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ABSTRACT
The deposition of aggregated amyloid-b (Ab) peptides in the brain as senile
plaques is a pathological hallmark of Alzheimers disease (AD). Several lines of evidence indicate that
brillar and, in particular, soluble aggregates of these 40- and 42-residue peptides are important in
the etiology of AD. Recent studies also stress that amyloid aggregates are polymorphic and that a
single polypeptide can fold into multiple amyloid conformations. Here we review our recent reports
that Ab(1-40) in vitro can form soluble aggregates with predominant b-structures that differ in
stability and morphology. One class of aggregates involved soluble Ab protobrils, prepared by vigorous overnight agitation of monomeric Ab(1-40) in low ionic strength buffers. These aggregates
were quite stable and disaggregated to only a limited extent on dilution. A second class of soluble
Ab aggregates was generated at polarnonpolar interfaces. Aggregation in a two-phase system of
buffer over chloroform occurred more rapidly than in buffer alone. In buffered 2% hexauoroisopropanol (HFIP), microdroplets of HFIP were formed and the half-time for aggregation was less than
10 minutes. Like Ab protobrils, these interfacial aggregates showed increased thioavin T uorescence and were rich in b-structure by circular dichroism. However, electron microscopy and atomic
force microscopy revealed very different morphologies. The HFIP aggregates formed initial globular clusters that progressed over several days to soluble brous aggregates. When diluted out of
HFIP these aggregates initially were very unstable and disaggregated completely within 2 minutes.
However, their stability increased as they progressed to bers. It is important to determine
whether similar interfacial Ab aggregates are produced in vivo. Microsc. Res. Tech. 67:164174,
2005. ' 2005 Wiley-Liss, Inc.V 2004 Wiley-Liss, Inc.
C
INTRODUCTION
The brains of patients with Alzheimers disease (AD)
contain large numbers of amyloid deposits in the form
of senile plaques (Cohen and Calkins, 1959). The amyloid core of the plaques consists of interwoven brils,
each 79 nm in diameter (Terry et al., 1964), that can
be visualized by light microscopy after staining with
Congo Red or thioavin S (Terry, 1985). The brils are
composed of 40- and 42-residue peptides (Glenner and
Wong, 1984; Miller et al., 1993), denoted Ab(1-40) and
Ab(1-42). These peptides are produced by cleavage of
cellular amyloid precursor protein (APP) by two proteases called b- and g-secretase (reviewed in Selkoe,
2001). As originally suggested by the amyloid cascade
hypothesis (Hardy and Higgins, 1992), it appears likely
that Ab aggregates are important in the etiology of AD.
The most striking evidence supporting this hypothesis
comes from the identication of numerous mutations
linked to early-onset familial AD (FAD) (Selkoe and
Podlisny, 2002). These mutations are located within
the APP gene or the genes for presenilins 1 and 2,
which play an integral role in g-secretase activity. All
FAD mutations reported thus far increase either the
level of the more amyloidogenic Ab(1-42) peptide
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V
(reviewed in Selkoe and Podlisny, 2002) or the propensity of a mutated Ab peptide to form amyloid aggregates (Nilsberth et al., 2001).
The amyloid hypothesis for the etiology of AD is also
supported by early results from therapeutic strategies
that reduce the amount of aggregated Ab in the brain
(Lansbury, 1997). An extremely promising example is a
vaccine comprised of Ab(1-42) (Schenk et al., 1999).
While a clinical trial with this vaccine was halted
because of adverse side effects in 5% of individuals
(Schenk, 2002), the antibodies generated by patients in
this trial appeared to be effective. Twenty patients generated antibodies against Ab, as determined by tissue
amyloid plaque immunoreactivity assay. These
patients showed signicantly slower rates of decline of
cognitive functions and activities of daily living than
those observed with patients who did not produce such
antibodies (Hock et al., 2003).
Early evidence suggested that brillar deposits of Ab
initiate a cascade of events that result in neuronal cell
death and lead to the cognitive decline characteristic of
AD (Yankner, 1996). However, concerns were raised
because the number of amyloid deposits detected by
neuropathological analysis of postmortem brains does
not correlate well with the degree of cognitive impairment experienced by AD patients prior to death (Hardy
and Selkoe, 2002). A number of investigators now
propose that soluble aggregates of Ab (also called
oligomers or protobrils), rather than monomers or
insoluble amyloid brils, may be responsible for synaptic dysfunction in the brains of AD patients and AD
animal models (Hardy and Selkoe, 2002; Hartley et al.,
1999; Klein et al., 2001; Westerman et al., 2002;
Kawarabayashi et al., 2004). This proposal is supported by observations that soluble aggregates generated in vitro from synthetic Ab(1-40) and (1-42)
induced toxicity in cultured cells (Hartley et al., 1999;
Lambert et al., 1998), that soluble Ab aggregates produced in cell culture markedly inhibited hippocampal
long-term potentiation in rats in vivo (Walsh et al.,
2002), and that transgenic mice expressing human Ab
show functional decits that precede extracellular deposition of brillar Ab (Hsia et al., 1999; Westerman
et al., 2002).
A mechanistic understanding of the brillogenesis
process would be very useful in identifying individual
steps as therapeutic targets (Teplow, 1998). Since this
understanding is difcult to attain in the complex environment of living cells, it is fortunate that synthetic
Ab(1-40) and Ab(1-42) form both soluble aggregates
and amyloid brils in vitro that share many features
with the amyloid in AD plaques as well as the amyloid
brils formed by other proteins in a number of diseases. These brils contain peptide segments that align
to form b-sheets with extended peptide strands perpendicular to and interstrand H-bonds parallel to the bril
axis (Sunde et al., 1997; Serpell et al., 2000). This
cross-b structure may underlie common properties,
including the binding of dyes like thioavin T and
Congo Red and of antibodies that react with a common
epitope in several amyloidogenic proteins (ONuallain
and Wetzel, 2002; Kayed et al., 2003). However, recent
reports also indicate a fascinating degree of amyloid
polymorphism at the molecular level. This feature was
rst noted with mammalian and yeast prion proteins
when it was shown that a single polypeptide can misfold into multiple amyloid conformations (Chien et al.,
2004). Specically, the yeast Sup35p prion protein was
aggregated at different temperatures into amyloid conformations that could be distinguished by thermal
stability and EPR spectroscopy, and infection of yeast
with these different conformations led to different
propagating yeast [PSI] strains (Tanaka et al., 2004;
King and Diaz-Avalos, 2004). Ab brils also show
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bolus of bovine serum albumin (50 mg) in running buffer to block nonspecic binding of Ab protobrils to the
resin and occasionally were washed with 1 N NaOH.
Light-Scattering Measurements
Particle hydrodynamic radii (RH) were measured by
dynamic light scattering (DLS) at room temperature
with a DynaPro MSX instrument (Wyatt Technology,
Santa Barbara, CA). Total light scattering intensity at
a 908 angle in kilocounts/sec (kcts) was collected using
a 5-second acquisition time (Nichols et al., 2002).
Weight average molecular masses (Mw) were estimated
by MALS with a DAWN EOS instrument (Wyatt Technology, Santa Barbara, CA) in-line with a Superdex 75
SEC column (Nichols et al., 2002). This SEC-MALS
procedure is attractive because, in contrast to DLS, Mw
estimates can be made without assumptions of molecular or aggregate shape.
Circular Dichroism (CD)
Spectra were obtained on an Aviv Model 215 Circular
Dichroism Spectrometer with a 0.1 cm pathlength
quartz cuvette (Hellma, Mullheim, Germany). Buffer
control spectra were averaged and subtracted from the
average of triplicate scans of each Ab sample spectra,
and each resulting point ([y]obs, deg) was converted to
mean residue ellipticity ([y], deg cm2 dmol1) (Nichols
et al., 2005a).
Electron Microscopy (EM)
Samples of Ab aggregates were applied to 200-mesh
formvar-coated copper grids (Ernest F. Fullam,
Latham, NY) and incubated for 1015 minutes at room
temperature. The sample was then wicked off with lens
paper, washed briey by placing the grid face-down on
a wash droplet, and stained by transferring the grid
face-down to a droplet of 2% uranyl acetate (Polysciences, Warrington, PA) for 510 minutes before wicking off the solution and air drying. With samples generated in 2% HFIP, all wash and treatment solutions
applied to the grids also contained 2% HFIP. Grids
were visualized in a Philips EM208S transmission electron microscope.
Atomic Force Microscopy (AFM)
Samples were applied to freshly cleaved mica that
had been modied with 30 -aminopropyl-triethoxysilane (APTES) and incubated for 15 minutes (Nichols
et al., 2002, 2005a). The residual sample liquid was
aspirated off and the disk was then rinsed gently with
water and blown dry with compressed air. With aggregates generated in HFIP, the rinse included 2% HFIP.
A Nanoscope III controller with a Multimode AFM
(Digital Instruments, Santa Barbara, CA) was used for
imaging by ambient tapping mode. Images were
obtained in either amplitude mode or height mode,
where increasing brightness indicates greater damping
of cantilever oscillation (Harper et al., 1999) or increasing feature height, respectively. Height mode images
were attened prior to measurement of particle
height distributions with NanoScope (R) III software
(v. 5.13r5, Digital Instruments).
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Fig. 1. Protobril growth arising from elongation by added monomer or direct protobril association as monitored by AFM. Ab(1-40)
protobrils were isolated by SEC in 5 mM Tris-EDTA and diluted to
1 mM (in Ab monomer units) in 50 mM Tris-EDTA. The dilution for
the elongation reaction included 30 mM Ab(1-40) monomer and after
1 hour the mixture was fractionated by SEC to isolate a subpopulation of elongated protobrils. The dilution for the association reaction
included 150 mM NaCl, but low recoveries precluded further fractionation by SEC. Aliquots (100 ml) from each of the three samples were
applied to mica stubs and analyzed by AFM. Initial (A), elongated
(B), and associated (C) protobrils are shown as 2.5 2.5 mm images
(cropped from 5 5 mm elds). Images are presented in amplitude
mode. Slightly different images from the same 5 5 mm elds were
previously presented in height mode (Nichols et al., 2002).
Interfacial Ab Aggregation
The concept that Ab brillogenesis is a nucleationdependent polymerization process (Jarrett et al., 1993;
Walsh et al., 1997) has provided a useful framework for
in vitro Ab aggregation studies. Nucleus formation is
considered rate-limiting in this process, but this rate
(as measured by the delay or lag time to the maximal
rate of aggregation) is highly variable even in homogeneous aqueous solutions and is sensitive to many conditions including pH, ionic strength, temperature, and
agitation (Wood et al., 1996; Harper et al., 1999; Nichols et al., 2002). Interfaces including phospholipid
membranes may play important roles in promoting
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Stabilities of Ab Aggregates
The stability of soluble Ab aggregates has received
much less attention than their assembly pathways.
Virtually the only quantitative estimate of an in vitro
protobril disaggregation rate involved Ab(1-40) protobrils trace-labeled with 125I-Ab(1-40). The dialyzed
protobrils released only about 30% of their radioactivity, mostly over the rst 2 days of dialysis (Walsh et al.,
1999). We measured the stability of our Ab(1-40) protobril preparations by diluting Ab(1-40) aggregation
reactions that were optimized for Ab protobril formation as outlined above. Large dilutions were necessary
to minimize further monomer deposition and reveal
disaggregation, indicated by a progressive decrease in
thioavin T uorescence (Fig. 4A). The uorescence
decrease occurred in at least two kinetic phases, with
initial disaggregation followed by a much slower secondary phase. Less than half of the initial protobrils disaggregated over a 16-hour period, an observation quite
compatible with the extent of disaggregation reported
for dialyzed 125I-Ab(1-40) protobrils (Walsh et al.,
1999). Continued incubation of the aggregation reactions revealed a progressive stabilization of the Ab protobrils, as demonstrated by decreased disaggregation
rate constants (Nichols et al., 2005a) and increased
percentages of stable aggregates after dilution at longer incubation times. In Figure 4A, no disaggregation
could be detected after 5 days of incubation. Ab(1-40)
protobrils isolated after SEC also were completely
169
8.0) was incubated over chloroform for 30 hours and aliquots were
removed from the aqueous phase and either diluted 15-fold into the
same buffer containing 5 mM thioavin T (left) or analyzed without
dilution (right). C: (Nichols et al., 2005a) Ab (20 mM) was aggregated
for 30 minutes without agitation in a solution containing 2% HFIP
and 5 mM thioavin T in 5 mM Tris-EDTA and aliquots were diluted
15-fold into the same thioavin T and buffer without HFIP. The
9 hours disaggregation curve from A (PF) is reproduced to allow comparison with Ab(1-40) protobrils.
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clear: the two-phase aggregates included no rod-like laments, and the elongated protobrils showed no
chain-like alignment of globules. Furthermore, Figure
5F showed discontinuities within central sections of
several bers that appeared to reect disaggregation of
individual globular components of the bers after
adsorption of the diluted sample and washing of the
AFM disk. Such rapid disaggregation is consistent with
the ber instability in the aqueous phase shown in
Figure 4B. One limitation of the two-phase chloroform/
buffer system in our analysis of interfacial Ab aggregation was the continued growth of the aggregates. Maximum aggregate levels were obtained after 1-4 days,
and this peak was followed by a rapid decline in aggregate concentration in the aqueous phase and formation
of a cloudy white precipitate hovering at and just above
the aqueous/chloroform interface (Nichols et al.,
2005b). This precipitation prevented us from examining long-term changes in the stability of the aggregates. In contrast, HFIP-induced Ab aggregates
remained soluble for several days, allowing the progressive stabilization of the aggregates to be discerned
(Fig. 4C).
EM analyses, conducted following negative staining
of samples with uranyl acetate, initially were useful in
clarifying the disperse state of dilute HFIP itself. Control samples of freshly diluted 2% HFIP showed no features in EM images, but addition of bovine serum albumin to the diluted HFIP resulted in the appearance of
circular features (Fig. 6A). The number of these fea-
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Fig. 6. Electron micrographs of HFIP-induced Ab(1-40) aggregates reveal progressive changes in morphology (Nichols et al.,
2005a). Bovine serum albumin (0.25 mg/ml) (A) or SEC-puried
monomeric Ab(1-40) (40 mM) (BE) were incubated in 5 mM thioavin
T, 5 mM Tris-HCl (pH 8.0), and 2% HFIP (neat) at room temperature.
After 0.1 hours a 10-ml sample of the albumin solution was removed
and at the indicated times aliquots of the Ab(1-40) solution were centrifuged at 18,000g for 10 minutes and supernatant samples (10 ml)
deposits differed from the albumin-coated microdroplets by remaining soluble after centrifugation at
18,000g. Two days later the clustered globular structures were incorporated into a mesh or lattice of berlike elements (Fig. 6C), and after 9 days more distinct
bers were apparent (Fig. 6D). These included long
bers from which many short bers branched and, very
rarely, structures resembling initial microdroplets
on which bers had formed (Fig. 6D, inset). After 23
days (Fig. 6E) the HFIP-induced bers appeared similar to control brils produced by elongation of Ab(1-40)
protobrils (Fig. 6F), but numerous short branches
remained that were not found on the control brils.
The bers at this time still remained soluble, as they
failed to sediment during 18,000g centrifugation.
AFM images supported and extended the features of
HFIP-induced Ab aggregates observed by EM. The
same mixture of clustered globular structures and individual globules was observed 1 hour after adding Ab(140) to dilute HFIP (Fig. 7A and inset). The heights of
the clustered structures typically ranged from 10
15 nm but in some cases extended to over 50 nm. After
23 days, bers with heights of 45 nm had appeared
and numerous very short rods with heights of 2.5
4 nm had emerged that were barely evident in the 1hour sample (Fig. 7B). These short rods were less clear
in the EM images, perhaps because of differences in
retention on the EM grid and AFM mica surfaces or
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well-formed bers on spherical structures that resemble microdroplets (Fig. 6D, inset) suggest that some
reorganization of the initial b-structured aggregates
can occur directly on the microdroplet surface.
Ab Aggregate Polymorphism
Since the interfacial Ab aggregates in this report differ from Ab protobrils in morphology and stability,
their molecular structures must differ at least in subtle
ways. One measure of this conformational specicity is
the efciency with which these aggregates acted as
nuclei or seeds for elongation by monomeric Ab
(ONuallain et al., 2004). HFIP-induced Ab aggregates
that had stabilized for 3 days and Ab(1-40) protobril
controls were diluted to equivalent concentrations
(1 mM) and incubated with 60 mM Ab(1-40). The net
elongation rate of the protobrils as measured by thioavin T uorescence was almost 30 times faster than
that of the HFIP-induced Ab aggregates (Nichols et al.,
2005a). The difference in elongation rates was not due
to a difference in aggregate and protobril sizes, as the
RH values measured by DLS were similar. We concluded that the seeding efciency of the HFIP-induced
aggregates was much lower than that of protobrils, at
least in aqueous buffers without HFIP.
Further studies are required to determine the molecular interactions in these aggregates, but models of
amyloid brils suggest ways in which differences could
arise. In one structural model for Ab(1-40) based on
solid-state NMR data, a cross-b unit is composed of two
b-strand segments involving residues 12-24 and 30-40
separated by a segment with a bend angle of 1808,
allowing interpeptide hydrogen bonding and separate
parallel b-sheet formation from both b-strands (Tycko,
2003). In contrast, low-angle X-ray diffraction analysis
of Ab(11-25) brils indicated an antiparallel alignment
of fully extended b-strands (Sikorski et al., 2003). In
these brils the b-sheets appeared to stack by slipping
relative to each other by the length of two amino acid
units (Sikorski et al., 2003), and the registry of the
hydrogen bonding appeared to vary with pH (Tycko,
2003). It is possible then that Ab(1-40) protobrils and
HFIP-induced aggregates differ by their b-strand
alignments or the registry of their b-sheets or hydrogen
bonding, and solid-state NMR measurements will be
required to resolve this issue.
It will also be of interest to determine whether
agents or conditions that favor rapid interfacial formation of unstable Ab aggregates occur in vivo. Analogs of
biological membranes, namely, GM1 ganglioside
micelles and articial lipid rafts that contain GM1
(Kakio et al., 2002), as well as lipoprotein particles
(Chan et al., 1996) have been reported to bind Ab peptides in a saturable manner and to convert the peptide
conformations to b-structures in the complexes. The
stability of these aggregates has not been investigated.
A recent study has also found that inhaled anesthetics
promote Ab aggregation (Eckenhoff et al., 2004). The
haloalkane halothane and the haloether isourane,
like HFIP, are highly uorinated, and both increased
the rates of Ab aggregation as measured by thioavin
T uorescence. Furthermore, an EM image of the Ab
aggregates induced by halothane at concentrations
173
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