Co-incorporationofAb40andAb42toformmixed pre-fibrillar
aggregates
David Frost
1
, Paul M. Gorman
1
, Christopher M. Yip
2
and Avijit Chakrabartty
1
1
Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, and
2
Department of Chemical Engineering and Applied Chemistry, Institute of Biomaterials and Biomedical Engineering,
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
Senile plaques, the invariable hallmark and likely proximal
cause of Alzheimer’s disease (AD), are structured deposi-
tions of the 40- and 42-residue forms of the Ab peptide.
Conversely, diffuse plaques, which are not associated with
neurodegeneration, consist mainly of unstructured Ab42.
We have investigated the interaction between Ab40 and
Ab42 through an assay, which involves labeling both vari-
ants with an environment-sensitive fluorophore. We have
monitored association of Ab without fibrillar seeds, which
allows investigation of molecular species preceding fibrils.
Immediately upon mixture, Ab40andAb42 associate into
mixed aggregates, in which the peptides are unstructured and
relatively accessible to water. When left to incubate for an
extended period, larger, more tightly packed aggregates,
which show secondary structure, replace the small,
unstructured aggregates formed earlier. Our results show
that in vitro the two Ab variants coassemble early in the
fibrillogenesis pathway. The ease of formation for mixed and
homogeneous aggregates is similar. A change in the local Ab
variant ratio can therefore have a significant impact on Ab
aggregation; indeed such a change has been reported in some
types of familial AD.
Keywords: Alzheimer’s disease; amyloid; fibril; peptide;
fluorescence.
Alzheimer’s disease (AD) is a significant and increasing
health concern. Neurohistological studies have uncovered
several hallmarks that distinguish the AD brain from its
normal counterpart. Chief among these are neurofibrillary
tangles (NFTs) andsenile plaques (SPs). NFTs are composed
of paired helical filaments of the (normally) microtuble-
associated Tau protein, while senile plaques are primarily
comprised of the 40- and 42-residue forms of the Ab peptide
[1,2]. The interaction between Ab40and Ab42, the two major
variants of the Ab peptide, is the subject of this study.
The Ab family of peptides is enzymatically cleaved from
the amyloid precursor protein (APP), a 563–770 residue
membrane protein that is expressed in both neuronal and
non-neuronal tissue [3,4]. Ab40, andto a lesser extent
Ab42, are normal constituents of cerebrospinal fluid
[5–8]. Both forms are capable of assembling into
60–100 A
˚
diameter b-sheet fibrils, which form the core of
the aforementioned senile plaques.
An impressive body of evidence points to Ab depo-
sition in senile plaques as the causal event in AD
pathology. Upon postmortem examination of AD brains,
senile plaques are invariably found in the limbic and
association cortices, surrounded by dead or dying
neurons, as well as activated microglia and reactive
astrocytes [1]. In several forms of familial AD, mutations
in the APP gene have been identified [9,10]. Also,
mutations of the presenilin genes have been linked to
familial AD, and appear to lead to an increase in the
ratio ofAb42toAb40 [11]. Transgenic mice over
expressing a mutant formof APP develop neurohistolo-
gical characteristics similar to those of AD patients
[12–14]. Perhaps most convincingly, Down’s syndrome
patients, who receive a triple dose of the genes present on
chromosome 21, including the APP gene, often show
senile plaque deposition and classical AD neurohistology
in their late 20s or early 30s, followed by progressive
cognitive and behavioral dysfunction in their mid 30s [15].
Unlike senile plaques, diffuse plaques are more loosely
packed depositions of mostly unstructured Ab42 [1].
Diffuse plaques are not associated with dead or dying
neurons, and have been found upon post mortem exami-
nation of the brains of elderly people who had not
exhibited AD symptoms [16–20]. Diffuse plaques are also
referred to as Ôpreamyloid plaquesÕ because of several lines
of evidence that point to them as precursors to senile
plaques. In the Down’s syndrome patients discussed earlier,
diffuse plaques are observed as early as age 12 years [21].
Similarly, mice transgenic for mutant human APP also
develop diffuse Ab42 plaques before fibrillar plaques
surrounded by dead and dying neurons [12–14].
Correspondence to A. Chakrabartty, Division of Molecular and
Structural Biology, Ontario Cancer Institute and Department of
Medical Biophysics, University of Toronto, 610 University Avenue,
Toronto, Ontario, Canada, M5G 2M9.
Fax: + 416 9466529, Tel.: + 416 9464501 ext. 4910,
E-mail: chakrab@uhnres.utoronto.ca
Abbreviations:Ab, Alzheimer beta amyloid; AD, Alzheimer’s disease;
AFM, atomic force microscopy; APP, amyloid precursor protein;
EDANS, ethyldiaminonaphthalene-1-sulfonic acid;
NFT, neurofibrillary tangle; SP, senile plaque.
(Received 13 September 2002, revised 27 November 2002,
accepted 5 December 2002)
Eur. J. Biochem. 270, 654–663 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03415.x
The observation that Ab42 diffuse plaques lead to senile
plaques, consisting of both Ab variants, and the apparent
importance of the Ab40/Ab42ratioinAD[22]haveledus
to investigate the interaction between these two peptides.
Studies by Hasegawa et al.[23]havedemonstratedthe
ability of preformed Ab42 fibrils to seed the fibrillogenesis of
Ab40, as well as the ability ofAb40to seed Ab42
fibrillogenesis. However, with little conclusive evidence
that Ab fibrils are neurotoxic, more attention is being
paid to prefibrillar Ab species in the search for a clear
culprit in AD pathology [24]. Recent work by our group
[25] as well as others [26–28], has shown a complex series
of reactions, which precedes the formation of mature
fibrils. To our knowledge, no study has been undertaken
to determine at which point in the fibrillogenesis pathway
Ab40 andAb42 can formmixed molecular species, and
the relative ease of formation ofmixed vs. homogenous
species.
We believe that the approach of studying fibrillogenesis in
the context ofAb40andAb42 mixtures is advantageous.
Studies are usually of either Ab40 or Ab42, while it is known
that in vivo, both Ab40andAb42 are present, and their
interaction may play a key role in the transition between
relatively innocuous diffuse plaques and possibly neurotoxic
senile plaques. Furthermore, many in vitro studies examine
fibril formation without rigorously removing all fibril seeds,
thereby making it impossible to characterize all species
preceding fibrils. In the present study, we ensure a homo-
geneous starting solution of monomeric Ab peptides,
thereby permitting an examination of the interaction
between Ab40andAb42 throughout the fibrillogenic
pathway.
Materials and methods
Peptide synthesis
A PerSeptive Biosystems 9050 Plus peptide synthesizer
was used to separately prepare both Ab40andAb42 by
solid phase peptide synthesis. An active ester coupling
procedure, employing O-(7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate of 9-fluorenyl-
methoxycarbonyl amino acids was used. The magnitude
of the syntheses was 0.05 mmol, and a three times excess
of reagents was used. The peptides were cleaved from the
resin with 95 : 5 trifluoroacetic acid and anisol mixture.
The cleavage mixture was incubated at room temperature
for 30 min, and the resin removed by filtration. Bromo-
trimethylsilane was added to a final concentration of
12.5% (v/v). The peptides were then precipitated and
washed five times in cold ether. The peptides were
removed from the ether and dissolved in 6
M
guanidine
hydrochloride, 0.15% NH
4
OH (pH 10) and purified by
HPLC. The purified peptide was chromatographed on a
Sephadex G-75 column (Amersham Pharmacia Biotech)
and the fractions corresponding to the correct monomer
molecular mass collected. Electrospray mass spectrometry
confirmed the presence of the correct molecular mass, and
purity was determined by six cycles of PTH peptide
sequencing by the Edman degradation reaction (Porton
gas-phase Microsequencer, model 2090), which revealed
that the purified peptides had the correct sequence.
Sequencing proceeds from the N- to the C-terminus,
while automated synthesis proceeds in the opposite
direction. By confirming that the main peptide present
has the correct N-terminal sequence, purity is established,
as any errors in synthesis usually result in a truncated
N-terminus.
Fluorescent labeling
A glycine residue was added to the N-terminus of both
Ab40 andAb42 prior to addition of the fluorophore. This
acts as a flexible linker to prevent the fluorophore from
interfering with the normal behavior of the peptides.
Ethyldiaminonaphthalene-1-sulfonic acid (EDANS; Mole-
cular Probes) was then coupled to the glycine linker.
Purification was performed as above, and sequencing
revealed the major peptide present in each synthesis to be
the correct labeled Ab, with a minor contaminant of
unlabeled Ab.
Preparation of stock peptide solutions without fibril
seeds
After chromatographic separation as described above, both
the labeled and unlabeled peptides were separately stored
at pH 10 at 4 °C until use. This method of stock storage
has previously been used by us [25] and others [29] to
successfully prevent the formation of fibril seeds.
Peptide concentration determination
For unlabeled Ab peptides, tyrosine absorbance of UV
light (275 nm) was used to determine concentration
in 0.15% NH
4
OH by Beer–Lambert law (e ¼ 1390
cm
)1
Æ
M
)1
[30]). Each concentration obtained was multi-
plied by the appropriate dilution factor to obtain stock
concentrations. The EDANS-labeled peptide stock con-
centration was determined by EDANS absorbance at
338 nm (e ¼ 6500 cm
)1
Æ
M
)1
[31]). This method of con-
centration determination was used because it ensures the
correct concentration of labeled peptide is obtained, and
is not affected by the minor unlabeled Ab contaminant
described above. All absorbance measurements were
made on a Milton-Roy Spectronic 3000 spectrophoto-
meter.
Sample preparation
All samples were measured at pH 7 with 40 m
M
phos-
phate buffer. Into Eppendorf tubes, first the amount of
unlabeled Ab stock (stored at pH 10, 4 °C) appropriate
for each concentration tested was added. Stock peptide
concentrations were 0.212 m
M
for Ab40and 0.165 m
M
for Ab42. As the final solution volume was 500 lL, each
increment of 10 l
M
unlabeled Ab40 required the addition
of 23.6 lL of stock. Similarly, each 10 l
M
increment of
unlabeled Ab42 required 30.3 lL of stock. Next, the
labeled Ab was added to the solution. Stock concentra-
tions were 8.65 l
M
for EDANS-Ab40 and 31.9 l
M
for
EDANS-Ab42, both stored at pH 10, 4 °C. All solutions
tested that included labeled Ab had a concentration of
0.1 l
M
EDANS-Ab. Therefore, for the solutions with
Ó FEBS 2003 Pre-fibrillary association between Ab40andAb42 (Eur. J. Biochem. 270) 655
EDANS-Ab40, 5.8 lL of stock was added. Similarly, for
solutions with EDANS-Ab42, 1.6 lL of stock was added.
Deionized water was then added to each sample, such
that the final volume was 300 lL. A 100-m
M
phosphate-
buffered solution was prepared, and separated into
aliquots, with pHs ranging from 6.8 to 7.0 in increments
of 0.05 pH units. Two hundred microliters of the buffer
with appropriate pH to make the final pH near 7.0 (i.e.
compensate for the addition of pH 10 Ab stocks) was
added, and 0.1 m
M
NaOH or HCl was added to make
the pH exactly 7.0, just prior to measurement. In most
cases the final adjustment involved no more than 1–2 lL
of acid or base, and therefore had negligible effect on the
final volume. This pH adjustment just prior to measure-
ment ensures that the fibrillogenesis process does not start
before measurements are taken. Controls (i.e. unlabeled
Ab alone and EDANS-Ab + hen lysozyme) were pre-
pared exactly as above, with the exception that the hen
lysozyme (Sigma Chemical) stock was kept at pH 7, at
1.57 m
M
,4°C. As the hen lysozyme stock was kept at
pH 7, a phosphate buffer of the same pH was added for
all samples.
Sample incubation
All samples were incubated for approximately 3 months
at room temperature. They were kept in a dark area to
prevent photobleaching of EDANS.
Fluorescence spectroscopy
Fluorescence assays were carried out at room temperature
using a Photon Technology International QM-1 fluores-
cence spectrophotometer equipped with excitation intensity
correction and a magnetic stirrer. All samples were scanned
in a quartz cuvette with 2 mm path length in the excitation
direction and 1 cm path length in the emission direction.
Total sample volume was 0.5 mL. All constituents of the
samples (i.e. buffer, water, unlabeled Ab and labeled Ab)
were first screened for the presence of fluorescent contami-
nants, and only the labeled Ab stocks exhibited EDANS
fluorescence. To measure EDANS fluorescence, emission
spectra were collected from 360 to 600 nm (k
ex
¼ 350 nm;
step size ¼ 1nm; 2sÆnm
)1
; bandpass ¼ 2nm). After
obtaining the spectra, the control of unlabeled Ab alone (of
appropriate concentration) was subtracted in order to
correct for the effect of light scattering by large aggregates.
The resultant spectrum was then integrated over the
wavelengths of 400–550 nm. In order to correct for daily
variation in the UV lamp and slight variations in bandpass,
as well as the minor unlabeled contaminant of EDANS-Ab
stocks described above, the fluorescence of EDANS-Ab
alone was subtracted from all other measurements, giving a
normalized measure of the fluorescence of EDANS-Ab at
different concentrations over time. All fluorescence experi-
ments were conducted in three different trials on different
days.
Circular dichroism spectroscopy
CD spectra were recorded on an Aviv Circular Dichroism
Spectrometer model 62DS at 25 °C. Spectra were obtained
from 190 to 260 nm (1 mm path length, 1 nm step size,
1 nm bandwidth).
Atomic force microscopy
All solution tapping atomic force microscopy images
were acquired using a combination contact/tapping mode
liquid cell fitted to a Digital Instruments Nanoscope IIIA
MultiMode scanning probe (Digital Instruments, Santa
Barbara, CA, USA). The AFM images were acquired
using the E scanning head, which has a maximum lateral
scan area of 14.6 · 14.6 lm. Samples were made by
diluting the appropriate Ab stocks with 100 m
M
phosphate buffer (pH 7). Five microliters of the mixed
sample solution were transferred onto a freshly cleaved
mica surface, and the sample was sealed in the liquid cell.
Sizes and volumes were calculated using Digital Instru-
ments’
NANOSCOPE
software (version 4.21) and the
shareware image analysis program
NIH
-
IMAGE
(version
1.62).
Results
Monitoring Ab association
We have employed a variation on the strategy used by
our group to monitor Ab40 fibrillogenesis [32]. First, the
Ab40 andAb42 peptides were synthesized separately.
EDANS, an environment-sensitive fluorophore, was
added to the N-terminus of aliquots of both Ab40 and
Ab42, separated from the rest of the sequence by a
glycine linker. Samples of 0.1 l
M
EDANS-labeled
Ab40 (AED-Ab40) and 0, 10, 20 and 30 l
M
unlabeled
Ab40 or Ab42 were separately prepared. Similarly,
samples of 0.1 l
M
AED-Ab42 were prepared with 0,
10, 20 and 30 l
M
unlabeled Ab40 or Ab42. Thus, every
combination ofAb40andAb42 heterogeneous associ-
ation, as well as homogeneous association was examined.
Given that the threshold concentration for fibril forma-
tion ofAb40 at neutral pH is between 10 and 40 l
M
[27],
0, 10, 20 and 30 l
M
Ab are ideal concentrations to
monitor prefibrillar species.
To start the fibrillogenesis process, the pH of the
solution is lowered from 10 to 7 by addition of
phosphate buffer. The AEDANS fluorophore absorbs
at approximately 350 nm, and emits at approximately
480 nm. In samples with only AED-Ab, fluorescence at
480 nm is relatively low due to fluorescence quenching
by water (Fig. 1). As the label is sequestered by
unlabeled Ab, fluorescence increases. In order to control
for light scattering by the peptides as an explanation for
increased fluorescence readings, we also scanned 10, 20
and 30 l
M
unlabeled Ab over the same wavelengths, and
subtracted these spectra from the corresponding ones
with EDANS-Ab. In Fig. 3B, we show the unsubtracted
fluorescence for EDANS-Ab40 incorporating into
unlabeled Ab40 at the early time period, as well as the
subtracted fluorescence and the difference, over the three
concentrations tested. As a second control, we prepared
samples of EDANS-Ab40 or EDANS-Ab42 with 0, 10,
20 and 30 l
M
hen lysozyme. This is to ensure that any
observed association is Ab-specific, and not simply due
656 D. Frost et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Fig. 1. Assay for Ab association through the use of Ab labeled with an environment sensitive fluorophore (EDANS) and the characteristic spectra of the
various aggregate species. d, EDANS-labeled Ab; s,unlabeledAb.AstheEDANS-Ab peptides reorganize into the structure late aggregates, the
emission peak wavelength shifts to 420 nm from the characteristic 480 nm.
Fig. 2. AFM image ofAb40 at early time period. Thesampleconsistsof30l
M
Ab40 in 40 m
M
phosphate, pH 7. The image was acquired
immediately after sample preparation. Homogeneous and heterogeneous mixtures ofAb40andAb42 formed these early aggregatesof similar
morphology and size.
Ó FEBS 2003 Pre-fibrillary association between Ab40andAb42 (Eur. J. Biochem. 270) 657
to hydrophobic interactions. Hen lysozyme is another
peptide that forms amyloid deposits [33].
Atomic force microscopy (AFM) at early time period
Immediately upon inducing fibrillogenesis by lowering
pH, small Ab aggregates, approximately 5–10 nm in
height, are visible across the freshly cleaved mica surface
(Fig. 2). Both mixedand homogeneous Ab solutions
form these aggregates, and both Ab variants form early
aggregates of similar morphology and size. These early
aggregates are similar to those identified in previous
studies of Ab40.
Fluorescence assay for association at early time period
Immediate incorporation of EDANS-Ab40 and EDANS-
Ab42 into unlabeled Ab40 or Ab42 occurs upon lowering
pH (Fig. 3). Addition of labeled Ab40to increasing
concentrations of both unlabeled Ab40 or unlabeled
Ab42 resulted in an increase in fluorescence intensity
indicating that labeled Ab40 incorporates into both
aggregates of unlabeled Ab40and Ab42. Similarly,
labeled Ab42 was found to incorporate into both
aggregates of unlabeled Ab40andAb42 (Fig. 3C).
Significantly, the observed incorporation is Ab-specific;
controls of EDANS-Ab mixed with the same concentra-
tions of hen lysozyme showed negligible incorporation.
Three trials were conducted on separate days, and yielded
these results consistently. The observed incorporation is
not due to light scattering from increasing protein
concentrations, as spectra of unlabeled peptide alone
were subtracted from their counterparts with EDANS-
labeled Ab to generate the data shown in Fig. 3. All
EDANS peaks in early spectra (i.e. upon mixture) occur
around the known maximum of approximately 480 nm
(Fig. 4).
CD spectroscopy at early time period
Immediately upon mixing, at the time point when
association between Ab species occurs and aggregates
are small and amorphous, CD shows a spectrum of a
random coil or unstructured conformation (data not
shown). The spectrum shows a minimum at approxi-
mately 190 nm. The presence of small aggregates in these
samples can confound the interpretation of CD spectra.
However, we are confident that light-scattering effects
have not adversely influenced the results because the
spectrum so closely resembles that of a typical random
coil.
AFM after extended incubation
After 3 months of incubation, spherical prefibrillar aggre-
gates (approximately 15 nm in height) have replaced the
unstructured aggregates observed initially (Fig. 5A). These
aggregates form in all samples examined, including samples
with mixedAb40and Ab42. AFM on the control EDANS-
Ab40 mixed with unlabeled hen lysozyme shows large
aggregates (Fig. 5B).
Fluorescence assay for association after extended
incubation
AsshowninFig.6,whenEDANS-Ab40 is allowed to
incorporate into unlabeled Ab40 or Ab42 for extended
time periods, it exhibits greater incorporation into Ab40,
although incorporation into Ab42 also occurs. However,
EDANS-Ab42 incorporates to a similar extent into either
unlabeled Ab40 or Ab42. These results suggest that Ab40
late aggregate formation displays a slight preference for
homogeneous vs. mixed aggregation, while Ab42 late
0
50000
100000
150000
200000
250000
300000
0 5 10 15 20 25 30 3
5
0
50000
100000
150000
200000
250000
300000
350000
0 5 10 15 20 25 30 35
[Unlabeled peptide] (µM)
[Unlabeled peptide] (µM)
Fluorescence (400-550nm)
Fluorescence (400-550nm)
0
50000
100000
150000
200000
250000
300000
350000
400000
10 20 30
Fluorescence (400-550nm)
[Unlabeled peptide] ( M)
A
B
C
Fig. 3. Fluorescence of EDANS-Ab40 with unlabeled Ab40, Ab42 or
hen lysozyme immediately after mixing. (A) Fluorescence of 0.1 l
M
EDANS-Ab40 with 0, 10, 20 and 30 l
M
unlabeled Ab40 (d),Ab42
(s) or hen lysozyme (j) immediately after mixing. (B) Fluorescence of
0.1 l
M
EDANS-Ab40 with 10, 20 and 30 l
M
unlabeled Ab40 imme-
diately after mixing. Data are shown before subtracting scattering
control of unlabeled Ab40 alone (h), after subtracting control of Ab40
alone (j) and the difference, which is signal due solely to scattering by
unlabeled Ab40 (filled grey square). (C) Fluorescence of 0.1 l
M
EDANS-Ab42 with 0, 10, 20 and 30 l
M
unlabeled Ab40 (d),Ab42
(s) or hen lysozyme (j) immediately after mixing. Samples were
scanned after initiating reaction by dropping pH from 10 to 7. Samples
were excited at 350 nm and scanned from 360 to 600 nm. The resultant
spectra were integrated over 400–550 nm. Scans of 0, 10, 20 and 30 l
M
unlabeled peptide alone over the same wavelengths were subtracted
from the EDANS spectra obtained.
658 D. Frost et al. (Eur. J. Biochem. 270) Ó FEBS 2003
aggregate formation does not display such a preference.
In all cases, incorporation into the lysozyme control
remains negligible even after extended incubation. The
lysozyme aggregates observed by AFM (Fig. 5B) there-
fore do not include the labeled Ab that was present in
the solution.
Significantly, the EDANS peak of approximately
480 nm shifts to approximately 420 nm, concomitant
with late aggregate formation. In addition, the magnitude
of EDANS fluorescence is approximately five- to 10-fold
higher with late aggregates relative to early aggregates,
indicating that the fluorophore is more sequestered in the
late aggregate. We have consistently observed that
structured aggregate formation is accompanied by a blue
shift and increased intensity in the EDANS spectrum.
Figure 7 demonstrates that a biphasic distribution of
EDANS fluorescence exists at certain Ab concentrations,
indicating a mixture of unstructured and structured Ab
aggregates in the solution. All fluorescence scans of
EDANS-Ab alone show maxima at 480 nm, indicating
no structured aggregate formation in these samples, as
expected by the trace concentration of labeled Ab (i.e.
0.1 l
M
). The unshifted spectrum of AEDANS-Ab alone
after extended incubation (Fig. 7) also eliminates the
possibility that the behavior of the EDANS fluorophore
changes due to the incubation itself rather than a change
in the aggregate species. As mentioned above, three trials
were conducted over separate days, and yielded similar
results.
CD spectroscopy after extended incubation
CD spectra of samples showing large, structured aggre-
gates and blue-shifted EDANS fluorescence were taken.
As mentioned above, large aggregates can confound CD
data, but the spectrum obtained shows definite secondary
structure. The spectrum is somewhat similar to the typical
b-sheet spectrum, with a positive band around 200 nm
and a negative band around 218 nm (data not shown).
Although not fully b-sheet, these blue-shifted, large late
aggregates clearly show secondary structure in the CD
spectrum, in stark contrast to the early aggregates which
are fully unstructured, both morphologically, shown
by AFM, and spectroscopically, shown by CD.
Discussion
With growing interest in the process preceding fibril forma-
tion in identifying a conclusively neurotoxic species, our
approach avoids the problem associated with nucleation-
extension studies, namely that only addition to a pre-existing
fibril is studied. By starting the fibrillogenesis pathway and
monitoring association of Ab as soon the conditions permit
association (i.e. lower pH from 10 to 7), we examine the
interaction between Ab40andAb42 throughout the entire
pathway. We have demonstrated that early aggregates form
in vitro at pH 7, andAb40andAb42 prefer to incorporate
into Ab40 at this stage. Our assay allows us to distinguish
unstructured aggregate from structured aggregate formation
Fig. 4. Fluorescence spectrum of EDANS-
Ab40 with unlabeled Ab40 immediately upon
mixing. (A) Fluorescence spectrum of 0.1 l
M
EDANS-Ab40 with 0 (h),10(j),20(s)
and 30 (d) l
M
unlabeled Ab40 immediately
upon mixing. Spectra of 0, 10, 20 and 30 l
M
unlabeled Ab40 alone subtracted. Peaks occur
at normal EDANS fluorescence maximum
of approximately 480 nm. (B) Fluorescence
spectrum of 0.1 l
M
EDANS-Ab40 with 0 (h),
10 (j),20(s) and 30 (d) l
M
unlabeled
Ab42 immediately upon mixing. Spectra of 0,
10, 20 and 30 l
M
unlabeled Ab42 alone
subtracted. Peaks occur at normal EDANS
fluorescence maximum of approximately
480 nm.
Ó FEBS 2003 Pre-fibrillary association between Ab40andAb42 (Eur. J. Biochem. 270) 659
because the EDANS spectrum shifts to a 420-nm peak when
structured aggregates are present. Hence, we are able to
detect the formation of structured aggregates after an
extended incubation period (3 months).
There is evidence to suggest that the aggregates formed
immediately upon lowering the pH are similar to the
diffuse plaques observed in vivo. The aggregates are
morphologically unstructured, andform a diffuse lawn
A
B
Fig. 5. AFM images ofAb40 (A) and hen
lysozyme (B) after extended incubation.
Samples consist of 30 l
M
Ab40 or hen lyso-
zyme in 40 m
M
phosphate, pH 7. Images were
acquired 3 months after sample preparation.
Homogeneous and heterogeneous mixtures of
Ab40 andAb42 formed these spherical prefi-
brillar aggregates with a uniform distribution
of morphology and size. CD spectroscopy
indicates that they contain secondary struc-
ture. Hen lysozyme mixed with labeled Ab
formed large aggregates (note that the scale of
the hen lysozyme image is five times larger
than that of the Ab40 image).
660 D. Frost et al. (Eur. J. Biochem. 270) Ó FEBS 2003
on the mica AFM surface. They are accessible to water
relative to the aggregates formed later (recall the 10-fold
increase in EDANS fluorescence after extended incuba-
tion). The fluorescence spectrum of the EDANS-labeled
Ab incorporated into them shows a distinct peak from
that which occurs after extended incubation, suggesting a
difference in the fluorescent behavior of the fluorophore
in each of the aggregates. Finally, CD spectroscopy
shows these early aggregatesto be random coil (i.e.
without secondary structure).
Similarly, evidence can link the late aggregatesto senile
plaques. AFM shows a large, well-defined spherical
structure, whose height is consistent with the diameter
of typical Ab amyloid fibrils. Fluorescence shows that the
peptides are highly sequestered from water, indicating
tighter packing. The fluorescence spectrum shifts to
420 nm from 480 nm, indicating a significant change in
fluorophore behavior. Finally, CD shows the aggregates
to be structured, with spectra similar to those of b-sheet
(Ab fibrils found in senile plaques also have b-sheet
secondary structure).
After 3 months of incubation, fibrils were not detected
by EM or AFM in any of the 0, 10, 20 and 30 l
M
Ab
samples tested. Because we undertook this study to
examine the early aggregation events, not the fibrils per
se,wehavechosenAb concentrations near or below the
known threshold for Ab40 fibril formation under the
conditions tested. It is therefore not surprising that
fibrils have not formed in these samples. It is also
important to note that fibril formation is quite difficult
to achieve de novo. As described in the Materials and
methods section, we have employed a rigorous proce-
dure to prevent the formation of fibrillar seeds in our
stock Ab solutions. This allows us to examine prefibril-
lar structures. We are confident therefore that by
examining concentrations at or near threshold for fibril
formation, prefibrillar structures are the major species
present.
After sufficient time for structured aggregatesto form,
we find that both homogeneous andmixed aggregates
have formed, but that Ab40 shows a slight bias towards
associating with Ab40toform spherical aggregates.
Significantly, Ab42 associates equivalently with itself or
Ab40 in these aggregates. Given that diffuse plaques
consist mostly of Ab42, and senile plaques of both
variants, the addition of monomer Ab42to local
Ab40 appears to favor the production of structured
aggregates, which could lead to senile plaques. It
appears less likely for these structured aggregates to
form when only Ab42 is present. In vivo,sucha
transition could conceivably be caused by an increase in
overall cerebral Ab, which would probably be mostly an
increase in Ab40, as this variant is generally more
abundant.
Mixed aggregates occur in vitro, and the association
between the Ab variants begins before fibrils form. The
transition between unstructured and structured aggregate
could be vital in the progression from diffuse to senile
plaque; this transition is unlikely to be direct, as solid-to-
solid transitions are rare, and usually require rather
extreme conditions (e.g. graphite to diamond). More
likely is that unstructured and structured aggregates are
alternate aggregation products of soluble Ab. This study
shows that the local Ab42/Ab40 ratio can significantly
influence the ease of formation of structured aggregates,
as in some cases mixedaggregatesform more easily than
homogeneous ones; indeed such a change in Ab42/Ab40
ratio has been identified in some forms of familial
AD [35]. The easier formation ofmixedaggregates in
some cases tested in vitro may also help explain the
difference in Ab variant content in diffuse vs. senile
plaques.
This work has demonstrated the possibility for Ab to
form both mixed early unstructured aggregates (similar to
diffuse plaques) and late structured aggregates (possibly
an intermediate in the transition to senile plaques), and
has shown that, in vitro, Ab40andAb42 associate early
in the fibrillogenesis pathway. We have also demonstra-
ted an interesting property of the EDANS fluorophore,
namely that its fluorescence spectrum shifts concomitant
with structured aggregate formation. This could be quite
useful in other fibrillogenesis studies. More work is
needed to elucidate not only the aggregation and
fibrillogenesis pathway of Ab40, which is an area of
much active research, but also the role that Ab42/Ab40
interaction plays in the formation of senile plaques. This
study provides a starting point for further investigation in
this regard.
Fig. 6. Spectra from samples excited at 350 nm and scanned from 360 to
600 nm. Samples were excited at 350 nm and scanned from 360 to
600 nm. The resultant spectra were integrated over 400–550 nm. Scans
of 0, 10, 20 and 30 l
M
unlabeled peptide alone over the same wave-
lengths were subtracted from the EDANS spectra obtained. (A)
Fluorescence of 0.1 l
M
EDANS-Ab40 with 0, 10, 20 and 30 l
M
unlabeled Ab40 (d),Ab42 (s) or hen lysozyme (j) after incubation
for approximately 3 months at pH 7. (B) Fluorescence of 0.1 l
M
EDANS-Ab42 with 0, 10, 20 and 30 l
M
unlabeled Ab40 (d),Ab42
(s) or hen lysozyme (j) after incubation for approximately 3 months
at pH 7.
Ó FEBS 2003 Pre-fibrillary association between Ab40andAb42 (Eur. J. Biochem. 270) 661
Acknowledgments
This work was supported by a grant to A.C. from the Canadian
Institutes for Health Research (CIHR) and by a grant to CMY from
CIHR. PMG acknowledges support from the Ontario Student
Opportunity Transfer Fund, a Scace Graduate Fellowship in Alzhei-
mer’s Research, and a Gamble Grant Graduate Fellowship. DF
acknowledges support from a Natural Science and Engineering
Research Council summer studentship.
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Ó FEBS 2003 Pre-fibrillary association between Ab40andAb42 (Eur. J. Biochem. 270) 663
. Ab40 incorporates into both
aggregates of unlabeled Ab40 and Ab42. Similarly,
labeled Ab42 was found to incorporate into both
aggregates of unlabeled Ab40. Co-incorporation of Ab40 and Ab42 to form mixed pre-fibrillar
aggregates
David Frost
1
, Paul M. Gorman
1
, Christopher M. Yip
2
and Avijit Chakrabartty
1
1
Division