From the Department of Medicine, Division of
Geriatrics, Evanston Northwestern Healthcare Research Institute,
Evanston, Illinois 60201 and Departments of § Molecular
Pharmacology and
Neurobiology and Physiology and
** Alzheimer's Disease Core Center, Northwestern University,
Chicago, Illinois 60611
Received for publication, October 4, 2002, and in revised form, December 18, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Extensive research causally links
amyloid- Amyloid plaques are one of the pathological hallmarks of
Alzheimer's disease (AD).1
Genetic findings over the past decade have further supported the
pivotal and likely causal role that amyloid- Soluble oligomers of A In light of genetic and in vivo evidence, we have chosen to
specifically focus on the longer, more hydrophobic A Considering the difficulties in detecting unaggregated and oligomeric
A These techniques were used to characterize the structural heterogeneity
present in lyophilized stocks of commercial A These results demonstrate the importance of a common homogeneous
starting material and how specific conditions determine the formation
of distinct conformations of A Initial Solubilization of A A CD Spectroscopy--
HFIP-treated A AFM--
Peptide solutions were characterized using a
NanoScope IIIa scanning probe work station equipped with a MultiMode
head using an E-series piezoceramic scanner (Digital Instruments, Santa
Barbara, CA). AFM probes were single-crystal silicon microcantilevers
with 300-kHz resonant frequency and 42 Newton/meter spring constant model OMCL-AC160TS-W2 (Olympus). Samples were imaged under dry helium.
10-50 µl of sample solution was spotted on freshly cleaved mica,
incubated at room temperature for 5 min, rinsed with 0.02 µm of
filtered (Whatman Anotop 10) deionized NANOpure water (Barnstad Thermoline), and blown dry with tetrafluoroethane (CleanTex MicroDuster III). Image data were acquired at scan rates between 1 and 2 Hz with
drive amplitude and contact force kept to a minimum.
Western Blot Analysis of SDS-PAGE--
Gel electrophoresis and
Western blot analysis were performed according to the manufacturer's
protocols (Invitrogen) as described previously (24). Briefly, unheated
samples in lithium dodecyl sulfate sample buffer were applied to 12%
bis-Tris NuPAGE gels were electrophoresed using MES running buffer and
transferred to 0.45-µm polyvinylidene difluoride membrane
(Invitrogen). Membranes were blocked in 5% non-fat dry milk in
Tris-buffered saline containing 0.0625% Tween 20. Blots were incubated
in the primary antibody 6E10 (mouse monoclonal against A Solubility Analysis--
A In vitro studies necessary to define the structure and
activity differences between small oligomeric and fibrillar assemblies of A HFIP Pretreatment Produces Uniform, Unaggregated Fields of
A
Fluorinated alcohols including HFIP and trifluoroethanol have been
shown to break down Detection of A Detection of A Solubilization of HFIP-treated Peptide in Me2SO
Produces a Uniform, Unaggregated Field of A Solution Conditions for the Formation of Oligomeric or Fibrillar
A
Immediately after resuspension under both the oligomer- and
fibril-forming conditions, the majority of A
Western blot analysis of SDS-PAGE revealed A
Western analysis of SDS-PAGE for A A
Immediately after dilution, 2-4-nm A
Under fibril-forming conditions, immediately after dilution A A
Under oligomer-forming conditions (4 °C), 100 µM
A
Under fibril-forming conditions (37 °C), 100 µM
A
Western blot analysis of SDS-PAGE of A A
Oligomer formation was favored at neutral pH and physiologic ionic
strength. However, some differences were present between A
Western blot analysis of SDS-PAGE revealed that samples prepared at
acidic pH and low ionic strength contained A Solubility of A
Approximately 90% of the total A A
Western blot analysis of SDS-PAGE for A The biological roles that A In the present study, A In vitro biological activity studies have demonstrated that
oligomeric (17-19, 21) and protofibrillar (20, 60) conformations of
A The variability in A The A Despite these limitations, some general trends were observed between
Western blot analysis of SDS-PAGE of A A more accurate representation of the molecular stoichiometry of the
different assemblies as they exist in solution may require covalent
cross-linking. This strategy was used recently (69) to demonstrate the
complexity and dynamic nature of the prenucleation phases of A In general, ionic interactions are influenced by pH. In this study,
solution pH significantly effected both oligomer and fibril assembly.
Acidic pH has been shown previously (36, 71, 72) to favor fibril
formation, possibly resulting from a partial denaturation state similar
to that observed with other amyloid-forming proteins (73). This may be
the result of protonation of the C-terminal carboxyl, N-terminal
Asp1, Glu23-Asp24,
His13-His14, or other Asp, Glu, or His
residues. These charged amino acids may be involved in the
stabilization of the local secondary structure that facilitates
fibrillogenesis. Studies of modified A In addition to ionic interactions influenced by solution pH,
hydrophobic interactions also appear to have a significant effect, particularly on fibril formation. At low ionic strength, A Further evidence supporting the role of hydrophobic interactions comes
from the dramatic differences between A Polarity changes induced by protein oxidation may influence both ionic
and hydrophobic interactions. In particular, oxidation of A The physical presence of amyloid plaques in vivo
demonstrates that at some point, favorable conditions exist for their
formation. However, in vitro fibril formation occurs in
solution conditions and at A Evidence in the literature continues to build supporting a central
causative role for A peptide (A
) to Alzheimer's disease, although the
pathologically relevant A
conformation remains unclear. A
spontaneously aggregates into the fibrils that deposit in senile
plaques. However, recent in vivo and in vitro
reports describe a potent biological activity for oligomeric assemblies
of A
. To consistently prepare in vitro oligomeric and
fibrillar forms of A
1-42, a detailed knowledge of how solution parameters influence structure is required. This manuscript represents the first study using a single chemically and structurally homogeneous unaggregated starting material to demonstrate that the formation of
oligomers, fibrils, and fibrillar aggregates is determined by time,
concentration, temperature, pH, ionic strength, and A
species. We
recently reported that oligomers inhibit neuronal viability 10-fold
more than fibrils and ~40-fold more than unaggregated peptide, with
oligomeric A
1-42-induced neurotoxicity significant at 10 nM. In addition, we were able to differentiate by structure and neurotoxic activity wild-type A
1-42 from isoforms containing familial mutations (Dahlgren, K. N., Manelli, A. M., Stine,
W. B., Jr., Baker, L. K., Krafft, G. A., and LaDu,
M. J. (2002) J. Biol. Chem. 277, 32046-32053).
Understanding the biological role of specific A
conformations may
define the link between A
and Alzheimer's disease, re-focusing
therapeutic approaches by identifying the pernicious species of A
ultimately responsible for the cognitive dysfunction that defines the disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptide (A
) plays in
the etiology of AD (1-4). This research demonstrates that autosomal
dominant mutations affecting the total amount or relative amount of the
42- versus 40-residue form of A
are sufficient to cause
the disease (for review, see Ref. 5). However, a direct causal
relationship between amyloid plaques and cognitive impairment remains
unclear (6, 7). This apparent disconnect between plaque burden and
neuronal dysfunction and loss has also been described in transgenic
mouse models of AD (8, 9). Recent theories that reconcile these
findings point to small soluble oligomeric or protofibrillar assemblies
of A
that would likely escape the immunostaining and
histopathological staining used to detect both diffuse amyloid deposits
and senile plaques (10-13).
have been isolated from brain, plasma,
cerebrospinal fluid (14, 15), transfected cells (16), and cells derived
from human brain (11, 17). In vitro, oligomeric and
protofibrillar forms of A
have been shown to be directly neurotoxic
and inhibit electrophysiologic activity that may be necessary for the
formation and maintenance of memory (15, 17-21). Experimental in
vivo and in vitro evidence linking oligomeric assemblies to neurodegeneration is reflected in a recent revision to
the amyloid hypothesis of AD (22) and other diseases involving amyloidogenic proteins (for review, see Ref. 23). A complete study of
A
, either in vivo or in vitro, requires an
understanding of the conditions that drive peptide assembly toward one
conformational state or another. Any change that affects the
conformation of A
likely affects its biological activity.
1-42 species. We have determined that the critical initial step in the controlled assembly of A
1-42 is to remove preexisting structure. Using this chemically and structurally uniform unaggregated starting material, we
demonstrate that changes in incubation time, concentration, temperature, ionic strength, and pH result in distinct structural species including soluble oligomers, protofibrils and short fibrils, extended fibrils, and insoluble fibrillar aggregates. We have reported
recently (24) that oligomers formed using these methods inhibit
neuronal viability 10-fold more than fibrils and ~40-fold more than
unaggregated peptide, with oligomeric A
1-42-induced inhibition
significant at 10 nM. In addition, we were able to differentiate by structure and neurotoxic activity wild-type A
1-42 from isoforms containing known familial mutations (24).
using traditional light and electron microscopy, atomic force
microscopy (AFM) was chosen for this study as the primary means of
characterizing A
assembly state. AFM has proven uniquely well suited
to the study of A
(15, 18, 25-28) and other amyloidogenic proteins
(29-31), because it generates detailed three-dimensional information
at a nanometer scale. This technique is capable of characterizing the
wide range of structures present in aggregated A
mixtures with
sufficient resolution to detect individual 0.9-nm structures in
unaggregated preparations that appear monomeric up to multi-micron
dense fibrillar aggregates. For the present study, samples were also
characterized by Western blot analysis of SDS-PAGE, a technique used in
previous publications to identify SDS-stable A
assemblies (11, 16,
17, 19, 24, 32).
peptide and define
conditions that eliminate any preformed peptide assemblies. Using this
common homogeneous unaggregated starting material we characterized how
concentration, time, temperature, pH, and ionic strength influence
A
1-42 assembly. In addition, the solubility of these A
1
42
preparations was estimated by centrifugation. Finally, solution
conditions that favored oligomer and fibril formation for A
1-42
were applied to A
1-40.
, thus providing the necessary
foundation for controlling their assembly. Understanding the biological
roles that specific conformations of A
play in the pathogenesis of
AD may define the link between A
and AD, re-focusing therapeutic
approaches by identifying the pernicious species of A
ultimately
responsible for the cognitive dysfunction that is the primary symptom
of the disease.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Peptide--
The peptide used in
these studies was produced either by chemical synthesis (American
Peptide, Sunnyvale, CA) or recombinant expression (Recombinant Peptide
Technologies, Athens, GA). Both sources yielded peptide of greater than
95% purity by reverse-phase high performance liquid chromatography and
demonstrated the correct molecular mass by mass spectrometry analysis.
Lyophilized peptide was stored in sealed glass vials in desiccated
containers at
80 °C. Prior to resuspension, each vial was allowed
to equilibrate to room temperature for 30 min to avoid
condensation upon opening the vial. The first step in resuspending the
lyophilized peptide was treatment in 1,1,1,3,3,3-hexafluoro-2-propanol
(HFIP; catalog number H8508; Sigma). All work with HFIP was done
in a chemical fume hood with adequate protection. Each vial of peptide
was diluted in 100% HFIP to 1 mM using a glass gas-tight
Hamilton syringe with a Teflon plunger. The clear solution containing
the dissolved peptide was then aliquoted in microcentrifuge tubes
(VWR, 20170-293) using a positive displacement repeat pipette
(Multipette; Eppendorf). The HFIP was allowed to evaporate in the fume
hood, and the resulting clear peptide films were dried under vacuum
(6.7 mtorr) in a SpeedVac (Savant Instruments) and stored desiccated at
20 °C. The HFIP-treated peptide aliquots were re-checked for
chemical purity by reverse-phase high performance liquid chromatography
and mass spectrometry. Immediately prior to use, the HFIP-treated
aliquots were carefully and completely resuspended to 5 mM
in anhydrous dimethyl sulfoxide (D2650; catalog number
D-2650; Sigma) by pipette mixing followed by bath
sonication for 10 min (model 2120; Branson).
1-42 Oligomer- and Fibril-forming Conditions--
A
1-42
oligomers were prepared by diluting 5 mM A
1-42 in
Me2SO to 100 µM in ice-cold cell culture
medium (phenol red-free Ham's F-12; BioSource), immediately vortexing
for 30 s, and incubating at 4 °C for 24 h (19). A
1-42
fibrils were prepared by diluting 5 mM A
1-42 in
Me2SO to 100 µM in 10 mM HCl,
immediately vortexing for 30 s, and incubating at 37 °C for
24 h.
(1-40 and 1-42)
samples prepared using the methods described above were diluted to 50 µM with HFIP. CD spectra were collected on a Jasco J-715
spectropolarimeter. Spectra were obtained from 190 to 260 nm (1-nm
steps, 1-nm bandwidth, 20-millidegree sensitivity) in a 0.1-cm path
length quartz cell fitted with a Teflon stopper to control for
evaporation. All spectra were solvent-subtracted and smoothed with the
Savitzky-Golay algorithm according to the manufacturer's instructions
provided by Jasco. Spectra were then converted to mean residue
ellipticity and fitted by using a linear least squares method with the
program CDFIT for Jasco data (JFIT; written by Bernhard Rupp, 1997. This software was provided by Lawrence Livermore National Laboratory,
and more information can be found at
www-structure.llnl.gov/cd/cdtutorial.htm#Program%20CDFIT).
residues
1-16; Signet, Dedham, MA) or 4G8 (mouse monoclonal against A
residues 17-24; Signet, Dedham, MA). Immunoreactivity was detected
using enhanced chemiluminescence (ECL; Amersham Biosciences) and
imaged on an Eastman Kodak Co. Image Station 440CF. Molecular weight
values were estimated using rainbow pre-stained molecular weight
markers (Amersham Biosciences).
1-42 preparations were centrifuged
for 30 min at 4 °C at either 16,000 × g in an
Eppendorf 5415D microcentrifuge (Brinkmann Instruments) or at
100,000 × g in a Beckman Optima MAX ultracentrifuge using a TLA 120.1 rotor in 8 × 34-mm polycarbonate
ultracentrifuge tubes (Beckman Coulter Instruments). A
supernatant
and pellet fractions were extracted by dilution to 70% formic acid
(EM Science), bath sonicated (model 2120; Branson) for 5 min,
and incubated for 1 h at room temperature. Control samples were
prepared in an identical manner using the same tubes and solutions in
the absence of centrifugation. Values from these control samples were used to determine non-sedimenting background and were subtracted from
test samples. For dot blot analysis, A
supernatant and pellet fractions were diluted to 23.3% formic acid in deionized water. Quantitative dot blot analysis was performed using a series 1055 96-well vacuum dot blot apparatus (Invitrogen) fitted with a
0.45-µm Immobilon-P membrane (Millipore). 40 µl of each diluted
fraction was applied to the membrane and developed using the same
methods described for Western blot analysis. Quantitation of
immunoreactivity was determined using Kodak one-dimensional image
analysis software (version 3.5.4).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
need to be based on procedures that consistently produce fully
characterized, homogeneous structural populations. To achieve this
goal, the first point to address is the intrinsic structural variability between different lots of A
peptide. This variability can exist between chemically identical peptide lots and often results
in changes in biological activity in vitro (33, 34).
1-42--
Removal of any preexisting structures in lyophilized
stocks of A
1
42 peptide is required for controlled aggregation
studies. In this study, lyophilized A
directly dissolved to 5 mM in 100% HFIP was characterized by AFM. This treatment
resulted in a dense, homogeneous field of unaggregated peptide
immediately after resuspension (Fig.
1A). After a 24-h incubation
at room temperature, no aggregates, fibrils, or protofibrils were
detected in these HFIP A
solutions. The z-height value for
the individual peptide structures as measured by AFM was 1.0 (± 0.3)
nm, which agrees the expected size of a single A
monomer. However
this technique is not capable of absolute molecular weight
determination and therefore does not provide proof that these
structures are monomeric. HFIP is a corrosive alcohol and is not
compatible with in vitro and cell-based assays. For this
reason, it was removed by evaporation in a fume hood followed by
incubation in a 6.7-mtorr vacuum, and samples were stored as
peptide films.
View larger version (104K):
[in a new window]
Fig. 1.
AFM analysis of
A 1-42 solubilized in HFIP, Me2SO,
and H2O. A, lyophilized synthetic
A
1-42 was solubilized to 5 mM in 100% HFIP,
Me2SO, or deionized H2O. 5 mM stock
solutions were incubated for 24 h at room temperature. Samples
before (0-h) and after incubation (24-h) were
mounted for AFM analysis at 10 µM. Representative 1 × 1-µm x-y, 5-nm total z-range AFM images are shown. Inset
image, 390 × 390 nm x-y, 5-nm total z-range. B,
HFIP-treated lyophilized peptide stocks were resuspended to 5 mM in Me2SO and diluted to 10 µM
for AFM analysis. Representative 2 × 2-µm x-y, 5-nm total
z-range AFM image is shown. Inset image, 200 × 200-nm
x-y, 5-nm total z-range.
-sheet structure, disrupt hydrophobic forces in
aggregated amyloid preparations, and promote
-helical secondary
structure (35-37). CD is commonly used to determine changes in
secondary structure averaged for a given sample solution. Thus, CD
spectroscopy is a bulk solution technique that might not detect small
quantities of fibrillar seeds that differ in secondary structure. A
preparations containing primarily fibrillar peptide as determined by
electron microscopy produce a CD spectra consistent with a predominately
-sheet conformation (38). However, despite random coil
CD spectra, aggregates can be detected by AFM in A
1-40 directly resuspended in dH2O (39). In the present study, using 100%
HFIP to remove structural history resulted in CD spectra for A
1-42 and A
1-40 solutions indicating a secondary structure almost
entirely
-helical (~50-70%) and random coil (~30-50%), with
little
-sheet (<1%) (Table
I). These data are in agreement with
previous CD results using either HFIP or trifluoroethanol to induce
-helical structure (36, 40). Taken together, these CD studies
indicate that dissolving A
1-42 in 100% HFIP removes any
preexisting
-sheet secondary structure, yielding predominately
-helix and random coil. These results are also in agreement with the
AFM analysis of A
1-42 solutions in HFIP that demonstrate the
peptide assumes a uniform, unaggregated conformation that shows no
signs of aggregation after incubation for 24 h (Fig.
1A).
CD spectroscopy of A1-40 and A
1-42 in HFIP
1-40 and A
1-42 were acquired in
100% HFIP at room temperature. Secondary structure values were
estimated by spectral deconvolution as described under "Experimental
Procedures."
1-42 Fibrils after Direct Resuspension in
Me2SO--
Me2SO is a highly polar,
water-soluble organic solvent commonly used to solubilize hydrophobic
peptides, including A
(41), and is recommended by commercial
suppliers (Bachem). Analytical ultracentrifugation studies have
demonstrated that A
1-40 and A
1-42 are primarily monomeric in
100% anhydrous Me2SO (42, 43). In the current study, AFM
analysis of different commercial lots of synthetic and recombinant
A
1-42 directly resuspended to 5 mM in 100% anhydrous
Me2SO without HFIP pretreatment demonstrated that the
peptide appears primarily unaggregated immediately after solubilization, with structures measuring 1.1 (± 0.3) nm (Fig. 1A). However, in some cases small diameter (~2-nm) fibrils
were observed (Fig. 1A, inset). After 24 h
at room temperature, additional small A
fibrils were detected in
A
1-42 Me2SO solutions, even in preparations that were
initially fibril-free. These small fibrillar structures were stable in
Me2SO for several weeks and formed even after HFIP
pretreatment (data not shown). These results indicate that 100%
anhydrous Me2SO alone is not a sufficiently strong solvent to remove all structural history present in lyophilized A
or maintain a concentrated unaggregated peptide solution.
1-42 Fibrils, Protofibrils, Large Aggregates, and
Oligomers after Direct Resuspension in Water--
Resuspension of
lyophilized A
1
42 directly in dH2O or aqueous buffer is
also used by investigators (44, 45) and recommended by commercial
manufacturers (AnaSpec, Bachem, U.S. Peptide, Inc.). To
determine the extent of structural heterogeneity present in lyophilized
stocks of A
1-42, peptide was directly resuspended to 5 mM in dH2O and characterized by AFM. Analysis
of these solutions revealed a wide size range of peptide aggregates and
extended fibrils immediately after resuspension (Fig. 1A).
These structures included large (10-15-nm z-height) and small (2-5-nm
z-height) amorphous peptide aggregates and intermediate diameter
fibrils (4-6 nm) greater than 1 µm in length. These structures
persisted after 24 h at room temperature, in addition to an
increase in the number of 2-5-nm globular aggregates. However, the
extent of all the structures observed under these conditions is highly dependent on seeded aggregation and thus dependent on the initial lyophilized peptide. These data indicate that direct resuspension of
lyophilized A
1-42 in dH2O produces heterogeneous
structural populations incompatible with controlled aggregation
studies. Note that the z-height range for this H2O
preparation was maintained at 5 nm, resulting in a greater contrast
compared with images of HFIP- and Me2SO-solubilized peptide.
1
42--
To ensure
that the HFIP-treated A
1-42 peptide film was fully solubilized,
peptide stocks were resuspended to 5 mM in
Me2SO. The resulting solution contained unaggregated
structures (Fig. 1B) that measured 1.0 (± 0.3) nm by AFM
(Fig. 1B, inset), comparable with the
measurements for A
1-42 structures measured directly in HFIP or
Me2SO (Fig. 1A). Solubilization of the
HFIP-treated peptide directly into dH2O or aggregation
buffer does not produce a fully resuspended unaggregated solution. The
thin film resulting from the HFIP treatment is not easily resuspended
directly in dH2O without the use of sonication or other
methods. The peptide film is more soluble in Me2SO
facilitating complete resuspension of the HFIP-treated peptide (data
not shown).
1-42 Assemblies--
Once conditions were established that
consistently produce unaggregated A
preparations, two different
aggregation protocols were used with the HFIP-treated Me2SO
peptide stocks. Previous studies demonstrated the formation of small
soluble oligomeric assemblies derived from A
1-42 either incubated
in the presence of apolipoprotein J under physiologic conditions (18)
or in cell culture medium where the oligomeric assemblies were referred to as ADDLs (amyloid-derived
diffusible ligands) (19). For the studies
presented herein, the cell culture medium method with a 24-h incubation
at 4 °C was used for oligomer formation. For the fibril-forming
conditions, previous studies have indicated that acidic pH favors
fibrillogenesis for A
(46, 47) and other amyloid-forming proteins
(48-50). The acidic pH method using 10 mM HCl with a 24-h
incubation at 37 °C was chosen for fibril formation. The resulting
oligomeric and fibrillar A
1
42 structures were characterized by AFM
(Fig. 2A) and Western blot
analysis of SDS-PAGE (Fig. 2B).
View larger version (57K):
[in a new window]
Fig. 2.
Solution conditions for the formation of
oligomeric or fibrillar A 1-42
assemblies. A, AFM. 5 mM HFIP-treated
A
1-42 in Me2SO was diluted to 100 µM in
ice-cold F-12 culture medium for oligomers and 10 mM HCl
for fibrils. Oligomer and fibril preparations were incubated for
24 h at 4 and 37 °C, respectively. Samples before
(0-h) and after incubation (24-h) were mounted
for AFM analysis at 10 µM. Representative 2 × 2-µm x-y, 10-nm total z-range AFM images are shown. Inset
images, 200 × 200-nm x-y, 2-nm total z-range. B,
Western analysis of SDS-PAGE. Representative Western blots of A
1-42
oligomers and fibrils incubated for 0 and 24 h, separated by
SDS-PAGE on a 12% NuPAGE bis-Tris gel, and probed with the monoclonal
antibody 6E10 (recognizing residues 1-17 of A
) are shown.
Oligomeric and fibrillar preparations of A
1-42 were prepared as
described for A. Samples were visualized by enhanced
chemiluminescence. The representative figure shows 0- and 24-h
oligomers (lanes 1 and 2) and fibrils
(lanes 3 and 4).
1-42 remained as unassembled structures measuring ~1.0 nm (Fig. 2A,
insets), although a few oligomers measuring between 2 and 4 nm were also detected (Fig. 2A). After a 24-h incubation
under oligomer-forming conditions, A
1-42 assembled into
predominantly 2-4-nm oligomeric structures (Fig. 2A). No
fibril formation was detected under these conditions. Within 24 h
under fibril-forming conditions, A
1-42 converted to ~4-nm amyloid
fibrils that extended over several microns, some of which had a
semi-periodic structure along the fibril axis. As previously reported,
AFM measurements may underestimate the diameter of A
structures
because of sample compression by the AFM probe (25, 51).
1-42 monomer, trimer,
and tetramer for unaggregated (0 h) samples incubated under both
oligomer- and fibril-forming conditions (Fig. 2B,
lanes 1 and 3, respectively). Larger oligomeric
assemblies ranging from 30 to 60 kDa were detected after incubation for
24 h under oligomer-forming conditions. Additionally, there was an
increase in the trimer and tetramer bands, as well as smearing between
tetramer and monomer, possibly indicating interconversion between these
assemblies during electrophoresis (Fig. 2B, lane
2). After 24 h, A
1-42 samples incubated under
fibril-forming conditions contained high molecular weight
immunoreactive A
that remained in the well, less abundant 30-60-kDa
large oligomers, and a smear between tetramer and monomer (Fig.
2B, lane 4). The abundant tetramer detected after
24 h under oligomer-forming conditions was not present under
fibril-forming conditions, although the increase in the trimer band and
smearing between trimer and monomer were present in both preparations
at 24 h.
1-42 oligomers and fibrils was
variable. In our previous report we observed more SDS-stable structures
between monomer and tetramer, particularly dimer, in the 24-h fibril
preparations than reported herein (24). Common trends observed in both
studies for oligomeric preparations include detection of an abundant
tetramer and trimer. Fibril preparations typically include less
abundant tetramer and the presence of >60-kDa large aggregates,
including A
immunoreactive material that remains in the well of the
gel. Unaggregated (0 h) A
1-42 samples typically contain some trimer
and tetramer, in addition to monomer. Comparable results are obtained
with A
monoclonal antibodies 6E10, recognizing A
residues 1-16
(Fig. 2B), and 4G8, recognizing A
residues 17-24 (data
not shown). The NuPAGE gel system used in these studies yields slightly
different banding patterns with these two antibodies than Tris/Tricine
and Tris/glycine gels where more dimer is detected (data not shown).
Thus, in contrast to the limited and variable information gained by the
gel-dependent detection of SDS-stable A
structures, AFM
analysis clearly differentiates between oligomeric and fibrillar
A
1-42 structures that form under the conditions described for these experiments.
1-42 Assembly Is Dependent on Incubation Time and Peptide
Concentration--
Total peptide concentration is known to influence
the fibrillogenesis of A
(47). To determine how A
peptide
concentration affects assembly under oligomer- and fibril-forming
conditions, A
1-42 concentrations ranging from 10 to 100 µM were prepared and analyzed immediately after dilution
(0 h), after 24 h, and after 1 week.
1-42 oligomers formed rapidly
under the oligomer-forming conditions at concentrations greater than 25 µM (Fig. 3A,
0 h). In general, a concentration-dependent increase in the number of 2-4-nm oligomers was observed between 10 and
100 µM for all three incubation periods (Fig.
3A). After 24 h, a slight increase in the number of
2-4-nm oligomers, but no fibril formation, was detected at each of the
four concentrations (Fig. 3A, 24 h). After 1 week, a concentration-dependent increase in protofibrils
(measuring <200 nm) and some short fibrils both straight and curved
(measuring <1 µm) were observed in the oligomer preparations at 50 and 100 µM, with very few protofibrils in 25 µM samples, and no protofibrils in the 10 µM A
1-42 solutions (Fig. 3A, 1 week). Of interest, no mature fibrils (measuring >1 µm) were
detected at any of the time points or concentrations despite the
detection of protofibrils and short fibrils, particularly abundant at
100 µM and 1-week incubation time. These results
demonstrate that A
1-42 rapidly forms oligomers under these
conditions and that only after extended incubation at A
1
42
concentrations
25 µM were significant numbers of
protofibrils and slightly longer structures detected.
View larger version (108K):
[in a new window]
Fig. 3.
A 1-42
assembly is dependent on incubation time and peptide
concentration. 5 mM A
1-42 Me2SO stocks
were diluted to 10, 25, 50, or 100 µM under oligomer-
(A) or fibril-forming conditions, (B) as
described in the legend for Fig. 2. Samples were prepared for AFM
analysis immediately after dilution (0-h), after 24 h,
and after 1 week of incubation. Representative 2 × 2-µm x-y,
10-nm total z-range AFM images are shown.
1-42
forms 2-4-nm oligomers, in addition to some protofibrils at 50 and 100 µM (Fig. 3B, 0 h). After incubation
for 24 h, no fibril formation could be detected at 10 µM, and mixed individual 4-nm fibrils, protofibrils, and
oligomers were detected at 25 µM, 4-nm fibrils mixed with
some 2-4-nm oligomers were present at 50 µM, and dense
networks of ~4-nm fibrils were present at 100 µM. After
1 week, only oligomeric and protofibrillar structures could be detected
at 10 µM A
1-42. At 25 µM, mixed
oligomeric, protofibrillar, and fibrillar A
1-42 structures were
observed, with the fibrils appearing more rigid than at 24 h. At
both 50 and 100 µM, dense fields fibrils were present.
These results indicate that A
1-42 incubated under fibril-forming
conditions results in concentration- and time-dependent
fibril formation and that under these conditions, homogeneous fibril
populations form within 24 h at 100 µM A
1-42 and
are stable for at least 1 week.
1-42 Assembly Is Dependent on Temperature--
Temperature
has been shown previously to be an important factor in fibril assembly
(52). Here we investigated the effects of temperature on oligomer- and
fibril-forming conditions. Solutions of 100 µM A
1-42
incubated at 4 °C under the standard oligomer-forming conditions
were also incubated at room temperature and 37 °C. Solutions of 100 µM A
1-42 incubated at 37 °C under the standard fibril-forming conditions were also incubated at room temperature and
4 °C. The resulting structures were characterized by AFM (Fig. 4A) and Western blot analysis
of SDS-PAGE (Fig. 4B).
View larger version (46K):
[in a new window]
Fig. 4.
A 1-42 assembly is
dependent on temperature. A, AFM. A
1-42 oligomers
and fibrils prepared as described in the legend for Fig. 2 were
incubated at 4 °C, room temperature, or 37 °C for 24 h.
Representative 2 × 2-µm x-y, 10-nm total z-range AFM images are
shown. B, Western analysis of SDS-PAGE. Representative
Western blots of samples prepared as described for A and
analyzed as described in the legend for Fig. 2 are shown. The figure
shows oligomers and fibrils at 4 °C (lanes 1 and
4), room temperature (lanes 2 and 5),
and 37 °C (lanes 3 and 6), respectively.
1-42 incubated for 24 h produced homogeneous solutions of
2-4-nm oligomers comparable with Fig. 2A (Fig.
4A, 4 °C). At room temperature, the solution
remained predominately oligomeric with a low concentration of short
protofibrils (Fig. 4A, RT). At 37 °C, the
solution contained both oligomers and an increased number of
protofibrils (Fig. 4A, 37 °C). These results
indicate that uniform populations of oligomers form at 4 °C, and
increasing temperature produces limited protofibril formation within
24 h at 100 µM A
1-42. However, as with
incubation time and peptide concentration, although protofibrils were
present, no extended fibrils were observed with the oligomer-forming
conditions at any of the temperatures.
1-42 incubated for 24 h produced extended ~4-nm fibrils,
comparable with Fig. 2A (Fig. 4A,
37 °C). When the incubation temperature was lowered to
room temperature, A
1-42 continued to form extended fibrils,
although an increase in the oligomeric background was observed (Fig.
4A, RT). Solutions incubated at 4 °C contained predominately unassembled peptide and 2-4-nm oligomeric structures, in
addition to a sparse population of 2-4-nm-diameter fibrils less than 2 µm in length. In summary, incubation at 37 °C produces the most
uniform fibril populations, with fibril density decreasing significantly with temperature.
1-42 incubated under
oligomer-forming conditions detected SDS-stable tetramer, trimer, and
monomer (Fig. 4B, lane 1). When the incubation
temperature was raised to room temperature and 37 °C, there was a
decrease in the amount of monomer and an increase in the presence of
large oligomers (Fig. 4B, lanes 2 and
3). Under fibril-forming conditions, the majority of the
large fibril assemblies were not SDS-stable as A
appeared as
monomer, trimer, and tetramer, and decreasing temperature induced an
increase in the amount of trimer and tetramer (Fig. 4B,
lanes 4-6).
1-42 Assembly Is Dependent on Ionic Strength and pH--
Two
primary differences between the oligomer- and fibril-forming conditions
are ionic strength and pH. For the oligomer-forming conditions,
A
1-42 is incubated in cell culture medium at physiologic ionic
strength and neutral pH. For fibril-forming conditions, A
1-42 is
incubated in dilute HCl at low ionic strength and acidic pH. To
determine how the combination of ionic strength and pH influence the
assembly of A
1-42, 100 µM A
1-42 solutions were incubated at 37 °C at acidic pH and neutral pH and at both low and
physiologic ionic strength. At acidic pH and low ionic strength (standard fibril-forming conditions), extended ~40-nm fibrils were
detected by AFM, comparable with Fig. 2A (Fig.
5A). Adding 150 mM
NaCl while maintaining the acidic pH resulted in the formation of dense
fibrillar aggregates that vary in z-height from 5 to greater than 25 nm
(Fig. 5A). Note that the z-height range representation for
this image was increased from 10 to 25 nm because of the size of the
fibrillar aggregates. At neutral pH and low ionic strength, A
1-42
solutions remained primarily oligomeric comparable with Fig.
2A, in addition to several short fibrils (Fig.
5A). When the pH was neutral, and 150 mM NaCl
was added, A
1-42 oligomers coalesced into primarily small
oligomeric aggregates with z-height values measuring ~5-8 nm and
protofibrils.
View larger version (51K):
[in a new window]
Fig. 5.
A 1-42 assembly is
dependent on ionic strength and pH. A, AFM. 5 mM A
1-42 in Me2SO stocks were diluted to
100 µM in either 10 mM HCl (low ionic
strength, acidic pH), 10 mM HCl + 150 mM NaCl (acidic pH, physiologic ionic
strength), 10 mM Tris, pH 7.4 (neutral pH),
or 10 mM Tris, pH 7.4, + 150 mM NaCl
(neutral pH, physiologic ionic strength). Samples
were prepared after a 24-h incubation at 37 °C. Representative
2 × 2-µm x-y, 10-nm total z-range AFM images are shown, except
for the image of the acidic pH, physiologic ionic strength condition,
which is scaled to 2 × 2-µm x-y, 25-nm total z-range.
B, Western analysis of SDS-PAGE. Representative Western
blots of samples prepared as described for A and analyzed as
described in the legend for Fig. 2 are shown. The figure shows HCl
(lane 1), HCl + NaCl (lane 2), Tris (lane
3), and Tris + NaCl (lane 4).
1-42
assemblies formed in F-12 culture medium (Fig. 2A) and in a
buffered salt solution (Fig. 5A). In F-12 culture medium, more evenly dispersed fields of 2-4-nm oligomers were observed by AFM,
whereas A
1-42 incubated in buffered salt solutions yielded more
aggregated clusters of oligomers and protofibrils. This may result from
the differences in the inorganic salts or the presence of trace metals
and/or amino acids in F-12 culture medium, initially selected based on
oligomer-forming conditions reported in the literature (19). In an
effort to simplify the oligomer-forming solution, several solutions of
comparable pH and ionic strength were screened, including Dulbecco's
modified Eagle's medium, artificial cerebral spinal fluid, and
buffered salt solutions containing trace metals and
N,N-dimethylglycine in place of individual amino acids. Although A
1-42 formed oligomeric assemblies in each of these
solutions as determined by AFM (data not shown), globular structures
were present in solution controls in the absence of A
, confounding
the detection of A
oligomers prepared in these solutions.
Ultimately, the most consistent preparations with the lowest background
levels for AFM were obtained using F-12 cell culture medium.
1-42 tetramer, trimer,
and monomer (Fig. 5B, lane 1). The addition of
150 mM NaCl at acidic pH corresponded to a slight decrease
in trimer and a slight increase in large oligomers (Fig. 5B,
lane 2). When A
1-42 was incubated at neutral pH and low
ionic strength, a prominent smear of large oligomers was detected, in
addition to A
tetramer, trimer, and monomer. The addition of 150 mM NaCl at neutral pH resulted in a decrease in the large
oligomers (Fig. 5B, lane 4) relative to the same
sample incubated without additional NaCl. The relative similarity
between acidic pH samples analyzed by Western blot indicates that most
of the dramatic conformational differences detected by AFM cannot be
detected by Western analysis of SDS-PAGE.
1-42 Assemblies--
Sedimentation analysis has
been used previously (53, 54) to characterize the aggregation state of
A
under a variety of conditions. In the present study, the
solubilities of unaggregated A
1
42, oligomers, and fibrils (Fig. 2)
and the fibrillar aggregates (Fig. 5A, acidic pH
and physiologic ionic strength) were estimated after
centrifugation for 30 min at 16,000 and 100,000 × g.
The soluble A
1-42 structures remaining in the supernatants were
characterized by AFM. Unaggregated A
1-42 total and supernatant
fractions (Fig. 6) contained structures
that measured 1.0 (± 0.3) nm (Fig. 6, insets) and very few
oligomeric assemblies. The total oligomeric and fibrillar A
1-42, as
well as the 16,000 and 100,000 × g supernatants (Fig.
6), were comparable in size and morphology to the assemblies described
for Fig. 2A. A decrease in the number of fibrils was observed in the 100,000 × g supernatant fraction,
indicating that these fibrils may eventually sediment with prolonged
ultracentrifugation. In contrast, fibrillar aggregates could not be
detected by AFM in either 16,000 or 100,000 × g
supernatants. No unassembled or oligomeric structures were detected in
the fibrillar aggregate 100,000 × g supernatant
fraction at high resolution by AFM (Fig. 6, inset).
View larger version (134K):
[in a new window]
Fig. 6.
AFM analysis of the solubility of
A 1-42 assemblies. Oligomeric and
fibrillar A
1-42 samples were prepared as described in the legend
for Fig. 2. Unaggregated solutions of A
1-42 were prepared by
dilution of 5 mM A
1-42 Me2SO stock solution
to 10 µM in ice-cold dH2O. Fibrillar
aggregates of A
1-42 were prepared by incubation in 10 mM HCl + 150 mM NaCl for 24 h at 37 °C
as described in the legend for Fig. 5. 100 µM samples
were diluted 1:10 and either not centrifuged (total) or
centrifuged for 30 min at 16,000 or 100,000 × g.
Similar results were obtained if the preparations were diluted before
or after centrifugation. Diluting the samples prior to centrifugation
facilitated supernatant and pellet recovery. Total and supernatant
fractions were prepared for AFM analysis. Representative 2 × 2-µm x-y, 10-nm total z-range AFM images are shown. Inset
images, 200 × 200-nm x-y, 2-nm total z-range.
peptide remained in the
supernatants when unaggregated, oligomeric, and fibrillar preparations were centrifuged at 16,000 × g for 30 min
(Table II). Fibrillar aggregates
were more insoluble yielding ~23% recovery from the 16,000 × g supernatants. Unaggregated and oligomeric preparations remained in the supernatant at 100,000 × g, whereas
the fibrillar conformations began to sediment with ~51% remaining in
the supernatant. Fibrillar aggregates completely sedimented at
100,000 × g. These percent-recovery results (Table II)
agree with the presence of unaggregated, oligomeric, and fibrillar
assemblies detected by AFM in supernatant fractions (Fig. 6). In
addition, the absence of fibrillar aggregates in 16,000 and
100,000 × g supernatants (Fig. 6) coincides with low
percent-recovery data (Table II). Taken together, these data indicate
that unaggregated, oligomeric, and to some extent fibrillar,
preparations of A
1-42 are soluble and that fibrillar aggregates
formed at physiologic ionic strength and acidic pH are insoluble.
Quantitation of the solubility of A1-42 assemblies
1-42 were prepared as described in the legend for
Fig. 6A. Samples diluted 1:10 were either not centrifuged
(total) or centrifuged for 30 minutes at 16,000 × g or
100,000 × g. Total, supernatant, and pellet fractions
were extracted in 70% formic acid and quantitated by dot blot
analysis. Values represent means (± S.E.M) of the percent recovery of
the total (uncentrifuged) in the supernatant and pellet fractions.
Control samples were prepared in an identical manner using the same
tubes and solutions in the absence of centrifugation. Values from these
control samples were used to determine non-sedimenting background and
were subtracted from test samples.
1-40 Requires Longer Incubation Times to Form Oligomers and
Fibrils Comparable with A
1-42 Oligomers and Fibrils--
A
1-40
is the predominant species of A
produced from the variable
C-terminal cleavage of APP. The oligomer- and fibril-forming conditions
defined for A
1-42 were applied to A
1-40. AFM characterization of A
1-40 immediately upon dilution reveals a fairly homogeneous field of unaggregated peptide structures (Fig.
7A). High resolution scans
detect dense fields peptide measuring 1.0 (±0.3) nm (Fig. 7A, insets for 0 h). No significant
changes in A
1-40 assembly state could be detected under the
A
1-42 oligomer- or fibril-forming conditions by AFM after 24 h
(Fig. 7A), although some short, ~4-nm-diameter A
1-40
fibrils and 2-4-nm oligomers were detected under fibril-forming conditions. Both A
1-40 solutions were allowed to incubate for 6 weeks. At this time point, ~4-nm assemblies comparable in size to
24-h A
1-42 oligomers in Fig. 2A were detected under
oligomer-forming conditions (Fig. 7A). At 6-weeks, solutions
of A
1-40 incubated under fibril-forming conditions contained
several 2-6-nm oligomeric assemblies and ~4-6-nm-diameter fibrils
(Fig. 7A), slightly larger than 24-h A
1-42 fibrils (~4
nm) in Fig. 2A.
View larger version (56K):
[in a new window]
Fig. 7.
A 1-40 requires
longer incubation times to form oligomers and fibrils comparable with
A
1-42 oligomers and fibrils.
A, AFM. Oligomeric and fibrillar A
1-40 samples prepared
as described in the legend for Fig. 2 were mounted for AFM analysis
immediately after dilution (0-h), 24 h, and 6 weeks.
Representative 2 × 2-µm x-y, 10-nm total z-range AFM images are
shown. Inset images, 200 × 200-nm x-y, 2-nm total
z-range. B, Western analysis of SDS-PAGE. Representative
Western blots of A
1-40 samples prepared as described in
A were analyzed as described in the legend for Fig. 2.
Oligomers and fibrils were sampled at 0 h (lanes 1 and
3), 24 h (lanes 2 and 4), and 6 weeks (lanes 5 and 6), respectively.
1-40 incubated under
oligomer (Fig. 7B, lanes 1 and 2)- and
fibril (Fig. 7B, lanes 3 and
4)-forming conditions revealed A
tetramer and monomer in both preparations at 0 (Fig. 7B, lanes 1 and
3)- and 24 (Fig. 7B, lanes 2 and
4)-h time points. Faint bands of larger oligomers between 30 and 60 kDa are present in both 24-h samples, and a faint smear centered
at ~60 kDa can be detected in the 24-h sample incubated under
fibril-forming conditions. Western blot analysis of long-aged A
1-40
under oligomer-forming conditions (Fig. 7B, lane
5) reveals monomer and a smear around ~60 kDa. Long-aged A
1-40 solutions incubated under fibril-forming conditions (Fig. 7B, lane 6) contain a faint monomer band, a smear
around ~60 kDa, and large aggregates >60 kDa extending up to the
well of the gel.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
plays in normal humans or in those
that suffer from AD remain unclear. Definitive genetic studies, as well
as a large number of in vitro and in vivo
experiments, provide compelling evidence that A
1-42 is a direct
causal agent for AD. What remains unclear is the relevant
conformational assembly(s) of A
1
42 associated with the disease
pathology. Early studies provided evidence that A
had to assemble
into a fibrillar conformation to induce significant levels of
neurotoxicity in vitro (55-57) or A
-induced tau
phosphorylation (58). Recently, a number of studies have uncovered both
in vitro and in vivo biological activities associated with soluble oligomeric and protofibrillar conformations of
A
, challenging the idea that fibrillar amyloid is the causative pathogenic agent in AD. The first studies that characterized soluble oligomeric A
(18), a dimer-containing A
1-42 fraction (15), ADDLs
(amyloid-derived diffusible
ligands) (19), and protofibrils (51, 59) introduced the
concept that amyloid fibrils are not the only biologically relevant
A
conformation.
1-42 pretreated in HFIP and solubilized in
Me2SO produced uniform peptide solutions that were free from the structural history present in commercially available lyophilized stocks, laying the critical foundation for controlled aggregation studies. From this structurally and chemically identical starting material, conformationally distinct populations of oligomeric and fibrillar A
were produced by controlling the incubation time, concentration, temperature, pH, ionic strength, and A
species. Incubation time had a moderate effect on A
1
42 conformation in that
the majority of the structures that formed within 24 h remained for up to 1 week. Concentration also had a moderate effect on conformation, particularly fibril formation, as the critical
concentration for fibrillogenesis under these conditions appears to be
between 10 and 50 µM, whereas oligomer formation can
proceed at much lower concentrations (Fig. 3). Temperature also
appeared to influence the rate of fibril formation to a greater extent
than oligomer formation. Oligomer and protofibril formation was favored
at neutral pH, and fibril formation was favored at acidic pH. Ionic
strength had a significant effect on conformation, particularly the
coalescence of soluble fibrillar A
1-42 into compact insoluble
fibril aggregates at 150 mM NaCl. When these oligomer- and
fibril-forming conditions for A
1-42 were applied to A
1-40,
significantly longer periods of time were required for oligomeric and
fibrillar structures to form that resembled those formed by A
1-42
within 24 h. Taken together, these results define specific
conditions for reproducibly controlling the formation of unaggregated,
oligomeric, fibrillar, and fibrillar aggregate conformations of
A
1-42.
are neurotoxic and impair electrophysiologic activity. Although experiments using a single conformation of A
can be informative, comparative experiments using multiple conformations of A
derived from a single starting material will further our understanding of how
structure affects function. Using this approach, we demonstrated recently (24) a significant difference in neurotoxicity among unaggregated, oligomeric, and fibrillar A
1-42. Oligomers inhibited neuronal viability 10-fold more than fibrils and ~40-fold more than
unaggregated peptide, with oligomer-induced toxicity significant at 10 nM. In addition, we were able to differentiate by structure and neurotoxic activity wild-type A
1-42 from isoforms containing known familial mutations (24). An in vitro comparative
analysis using these preparations also demonstrated a significant
increase in glial activation by A
1-42 oligomers compared with
fibrils, as measured by morphological changes and increased expression of inflammatory markers including nitric oxide, inducible
nitric-oxide synthase, and
interlukin-1
.2
activity reported in the literature is likely
because of the structural heterogeneity present in commercial preparations that are chemically identical. Commercial sources of
synthetic A
suggest resuspension in either H2O or
Me2SO, and these solubilization procedures are reported
throughout the literature. In this study, AFM analysis of lyophilized
A
stocks directly resuspended in H2O detected a wide
variety of structures including oligomers, fibrils, and globular
aggregates. These structures could seed further aggregation, strongly
influencing the formation and biological activity of the resulting
solutions. Seed-free preparations of A
have been described using the
polar solvent Me2SO, in conjunction with bath sonication
and/or filtration (61, 62). Surprisingly, in this study AFM analysis of
A
solutions in Me2SO that appeared initially uniform and
unaggregated revealed fibril formation over time. Previous studies
characterizing the formation of A
protofibrils (59) and oligomers
(63) have emphasized the importance of removing preexisting seeds from
A
stocks by size-exclusion chromatography. Although this approach appears to be successful, it is not always practical for the routine preparation of the large number of small peptide aliquots frequently required for biological assays or screening. Centrifugation is another
method commonly used to separate soluble A
from insoluble fibrillar
amyloid in both extracted tissue (64, 65) and in vitro
aggregation preparations (66, 67). In this study, the fibrillar
aggregates formed at acidic pH and physiologic ionic strength were
insoluble and sedimented upon centrifugation at both 16,000 and
100,000 × g (see Fig. 6 and Table II). However, fibrillar preparations of A
1-42 remained in the supernatant at 16,000 × g and only partially sedimented at
100,000 × g (see Fig. 6 and Table II). Although these
results confirm the solubility of unaggregated and oligomeric A
1-42
preparations, and the insolubility of fibrillar aggregates, the
importance of differentiating between preparations defined as
"soluble" versus those that are truly "fibril-free" is emphasized by the solubility of fibril preparations.
structural conformations that formed under the conditions used
in this study were readily discriminated by AFM, whereas Western blot
analysis of SDS-PAGE provided only limited information. In light of the
structural differences detected by AFM, it is clear that some of the
A
1-42 and A
1-40 assemblies that form under the oligomer- and
fibril-forming conditions used in this study dissociate during
SDS-PAGE. In particular, the distinct structural morphologies in
A
1-42 resulting from changes in pH and ionic strength detected by
AFM (Fig. 5A) could not be differentiated clearly by Western
blot analysis of SDS-PAGE (Fig. 5B). In addition, the
electrophoretic process and the combination buffer, glycerol, and
detergent present in SDS sample buffer may actually induce the
formation of SDS-stable oligomers as reported previously (68). This may
account for the SDS-stable oligomers, trimers, and tetramers detected
in preparations of A
1-40 and A
1-42 that appeared unaggregated by AFM (see Figs. 2, 6, and 7). In addition, when distinct A
1-40 oligomers and fibrils were detected by AFM, no SDS-stable oligomeric bands were present.
1-42 preparations incubated
under oligomer- and fibril-forming conditions. Oligomers produced
slightly more SDS-stable tetramer and large oligomer bands than
fibrils, and both conditions showed more SDS-stable trimer and tetramer
than unaggregated peptide. Under fibril-forming conditions, the amount
of SDS-stable trimer and tetramer appears to vary and may be in
equilibrium with unstable assemblies that break down to monomer, the
primary conformation observed in fibril preparations in our previous
study (24). An indication of this molecular rearrangement between
monomer and tetramer was indicated by the pronounced boundary and smear
between these bands (Fig. 2B, lanes 2 and
4). These intermediates appear unstable during SDS-PAGE and
form under both oligomer- and fibril-forming conditions. Interestingly,
unaggregated preparations (Fig. 2B, lanes 1 and 3) that did contain some trimer and tetramer did not contain
a smear between tetramer and monomer.
1
40
assembly and specifically detected oligomeric intermediates that were
not SDS-stable. Further evidence that SDS-PAGE alone cannot detect
A
1
40 monomer-oligomer distribution as it exists in solution comes
from studies using analytical ultracentrifugation (63) and mass
spectrometry (70). These studies, like the covalent cross-linking
study, revealed a more complex, dynamic equilibrium between monomer and
oligomer. Thus, it does not appear possible to equate an aggregate
conformation of A
1-40 or 1-42 as visualized by AFM to a particular
molecular weight band from Western blot analysis of SDS-PAGE.
1-40, in which Asp or His
residues were replaced by Asn and Gln, demonstrated that a strong
relationship exists between the protonation state at these positions
and
-helical content (74). Fibrillogenesis is thought to proceed
through an
-helical intermediate, and stabilization of local helical
structure can accelerate this process (40). This may account for the
very rapid conversion of unassembled A
1-42 to extended fibrils
within 24 h of incubation at acidic pH as observed in this study.
1-42 fibrils form but remain as individual extended structures for at least
a week (Fig. 3B) and are resistant to sedimentation (see Fig. 6 and Table II). However, the salt-dependent
coalescence of A
1
42 fibrils into supramolecular fibril aggregates
(see Fig. 5A and Fig. 6) argues that exposed hydrophobic
patches present on the surface of A
1-42 fibrils can drive the
formation of dense, insoluble fibrillar aggregates. Alternatively, this
process may be the result of polar interactions screened by the
increased ionic strength, although it is more likely that the fibrillar aggregates resulted from non-polar surface interactions. Interestingly, the presence or absence of 150 mM NaCl did not have as
significant of an effect on the coalescence of primarily oligomeric
assemblies formed at neutral pH (Fig. 5A), indicating that
these oligomeric assemblies may have already sequestered non-polar
surfaces by adopting a favorable conformation.
1-42 and A
1-40 conformations that were observed by AFM under the A
1-42 fibril- and
oligomer-forming conditions. The additional hydrophobic Ile-Ala residues found at the C termini of A
1-42 were necessary for the fibril and oligomer formation within 24 h as detected by AFM, as
well as the different patterns of SDS-stable assemblies detected by
Western blot analysis of SDS-PAGE. Therefore, these hydrophobic residues in A
1-42 appear to play an important role in
vitro in the formation of stable oligomers fibrils. These findings
provide evidence that hydrophobic forces play an important role in both differentiating A
1-40 and A
1-42 species and in the formation of
oligomeric and fibrillar aggregates of A
1-42.
at
methionine 35 alters the polarity at that position and may influence
hydrophobic or ionic interactions necessary for fibril formation (28).
In this recent study, AFM analysis differentiated A
1-42 solutions
containing mixed short fibrils from A
1-42Met35ox
solutions containing only small, non-fibrillar aggregates. These results further emphasize how the distribution of oligomeric and fibrillar A
1-42 assemblies is affected by factors that influence molecular interactions.
concentrations that are decidedly
non-physiologic. The trends observed in this study suggest that at
lower concentrations and under physiologic solution conditions
A
1-42 adopts a favorable, stable oligomeric conformation. At higher
peptide concentrations and elevated temperature under oligomer-forming
conditions, soluble oligomeric, protofibrillar, and short fibrillar
conformations coexist, indicating that these structures may be in a
conformational equilibrium. However, it is unlikely that these soluble
assemblies represent on-pathway fibril intermediates under the
oligomer-forming conditions used in this study. If this were the case,
then increasing the peptide concentration, increasing temperature, or
extending the incubation time would result in the rapid conversion of
oligomeric, protofibrillar, and short fibrillar conformations to mature
fibrillar conformations >1 µm in length. This result was not
observed (see Fig. 3A and Fig. 4A). In
vitro studies characterizing A
protofibrils as metastable
fibril precursors demonstrated that in the absence of fibrillar seeds,
the conversion of protofibrils to mature amyloid fibrils is a slow
process (26). Additionally, the authors noted a disappearance of
protofibrils that preceded the appearance of mature fibrils (26, 51),
and in vitro dialysis experiments using radiolabeled A
indicate that protofibrils are in equilibrium with low molecular weight
A
(60). Whether in vivo oligomeric and protofibrillar
forms of A
1-42 represent stable end point assemblies that
dissociate to monomer before proceeding to form mature fibrils via a
nucleation-dependent polymerization mechanism remains to be
determined. Amyloid deposits that form in vivo may result
from microenvironments where nucleation-dependent
polymerization can proceed and are only indirectly responsible for
neurodegeneration and cognitive impairment in AD. However, a second and
potentially more biologically significant soluble pool of oligomeric
and/or protofibrillar A
favored by A
1-42 may play a more direct
and deleterious role in AD pathology. These remaining mechanistic questions can be addressed by additional kinetic studies.
in the neuropathology of AD. With increased
significance comes an increased need for a better understanding of how
A
structure affects function. The in vitro experiments reported in this study provide unique insights into the conditions that
control the complex process of A
assembly by utilizing a single,
homogenous starting material to produce multiple, distinct conformational species by varying the incubation conditions.
Understanding the assembly and biological activity of both oligomeric
and fibrillar forms of A
is important both mechanistically and
because it influences whether therapeutic strategies in AD are best
aimed at eliminating oligomeric assemblies of A
or plaque
deposition. Furthermore, therapeutic approaches solely focused on
fibril destabilization may have the undesirable side effect of
increasing a soluble pool of neurotoxic A
oligomers and
protofibrils. Experiments that take into account A
conformational
differences will yield more consistent and interpretable results,
providing the biological infrastructure for developing successful
therapeutics for the treatment of AD.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Andrew Murphy for help with the CD experiments and Arlene Manelli, Tom Brodowski, and Jillian Hibler for the Western blot experiments and solubility studies. We thankfully acknowledge the use of instruments in the Keck Biophysics Facility at Northwestern University.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institute of Health Grants AG19121 (to M. J. L. D.), AG13496 (to G. A. K.), and AG15501 (to G. A. K.), Alzheimer's Association Grant IIRG-01-3073 (to M. J. L. D.), and the Charles Walgreen, Jr. Fund (to M. J. L. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Acumen Pharmaceuticals, Inc., Glenview, IL.
To whom correspondence should be addressed: ENH Research Inst.,
1801 Maple Ave., Suite 6240, Evanston, IL 60201. Tel.: 847-467-5975; Fax: 847-467-7781; E-mail: m-ladu@Northwestern.edu.
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M210207200
2 M. J. LaDu and L. J. Van Eldik, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
A, amyloid-
;
AFM, atomic force microscopy;
HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol;
bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
MES, 4-morpholineethanesulfonic acid;
dH2O, distilled
H2O;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Goate, A., Chartier-Harlon, M. C., Mullan, M., Brown, J., Crawford, F., Fidani, L., Giuffra, L., Haynes, A., Irving, N., James, L., Mant, R., Newton, P., Roote, K., Roques, P., Talbot, C., Pericak-Vance, M., Roses, A., Williamson, R., Rossor, M., Owen, M., and Hardy, J. (1991) Nature 349, 704-706[CrossRef][Medline] [Order article via Infotrieve] |
2. | Citron, M., Oltersdorf, T., Haas, C., McConlogue, L., Hung, A. Y., Seubert, P., Vigo-Pelfrey, C., Lieberburg, I., and Selkoe, D. J. (1992) Nature 360, 672-674[CrossRef][Medline] [Order article via Infotrieve] |
3. | Cai, X.-D., Golde, T. E., and Younkin, S. G. (1993) Science 259, 514-516[Medline] [Order article via Infotrieve] |
4. | Citron, M., Westaway, D., Xia, W., Carlson, G., Diehl, T., Levesque, G., Johnson-Wood, K., Lee, M., Seubert, P., Davis, A., Kholodenko, D., Motter, R., Sherrington, R., Perry, B., Yao, H., Strome, R., Lieberburg, I., Rommens, J., Kim, S., Schenk, D., Fraser, P., St. George Hyslop, P., and Selkoe, D. J. (1997) Nature Med. 3, 67-72[Medline] [Order article via Infotrieve] |
5. | Selkoe, D. J., and Podlisny, M. B. (2002) Annu. Rev. Genomics Hum. Genet. 3, 67-99 |
6. | Cummings, B. J., and Cotman, C. W. (1995) Lancet 346, 1524-1528[CrossRef][Medline] [Order article via Infotrieve] |
7. |
Naslund, J.,
Haroutunian, V.,
Mohs, R.,
Davis, K. L.,
Davies, P.,
Greengard, P.,
and Buxbaum, J. D.
(2000)
JAMA
283,
1571-1577 |
8. |
Irizarry, M. C.,
Soriano, F.,
McNamara, M.,
Page, K. J.,
Schenk, D.,
Games, D.,
and Hyman, B. T.
(1997)
J. Neurosci.
17,
7053-7059 |
9. | Chui, D. H., Tanahashi, H., Ozawa, K., Ikeda, S., Checler, F., Ueda, O., Suzuki, H., Araki, W., Inoue, H., Shirotani, K., Takahashi, K., Gallyas, F., and Tabira, T. (1999) Nat. Med. 5, 560-564[CrossRef][Medline] [Order article via Infotrieve] |
10. | Klein, W. L. (2002) Neurobiol. Aging 23, 231-235[CrossRef][Medline] [Order article via Infotrieve] |
11. | Walsh, D. M., Tseng, B. P., Rydel, R. E., Podlisny, M. B., and Selkoe, D. J. (2000) Biochemistry 39, 10831-10839[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Hsia, A. Y.,
Masliah, E.,
McConlogue, L., Yu, G.-Q.,
Tatsuno, G.,
Hu, K.,
Kholodenko, D.,
Malenka, R. C.,
Nicoll, R. A.,
and Mucke, L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3228-3233 |
13. | Nilsberth, C., Westlind-Danielsson, A., Eckman, C. B., Condron, M. M., Azelman, K., Forsell, C., Stenh, C., Luthman, J., Teplow, D. B., Younkin, S. G., Naslund, J., and Lannfelt, L. (2001) Nat. Neurosci. 4, 887-893[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Kuo, Y. M.,
Emmerling, M. R.,
Vigo-Pelfrey, C.,
Kasunic, T. C.,
Kirkpatrick, J. B.,
Murdoch, G. H.,
Ball, M. J.,
and Roher, A. E.
(1996)
J. Biol. Chem.
271,
4077-4081 |
15. |
Roher, A. E.,
Chaney, M. O.,
Kuo, Y. M.,
Webster, S. D.,
Stine, W. B.,
Haverkamp, L. J.,
Woods, A. S.,
Cotter, R. J.,
Tuohy, J. M.,
Krafft, G. A.,
Bonnell, B. S.,
and Emmerling, M. R.
(1996)
J. Biol. Chem.
271,
20631-20635 |
16. | Podlisny, M. B., Walsh, D. M., Amarante, P., Ostaszewski, B. L., Stimson, E. R., Maggio, J. E., Teplow, D. B., and Selkoe, D. J. (1998) Biochemistry 37, 3602-3611[CrossRef][Medline] [Order article via Infotrieve] |
17. | Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., and Selkoe, D. J. (2002) Nature 416, 535-539[CrossRef][Medline] [Order article via Infotrieve] |
18. | Oda, T., Wals, P., Osterburg, H. H., Johnson, S. A., Pasinetti, G. M., Morgan, T. E., Rozovsky, I., Stine, W. B., Snyder, S. W., Holzman, T. F., Krafft, G. A., and Finch, C. E. (1995) Exp. Neurol. 136, 22-31[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Lambert, M. P.,
Barlow, A. K.,
Chromy, B. A.,
Edwards, C.,
Freed, R.,
Liosatos, M.,
Morgan, T. E.,
Rozovsky, I.,
Trommer, B.,
Viola, K. L.,
Wals, P.,
Zhang, C.,
Finch, C. E.,
Krafft, G. A.,
and Klein, W. L.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6448-6453 |
20. |
Hartley, D. M.,
Walsh, D. M.,
Ye, C. P.,
Diehl, T.,
Vasquez, S.,
Vassilev, P. M.,
Teplow, D. B.,
and Selkoe, D. J.
(1999)
J. Neurosci.
19,
8876-8884 |
21. | Wang, H. W., Pasternak, J. F., Kuo, H., Ristic, H., Lambert, M. P., Chromy, B., Viola, K. L., Klein, W. L., Stine, W. B., Krafft, G. A., and Trommer, B. L. (2002) Brain Res. 924, 133-140[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Hardy, J.,
and Selkoe, D. J.
(2002)
Science
297,
353-356 |
23. | Kirkitadze, M. D., Bitan, G., and Teplow, D. B. (2002) J. Neurosci. Res. 69, 567-577[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Dahlgren, K. N.,
Manelli, A. M.,
Stine, W. B., Jr.,
Baker, L. K.,
Krafft, G. A.,
and LaDu, M. J.
(2002)
J. Biol. Chem.
277,
32046-32053 |
25. | Stine, W. B., Jr., Snyder, S. W., Ladror, U. S., Wade, W. S., Miller, M. F., Perun, T. J., Holzman, T. F., and Krafft, G. A. (1996) J. Protein Chem. 15, 193-203[Medline] [Order article via Infotrieve] |
26. | Harper, J. D., Lieber, C. M., and Lansbury, P. T. (1997) Chem. Biol. 4, 951-959[Medline] [Order article via Infotrieve] |
27. | Goldsbury, C., Kistler, J., Aebi, U., Arvinte, T., and Cooper, G. J. (1999) J. Mol. Biol. 285, 33-39[CrossRef][Medline] [Order article via Infotrieve] |
28. | Hou, L., Kang, I., Marchant, R. E., and Zagorski, M. G. (2002) J. Biol. Chem. 26, 26 |
29. |
Conway, K. A.,
Lee, S. J.,
Rochet, J. C.,
Ding, T. T.,
Williamson, R. E.,
and Lansbury, P. T., Jr.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
571-576 |
30. |
Serio, T. R.,
Cashikar, A. G.,
Kowal, A. S.,
Sawicki, G. J.,
Moslehi, J. J.,
Serpell, L.,
Arnsdorf, M. F.,
and Lindquist, S. L.
(2000)
Science
289,
1317-1321 |
31. | DePace, A. H., and Weissman, J. S. (2002) Nat. Struct. Biol. 9, 389-396[Medline] [Order article via Infotrieve] |
32. | Walsh, D. M., Hartley, D. M., Condron, M. M., Selkoe, D. J., and Teplow, D. B. (2001) Biochem. J. 355, 869-877[Medline] [Order article via Infotrieve] |
33. | Howlett, D. R., Jennings, K. H., Lee, D. C., Clark, M. S., Brown, F., Wetzel, R., Wood, S. J., Camilleri, P., and Roberts, G. W. (1995) Neurodegeneration 4, 23-32[CrossRef][Medline] [Order article via Infotrieve] |
34. | Soto, C., Castano, E. M., Kumar, R. A., Beavis, R. C., and Frangione, B. (1995) Neurosci. Lett. 200, 105-108[CrossRef][Medline] [Order article via Infotrieve] |
35. | Barrow, C. J., and Zagorski, M. G. (1991) Science 253, 179-182[Medline] [Order article via Infotrieve] |
36. | Barrow, C. J., Yasuda, A., Kenny, P. T., and Zagorski, M. G. (1992) J. Mol. Biol. 225, 1075-1093[Medline] [Order article via Infotrieve] |
37. | Wood, S. J., Maleeff, B., Hart, T., and Wetzel, R. (1996) J. Mol. Biol. 256, 870-877[CrossRef][Medline] [Order article via Infotrieve] |
38. | Tomski, S. J., and Murphy, R. M. (1992) Arch. Biochem. Biophys. 294, 630-638[Medline] [Order article via Infotrieve] |
39. |
Yang, D. S.,
Yip, C. M.,
Huang, T. H.,
Chakrabartty, A.,
and Fraser, P. E.
(1999)
J. Biol. Chem.
274,
32970-32974 |
40. | Fezoui, Y., and Teplow, D. B. (2002) J. Biol. Chem. 30, 30 |
41. | Shen, C. L., and Murphy, R. M. (1995) Biophys. J. 69, 640-651[Abstract] |
42. | Snyder, S. W., Ladror, U. S., Wade, W. S., Wang, G. T., Barrett, L. W., Matayoshi, E. D., Huffaker, H. J., Krafft, G. A., and Holzman, T. F. (1994) Biophys. J. 67, 1216-1228[Abstract] |
43. | Holzman, T. F., and Snyder, S. W. (1994) in Modern Analytical Ultracentrifugation: Acquisition and Interpretation of Data for Biological and Synthetic Polymer Systems (Schuster, T. M. , and Laue, T. M., eds) , pp. 298-314, Birkhäuser, Boston, MA |
44. | Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Liao, Z., Lieberburg, I., Motter, R., Mutter, L., Soriano, F., Shopp, G., Vasquez, N., Vandevert, C., Walker, S., Wogulis, M., Yednock, T., Games, D., and Seubert, P. (1999) Nature 400, 173-177[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Gotz, J.,
Chen, F.,
van Dorpe, J.,
and Nitsch, R. M.
(2001)
Science
293,
1491-1495 |
46. | Fraser, P. E., Nguyen, J. T., Surewicz, W. K., and Kirschner, D. A. (1991) Biophys. J. 60, 1190-1201[Abstract] |
47. |
Lomakin, A.,
Chung, D.,
Benedek, G.,
Kirschner, D.,
and Teplow, D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
1125-1129 |
48. | Whittingham, J. L., Scott, D. J., Chance, K., Wilson, A., Finch, J., Brange, J., and Guy Dodson, G. (2002) J. Mol. Biol. 318, 479-490[CrossRef][Medline] [Order article via Infotrieve] |
49. | Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M., and Stefani, M. (2002) Nature 416, 507-511[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Chiti, F.,
Bucciantini, M.,
Capanni, C.,
Taddei, N.,
Dobson, C. M.,
and Stefani, M.
(2001)
Protein Sci.
10,
2541-2547 |
51. | Harper, J. D., Wong, S. S., Lieber, C. M., and Lansbury, P. T. (1997) Chem. Biol. 4, 119-125[Medline] [Order article via Infotrieve] |
52. |
Kusumoto, Y.,
Lomakin, A.,
Teplow, D. B.,
and Benedek, G. B.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12277-12282 |
53. |
Pike, C. J.,
Overman, M. J.,
and Cotman, C. W.
(1995)
J. Biol. Chem.
270,
23895-23898 |
54. |
Atwood, C. S.,
Moir, R. D.,
Huang, X.,
Scarpa, R. C.,
Bacarra, N. M.,
Romano, D. M.,
Hartshorn, M. A.,
Tanzi, R. E.,
and Bush, A. I.
(1998)
J. Biol. Chem.
273,
12817-12826 |
55. | Pike, C. J., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1991) Brain Res. 563 (1-2), 311-314[CrossRef][Medline] [Order article via Infotrieve] |
56. | Pike, C. J., Burdick, D., Walencewicz, A. J., Glabe, C. G., and Cotman, C. W. (1993) J. Neurosci. 13, 1676-1687[Abstract] |
57. |
Lorenzo, A.,
and Yankner, B. A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12243-12247 |
58. | Busciglio, J., Lorenzo, A., Yeh, J., and Yankner, B. A. (1995) Neuron 14, 879-888[Medline] [Order article via Infotrieve] |
59. |
Walsh, D. M.,
Lomakin, A.,
Benedek, G. B.,
Condron, M. M.,
and Teplow, D. B.
(1997)
J. Biol. Chem.
272,
22364-22372 |
60. |
Walsh, D. M.,
Hartley, D. M.,
Kusumoto, Y.,
Fezoui, Y.,
Condron, M. M.,
Lomakin, A.,
Benedek, G. B.,
Selkoe, D. J.,
and Teplow, D. B.
(1999)
J. Biol. Chem.
274,
25945-25952 |
61. | Harper, J. D., Wong, S. S., Lieber, C. M., and Lansbury, P. T., Jr. (1999) Biochemistry 38, 8972-8980[CrossRef][Medline] [Order article via Infotrieve] |
62. |
Kowalewski, T.,
and Holtzman, D. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
3688-3693 |
63. | Huang, T. H., Yang, D. S., Plaskos, N. P., Go, S., Yip, C. M., Fraser, P. E., and Chakrabartty, A. (2000) J. Mol. Biol. 297, 73-87[CrossRef][Medline] [Order article via Infotrieve] |
64. | Wang, J., Dickson, D. W., Trojanowski, J. Q., and Lee, V. M. (1999) Exp. Neurol. 158, 328-337[CrossRef][Medline] [Order article via Infotrieve] |
65. |
Lue, L. F.,
Kuo, Y. M.,
Roher, A. E.,
Brachova, L.,
Shen, Y.,
Sue, L.,
Beach, T.,
Kurth, J. H.,
Rydel, R. E.,
and Rogers, J.
(1999)
Am. J. Pathol.
155,
853-862 |
66. |
Solomon, B.,
Koppel, R.,
Frankel, D.,
and Hanan-Aharon, E.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
4109-4112 |
67. |
Chung, H.,
Brazil, M. I.,
Soe, T. T.,
and Maxfield, F. R.
(1999)
J. Biol. Chem.
274,
32301-32308 |
68. | Levine, H., III (1995) Neurobiol. Aging 16, 755-764[CrossRef][Medline] [Order article via Infotrieve] |
69. |
Bitan, G.,
Lomakin, A.,
and Teplow, D. B.
(2001)
J. Biol. Chem.
276,
35176-35184 |
70. |
Palmblad, M.,
Westlind-Danielsson, A.,
and Bergquist, J.
(2002)
J. Biol. Chem.
277,
19506-19510 |
71. |
Burdick, D.,
Soreghan, B.,
Kwon, M.,
Kosmoski, J.,
Knauer, M.,
Henschen, A.,
Yates, J.,
Cotman, C.,
and Glabe, C.
(1992)
J. Biol. Chem.
267,
546-554 |
72. | Shen, C. L., Fitzgerald, M. C., and Murphy, R. M. (1994) Biophys. J. 67, 1238-1246[Abstract] |
73. | Kayed, R., Bernhagen, J., Greenfield, N., Sweimeh, K., Brunner, H., Voelter, W., and Kapurniotu, A. (1999) J. Mol. Biol. 287, 781-796[CrossRef][Medline] [Order article via Infotrieve] |
74. | Kirkitadze, M. D., Condron, M. M., and Teplow, D. B. (2001) J. Mol. Biol. 312, 1103-1119[CrossRef][Medline] [Order article via Infotrieve] |