coexpres-
sion with the ZAβ3 Affibody (20). This Affibody binds Aβ in the
hairpin conformation, which allows for the formation and purifi-
cation of monomeric AβCC with an intramolecular disulfide bond.
Recombinant Aβ produced in this way contains an N-terminal
methionine residue, but this does not affect its biophysical prop-
Author contributions: A.S. and T.H. designed research; A.S., L.M.L., S.S., T.P.d.B., B.M.,
T.P.J.K., H.B., C.L., and A.D. performed research; F.E.-P. and L.L. contributed new re-
agents/analytic tools; A.S., L.M.L., S.S., T.P.d.B., T.P.J.K., H.B., C.L., A.D., and T.H. analyzed
data; and A.S., L.M.L., L.L., C.M.D., and T.H. wrote the paper.
Conflict of interest statement: A.S. and T.H. are shareholders of MIVAC Development AB,
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: Torleif.Hard@molbio.slu.se.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 31, 2010
| vol. 107
| no. 35
AβCC with an Intact Disulfide Bond Cannot Form Amyloid Fibrils. Our
hairpin hypothesis postulates that AβCC should not form amyloid
fibrils as long as the intramolecular disulfide is intact, and experi-
ments show that this is indeed the case. Thioflavin T (ThT) fluo-
of tris-2-carboxyethyl-phosphine (TCEP) reducing agent to break
electron microscopy (TEM) confirms that oxidized Aβ40CC forms
oligomeric aggregates, whereas reduced Aβ40CC forms fibrils (Fig.
oxidized state (Fig. S1) but, as described below, instead forms
β-sheet containing oligomers and protofibrils.
Formation of Oligomers with Different Secondary-Structure Content.
Blocking of fibril formation allows for the enrichment of oligo-
meric aggregates that are otherwise only transiently formed by
wild-type Aβ (1, 2). Purified Aβ40CC and Aβ42CC were denatured
into monomeric form in 7 M guanidinium chloride and subjected
to size exclusion chromatography (SEC) with native (nonde-
naturing) phosphate buffer at pH 7.2 as running buffer. Different
oligomers then form spontaneously during SEC. The SEC chro-
matograms of Aβ40CC and Aβ42CC reveal the separation of dis-
tinct oligomeric species with apparent molecular weights of ∼16,
∼30, ∼50, and ∼100 kDa, in addition to monomer (at 8–10 kDa)
kDa) eluting at the void volume (Fig. 2 A and B). Aβ42CC and
Aβ40CC form oligomers with similar molecular weights and higher
concentration favors the formation of larger oligomers of both
species, as expected (Fig. S2). Sometimes, ∼185-kDa oligomers
are also observed; these are not well separated in SEC, but can be
distinguished in the SEC profiles in Fig. 2B and Fig. S1A.
Circular dichroism (CD) spectroscopy (Fig. 2C) reveals that
Aβ40CC and Aβ42CC oligomers with an apparent molecular weight
of ∼100 kDa contain ∼40% β-sheet secondary structure (β-sheet
oligomers). Low molecular-weight (LMW) oligomers display
a “random coil” CD spectrum similar to that of a disordered pep-
tide and therefore lack regular secondary structure. LMW oligo-
mers may, however, convert into either β-sheet oligomers or
HMW aggregates and β-sheet oligomers may assemble further
into protofibrils (Fig. 2D), as described below. The presence of
antiparallel β-sheet in the larger oligomers was confirmed by
Fourier transform infrared (FTIR) spectroscopy (Fig. S3).
Differences Between Aβ40CC and Aβ42CC Oligomerization. There is
aclear difference betweenthe aggregation patterns ofAβ40CCand
and Fig. S2). However, Aβ40CC LMW oligomers form β-sheet
oligomers when they are concentrated to ∼0.6 mM and subjected
to a 10-min heat treatment at 60 °C (Fig. 2C and Fig. S4). Hence,
a kinetic barrier for formation of β-sheet oligomers is more eas-
ily overcome by Aβ42CC than by Aβ40CC. This finding explains
the different size distributions observed in SEC of Aβ42CC and
Aβ40CC (Fig. 2A) and it may also explain differences in toxicity, as
described in the Discussion.
β-Sheet Oligomers Contain SDS-Stable Dimers and Trimers. Aggre-
gated Aβ isolated from the brains of AD patients contains neu-
rotoxic dimeric and trimeric species that are either formed in
SDS-containing solutions or are resistant to denaturation in SDS/
PAGE electrophoresis experiments. It has been suggested that
such species may constitute building blocks of toxic Aβ aggregates
(3, 18). If this suggestion is true and if the aggregates formed by
AβCC are structurally identical to wild-type aggregates, then one
would expect AβCC to form SDS-stable dimers. We find that di-
meric and trimeric bands of purified but unfractionated Aβ42CC
are more prominent in SDS/PAGE than those of wild-type Aβ42.
But more importantly, ∼100 kDa β-sheet oligomer fractions of
Aβ42CC areonlyobserved asdimer andtrimerunits inSDS/PAGE
with Coomassie staining (Fig. 2E). The absence of a monomer
band implies that dimers and trimers form in the oligomers and
remain stable in SDS-containing buffers.
β-Sheet Oligomers Can Form Protofibrils. Pooling and concentrating
SEC fractions containing Aβ42CC or Aβ40CC β-sheet oligomers
results in the formation of higher-order aggregates (Fig. 2D),
which are distinct from the HMW aggregates observed to elute in
the void of the SEC profiles in Fig. 2A and B (also shown by
antibody assays below). TEM images of such samples (Fig. 2F)
show spherical oligomers with an average diameter of ∼6 nm, as
well as longer and curved aggregates with the same diameter. The
morphology of the larger aggregates is such that they appear to be
assemblies of the 6-nm oligomers. Their size and appearance are
also very similar, if not indistinguishable, to those of protofibrils
figure 1C in the work of Goldsbury et al. (figure 1C in ref. 21).
A globular protein, which in SEC elutes at a volume corre-
sponding to that of the β-sheet oligomers (∼100 kDa) will have
a diameter of ∼6 nm, in agreement with the size of the small
spherical oligomers. A molecular weight of ∼100 kDa per 6-nm
oligomer subunit in AβCC protofibrils is also close to the observed
mass per length of wild-type Aβ protofibrils: 114 ± 12 kDa per
6 nm (21). Hence, it appears that the smaller particles in Fig. 2F
are β-sheet oligomers and that these associate to form proto-
fibrils. Aβ40CC β-sheet oligomers also form protofibrils upon con-
Wild type Aβ
Aβ40CC + 5 mM TCEP
Wild type Aβ40
(Aβ40CC + 10 mM TCEP)
observed in complex with an Affibody binding protein (17). Nonpolar side
chains at the two hydrophobic faces are shown as sticks and colored yellow
and orange, respectively. The Ala21 and Ala30 methyls are located in close
proximity on opposite β-strands. (Right) Model of the AβA21C/A30C double
mutant (AβCC) in which the β-hairpin conformation is locked by a disulfide
bond. (B) ThT fluorescence assays of Aβ40CC aggregation in the absence or
presence of TCEP reducing agent compared with wild-type Aβ40aggrega-
tion. (C) TEM micrographs of β-sheet oligomers of Aβ40CC (Left) and of fibrils
formed in presence of TCEP (Right).
Protein engineering. (A) (Left) The β-hairpin conformation of Aβ40
| www.pnas.org/cgi/doi/10.1073/pnas.1001740107 Sandberg et al.
centration. Dilution of protofibril samples followed by SEC sug-
gest that they are stable (Fig. 2D). Such diluted samples, which we
neurotoxicity experiments described in the following section.
Recognition of AβCC by a Physiologically Relevant Conformation-
Specific Antibody. We used two different conformation-specific
antibodies that bind wild-type Aβ aggregates to examine if their
binding epitopes are preserved in AβCC and to shed light on how
the many AβCC aggregates are related. The mAb158 monoclonal
antibody (22) was selected based on its affinity for protofibrils of
Aβ42carrying the Arctic mutation (11). The mAb158 antibody
alsorecognizesprotofibrils ofwild-type Aβ42,aprotofibrillar form
that is present in the medium of APP-expressing cells, and Aβ
aggregates in brains of transgenic mice (22). ELISA experiments
show that β-sheet oligomers/protofibrils of Aβ42CC bind mAb158
with the same affinity, or better, than wild-type protofibrils (Fig.
3A). This assay is conformation-specific because monomeric
Aβ42CC or SDS-treated β-sheet oligomers are not recognized by
mAb158 (Fig. 3B). β-Sheet oligomers of Aβ40CC that have been
converted from LMW oligomers by concentration and heat treat-
ment are also recognized by mAb158 (Fig. S4). Hence, β-sheet
AβCC oligomers expose a mAb158 conformational epitope that is
also present in wild-type Aβ protofibrils in vitro and in vivo.
AβCC Can Access Two Distinct Aggregation Pathways. In addition to
oligomers and protofibrils, AβCC also forms HMW aggregates
contain both large amorphous aggregates and long protofibrils
(Fig. S5), the latter of which may account for mAb158 binding
observed for this fraction (Fig. 3B). Interestingly, the HMW SEC
fractions also bind the A11 antibody (Fig. S6), which also is
conformation-specific (23). However, β-sheet oligomers/proto-
fibrils of AβCC that bind mAb158 do not bind A11, and these are
therefore structurally distinct from the HMW aggregates. To id-
entify which oligomeric species represent the precursors of the
A11 binding HMW aggregates of AβCC, we aged both LMW and
β-sheet oligomer fractions of Aβ42CC at 37 °C. (LMW oligomers
were kept in diluted form to avoid the transformation into β-sheet
oligomers). Within a few days, LMW oligomer samples of Aβ42CC
assemble into large, stable A11-positive aggregates with a di-
ameter of 19 to 25 nm, whereas β-sheet oligomers do not form
A11-binding species even after 8 wk of aging (Fig. 3C and Fig. S6).
is different from that of mAb158: mAb158 binds an epitope pre-
Absorbance at 280 nm (mAU)
Absorbance at 280 nm (mAU)
Apparent molecular weight (kDa)
Elution volume (mL)
Absorbance at 280 nm (mAU)
Apparent molecular weight (kDa)
Elution volume (mL)
190 200210 220230240250
Mean residue ellipticity (deg cm2 dmol-1)
ca. 650 kDa
LMW LMW LMW HMW
oligomers during SEC on a Superdex 200 PG 16/600 column. Monomer peptide samples were loaded in denaturing buffer and eluted with native phosphate
buffer at pH 7.2. Apparent molecular weights and classification of eluted oligomers have been indicated. HMW aggregates elute with the void volume.
Sample amounts: 2.7 mg Aβ40CC and 1.7 mg Aβ42CC; see also Fig. S2. (B) SEC as in A showing separation of Aβ40CC oligomers on a Superdex 75 PG 16/60 column
on which smaller aggregates become better separated. Sample amount: ∼ 3 mg. (C) CD (mean residue ellipticity) of SEC fractions pooled as indicated by
shaded areas in A and B. Dashed lines: 8-kDa monomer (gray, 12 μM), 30-kDa LMW oligomers (blue, 16 μM), and 96-kDa β-sheet oligomers (red, 8 μM) of
Aβ40CC. Green: ∼100-kDa β-sheet oligomers of Aβ42CC (13 μM). Solid blue line: the 30-kDa LMW oligomer fraction of Aβ40CC after concentration and heat
treatment showing formation of β-sheet oligomers. (D) SEC of concentrated Aβ42CC β-sheet oligomers (1 mL, 145 μM), which form protofibrils with an average
apparent molecular weight of ∼ 650 kDa. The dotted line shows that these are smaller than HMW aggregates in A. (E) SDS/PAGE of wild-type Aβ42(Left),
purified but unfractionated Aβ42CC (Center), and Aβ42CC β-sheet oligomers formed during SEC as in A (Right). The right lanes in all panels contain Aβ samples,
and other lanes contain high and low molecular weight standards (HMW and LMW). Weights corresponding to monomer and SDS-resistant dimers and
trimers have been indicated. The loading buffer contained 2.5 mM TCEP (heat-stable reducing agent) to completely break all disulfide bonds. (F) TEM mi-
crograph of a concentrated sample of Aβ42CC β-sheet oligomers (190-μM monomer concentration) showing assembly of β-sheet oligomers into protofibrils.
Biophysical and biochemical characterization of AβCC oligomers and protofibrils. (A) Formation and separation of Aβ42CC (black) and Aβ40CC (red)
Sandberg et al.PNAS
| August 31, 2010
| vol. 107
| no. 35
sent in aggregates containing Aβ42CC or Aβ40CC β-sheet oligomers,
whereas A11 binds aggregates that are formed by LMWAβ40CC or
two aggregation pathways for AβCC with different end products:
the β-sheet pathway, involving aggregation of β-sheet oligomers
into protofibrils (which are detected in the mAb158 ELISA), and
the coil pathway, involving aggregation of monomer or disordered
LMW oligomers into HMW A11-binding aggregates (which are
also detectable in vivo)(23). (The naming of theaggregation path-
ways reflects the secondary-structure content of the originating
Neurotoxicity of AβCC Oligomers. The neurotoxicity of different
AβCC aggregates was assayed by measuring their ability to induce
apoptosis, indicated by the level ofcaspase-3/7 activity in a human
neuroblastoma cell line, SH-SY5Y. Aβ42CC aggregates pooled
from SEC all induce apoptosis 24 h after addition to the cells, but
or HMWfractions ofAβ42CC.In fact, the toxicity is comparable or
higher than that of wild-type Aβ42oligomers, as one would expect
for a stabilized toxic aggregate (Fig. 4A). Nerve cell apoptosis is
dose-dependent and measurable after 24 h of treatment at 1-μM
peptide concentrations and the β-sheet Aβ42CC oligomers are 50
times more potent than wild-type Aβ42monomer or amyloid fib-
ril samples (Fig. 4B). In contrast, the equivalent monomer and
LMW oligomer fractions of Aβ40CC do not induce apoptosis.
However, when Aβ40CC LMW oligomers are transformed to
β-sheet oligomers/protofibrils, as described above, these start to
induce caspase activity, but at lower levels compared with Aβ42CC
(Fig. 4C). The neurotoxicity experiments therefore suggest that
the actively toxic species are β-sheet oligomers of Aβ42CC and
Aβ40CC, and larger protofibrillar aggregates that form when these
are concentrated, as they have to be before the apoptosis assays.
The results are also consistent with the lower propensity of
Aβ40CC monomers to form β-sheet oligomers and these are, once
formed, intrinsically less toxic than those of Aβ42CC. The fact that
LMW oligomers and HMW aggregates of Aβ42CC also induce
apoptosis (as shown in Fig. 4A) does not contradict that β-sheet
oligomers/protofibrils are the primary toxic species, because LMW
and β-sheet oligomers are not completely separated in SEC
(Fig. 2 A and B) and large protofibrils are present in HMW
fractions (Fig. S5). Overall, the neurotoxicity of different Aβ42CC
and Aβ40CC aggregates (Fig. 4) matches specific recognition in the
mAb158 ELISA(Fig. 3Aand B,andFig. S4),indicating that toxic
aggregates formed by AβCC are to be found along the β-sheet
Do AβCC Oligomers Mimic Wild-Type Aβ Oligomers? AβCC contains
alanine to cysteine replacements, at positions 21 and 30, designed
tostabilizea β-hairpin conformationby forming anintramolecular
disulfide cross-link. The hairpin is predicted to comprise residues
17 to 23 and 30 to 36 as antiparallel β-strands connected by a turn
involving residues 25 to 29. There is considerable evidence that
such a conformation is accessible in monomeric Aβ: (i) it forms in
complex with binding proteins (16, 17), (ii) its secondary structure
elements persistently appear in computer simulations of different
Aβ fragments (15, 24, 25), and (iii) NMR data suggest that turn
previously postulated that metastable Aβ oligomers contain hair-
pin subunits and that conversion into a related cross-β conforma-
runaway aggregation that is typical of amyloid fibril formation.
Here we test if soluble Aβ oligomers indeed consist of hairpins
as constituent monomer building blocks and, if this is the case, if
hairpin stabilization then provides a method for stabilization of
physiologically relevant oligomers. We find that a Cys21-Cys30
disulfide prevents the formation of amyloid fibrils, resulting in-
stead in the formation of stable oligomeric aggregates and pro-
tofibrils. These aggregates are indistinguishable from wild-type
Aβ aggregates in TEM and they contain the dimeric and trimeric
SDS-resistant units that are regarded as fingerprints ofneurotoxic
Aβ in vivo. Conformation-specific antibodies that recognize wild-
typeAβin thebrainsofAD patients(23)andtransgenic mice(22)
also recognize AβCC oligomers. Finally, β-sheet oligomers and
protofibrillar species formed by Aβ42CC or Aβ40CC are powerful
inducers of apoptosis in neuronal cells in culture. AβCC oligomers
are therefore biologically functional, and we propose that they
constitute an appropriate model for structural and functional
studies of oligomers relevant to the pathogenesis of AD. AβCC
may potentially also be used for therapy development based on
immunization or for small-molecule drug discovery.
Architecture of Neurotoxic Aβ Oligomers and Protofibrils. The con-
formation of AβCC in toxic β-sheet oligomers is most likely a
β-hairpin, as shown in Fig. 1A, but β-hairpin conformations in
which the turn is shifted from residues 25 to 29 toward, for in-
stance, 24 to 28 or 23 to 27 may in fact also be compatible with
a Cys21/Cys30 disulfide. The proposition that toxic Aβ oligomers
are built of hairpin monomer subunits arranged to form larger
050 100 150200250
124 8 weeks
20 uM Aβ42CC β-sheet oligomers
014 7 days
50 uM Aβ42CC LMW oligomers
specific for wild-type Aβ aggregates. (A) mAb158 antibody sandwich ELISA
detection (22) of Aβ42CC β-sheet oligomers/protofibrils compared with de-
tection of wild-type Aβ42protofibrils. (B) mAb158 ELISA analysis of different
Aβ42CC species at 83 pM total peptide concentrations. (C) Aging of Aβ42CC
LMW and β-sheet oligomers at 37 °C followed by A11 antibody dot blot
analysis showing that A11 binding aggregates form from LMW oligomers
but not from β-sheet oligomers. Concentrations were equalized before
Recognition of AβCC oligomers by antibodies that are conformation
| www.pnas.org/cgi/doi/10.1073/pnas.1001740107Sandberg et al.
units of antiparallel β-sheet secondary structure is in agreement
with other observations. Infrared spectroscopy shows that anti-
parallel (and not parallel) β-sheet is present in oligomeric ag-
gregates (19, 27). A β-hairpin similar to what is described here is
also observed in synthetic Aβ globulomers (18). Aβ species with
a mass equivalent to that of a dimer has been directly linked to
disease (3). It is not certain that these are chemically identical to
dimers observed in vitro, but the present data linking neuro-
toxicity, β-sheet oligomers, and SDS-resistant dimers and trimers
are consistent with such a view.
Our experiments are not conclusive with regard to the precise
molecular weight of a minimal β-sheet oligomer from which pro-
tofibrils assemble. SEC elution volumes suggest ∼100 kDa, but
SEC columns are calibrated with globular protein standards and
any disorder will result in a larger Stokes radius and an over-
estimate of the molecular weight. For example, the disordered
nature of monomeric Aβ makes it appear larger in SEC than
tetramers, and hexamers of AβCC, respectively, or if some other
stoichiometry applies. However, when also considering that dis-
ordered regions may remain in the ∼100-kDa β-sheet oligomers,
these should contain at least 12 monomer subunits. Hence, a
dodecamer stoichiometry of toxic Aβ oligomers that has been fa-
vored in other research (4, 28–30) is consistent with our data.
Electron microscopy images indicate that β-sheet oligomers of
∼6 nm in diameter assemble into protofibrils of variable lengths,
thereby implying that the protofibrils are composed of β-sheet
oligomers. AβCC protofibrils are morphologically very similar
to wild-type protofibrils. These protofibrils are also recognized
in the mAb158 monoclonal antibody ELISA (22), which is
conformation-specific for protofibrils of wild-type Aβ42.
With regard to the structural differences between β-sheet and
LMW oligomers, it is possible that the latter either do not form
β-hairpins with intramolecular hydrogen bonds (despite the disul-
fide) or that hydrogen-bonded β-hairpins do not assemble into
larger β-sheet structures in LMW oligomers.
Differences Between Aβ40and Aβ42Aggregation and Toxicity. We
find that Aβ42CC more readily forms β-sheet oligomers than
Aβ40CC. This occurs as a result of the presence of a kinetic barrier
to oligomer formation that can only be overcome by Aβ40CC with
the aid of heating and concentration. Wild-type Aβ42is consid-
ered to be more toxic than Aβ40. It is possible that this difference
in toxicity reflectsdifferent barriersto β-sheetoligomer formation
that we observe here with AβCC.
ways of wild-type Aβ42and Aβ40(29) and multiple pathways for
in which monomeric Aβ can form disordered LMW oligomers or
larger β-sheet oligomers, and that LMW oligomers can convert
into β-sheet oligomers. β-Sheet oligomers can associate into pro-
tofibrils recognized in the mAb158 ELISA. LMW oligomers, on
Caspase-3/7 activity (x 104)
Change in caspase-3/7 activity
vs. buffer (%)
and HMW aggregates
Caspase-3/7 activity (x 104)
samples, except β-sheet oligomers/protofibrils in C, were prepared and iso-
lated by SEC, as in Fig 2A, concentrated to ∼75 to 250 μM in phosphate
buffer at pH 7.2, and added to cell cultures at 1-, 5-, or 10-μM concentrations.
Caspase-3/7 activity reporting on apoptosis was measured after 24 h of
treatment. (A) Aβ42CC induced apoptosis following treatment with 10 μM of
different species. Blue: comparison of different Aβ42CC species. Green: Aβ42CC
oligomers compared with wild-type Aβ42oligomers in another experiment.
(B) Dose-dependence of apoptosis induced by Aβ42CC species compared with
that of wild-type Aβ42monomer and fibrils and Aβ42E22G. (C) Apoptosis
induced by different Aβ40CC species and wild-type Aβ40. The sample marked
“Aβ40CC LMW oligomers” is a 75-kDa SEC oligomer fraction. The Aβ40CC
β-sheet oligomers/protofibrils were formed by pooling and concentrating
monomer and LMW oligomer SEC fractions and heating the concentrated
sample as described in the text and in Fig. S4.
Neurotoxicity of AβCC to SH-SY5Y human neuroblastoma cells. AβCC
oligomers without regular secondary structure and eventually large non-
fibrillar aggregates binding the A11 antibody (coil pathway; Upper) and the
other involves assembly into β-sheet oligomers, or coil oligomers that are
converted into β-sheet oligomers, which are building blocks of mAb158
binding protofibrils (β-sheet pathway; Lower). Neurotoxic Aβ aggregates are
formed along the β-sheet pathway. The scheme is consistent with the pres-
ent studies of AβCC and overall features can be reconciled with a large body
of work on wild-type and naturally occurring Aβ mutants as discussed in the
text. Red arrows reflect the interconversion of Aβ subunits from β-hairpin
conformation in soluble aggregates to cross-β structure in fibril seeds and
mature amyloid fibrils.
Aβ aggregation via two pathways. One pathway involves LMW
Sandberg et al. PNAS
| August 31, 2010
| vol. 107
| no. 35
in different populations of the two peptides (at a given concen-
tration) aggregating along the LMW oligomer → A11-binding
HMW-aggregate pathway and the mAb158-binding β-sheet oligo-
mer → protofibril pathway.
It is not clear from which aggregate fibril seeds are formed in
AβCC after the addition of reducing agent. However, protofibrils
have been suggested to constitute the penultimate intermediate
in amyloid fibril formation (1, 10), and fibril formation can also
occur in solutions containing A11-binding aggregates (31). In the
reaction scheme in Fig. 5, we therefore allow for seed formation
by both these aggregates. Still, the aggregation equilibria also
allow for other possibilities, such as seeding directly from small
β-sheet oligomers as suggested previously (17), or by secondary
nucleation events (32).
Possible Link Between β-Sheet Oligomer Formation and Early-Onset
AD. Arguments relating toxicity to aggregation pathway may be
extended to include inherited mutations in Aβ that are linked to
higher toxicity and early-onset AD. For example, Aβ with the
Arctic E22G mutation forms protofibrils more rapidly than wild-
type Aβ (11), which in our aggregation scheme is equivalent to
a higher rate of β-sheet oligomer formation (aggregation along
the β-sheet pathway). In accordance with such a mechanism, the
AβE22G peptide more easily forms SDS-resistant higher-order
aggregates (20), which we also associate to β-sheet oligomers. An
experimental linkage between kinetic partitioning of mutant Aβ
peptides into different aggregation pathways and their toxicity
would, if it can be further substantiated, constitute an attractive
mechanism to rationalize the relationship between inherited mu-
tations and early-onset AD.
We have engineered an Aβ peptide variant (AβCC) in which a
β-hairpin is stabilized by a disulfide bond. AβCC forms oligomeric
aggregates in which conformational and biological properties of
wild-type Aβ oligomers are preserved, and amyloid formation is
prevented. Aβ42CC and Aβ40CC both aggregate via two distinct
pathways that can be distinguished using conformation-specific
antibodies. The coil pathway involves formation of less structured
LMW oligomers with apparent molecular weights of 50 kDa or
less, which can aggregate further into nonfibrillar HMW species.
The β-sheet aggregation pathway involves initial formation of
100 kDa or more, which also contain SDS-stable dimeric and tri-
meric units. β-Sheet oligomers can associate into protofibrils that
are morphologically identical to wild-type protofibrils and detec-
ted in a mAb158 antibody ELISA, which is specific for wild-type
protofibrils. Aggregates on the β-sheet aggregation pathway are
neurotoxic and induce neuronal apoptosis. A kinetic barrier for
formation of β-sheet oligomers makes Aβ40CC more prone to ag-
of Aβ40CC induce neuronal apoptosis as well. Kinetic partitioning
into two aggregation pathways in which one contains the neuro-
toxic aggregates may explain why Aβ40is less toxic than Aβ42and
why certain Aβ mutations lead to early onset AD.
Materials and Methods
Complete discussions of molecular modeling, peptide production and puri-
fication, SDS/PAGE, fibril formation assays, CD, infrared spectroscopy, TEM,
atomic force microscopy, mAb158 ELISA, A11 dot blot, and neurotoxicity
assays are in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank Professor Andreas Barth at Stockholm
University for providing expertise on FTIR spectroscopy. This work was
supported by Grants 2008-3475 (to T.H.) and 2006-2822 (to L.L.) from the
Swedish Research Council, grants from the Medical Research Council and
Engineering and Physical Sciences Research Council (to C.M.D. and L.M.L.),
grants from the Wellcome Trust (to C.M.D), and grants from Hjärnfonden,
Alzheimerfonden, and Uppsala University Hospital (to L.L.). T.H. and A.S. are
part of the Mucosal Immunobiology and Vaccine Center supported by the
Swedish Foundation for Strategic Research.
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| www.pnas.org/cgi/doi/10.1073/pnas.1001740107Sandberg et al.