From the Longtine Center for Molecular Biology and
Genetics and the ¶ Civin Laboratory of Neuropathology, Sun Health
Research Institute, Sun City, Arizona 85351, the
§ Department of Microbiology, Midwestern University,
Glendale, Arizona 85308, the
Department of Chemistry and
Biochemistry, Arizona State University, Tempe, Arizona 85287-1604, and
** Novartis Pharma, Incorporated, CH-4002 Basel, Switzerland
Received for publication, August 28, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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We have undertaken an integrated chemical
and morphological comparison of the amyloid- Alzheimer's disease
(AD)1 is a progressive
neurodegenerative disorder characterized by the presence of
extracellular amyloid plaques composed principally of amyloid- The APP23 transgenic (tg) mice contain an APP751
cDNA with the Swedish familial AD mutation under the control of the
neuron-specific Thy-1 promoter and express this human gene
at levels 7-fold greater than endogenous murine APP (12). Longitudinal
studies of these mice have revealed that extracellular amyloid deposits
become evident as the APP23 tg mice age. These deposits exhibit, at
their earliest appearance, the Congo red birefringence characteristic of the dense core plaques of human AD (12). A gradual progression from
a diffuse deposit to a dense plaque is not a feature of the APP23 tg
mouse pathology, paralleling our previous finding (13) that the diffuse
amyloid deposits of AD do not represent a precursor developmental stage
of senile plaques.
A transgenic mouse model system that faithfully mimics every aspect of
AD has not been developed. The APP23 tg mouse model reproduces some of
the neuropathological changes associated with AD such as amyloid
plaques with a core of amyloid, neuritic alterations, and neuron loss
(15) as well as astrogliosis, microglial activation, and deposition of
a cerebrovascular amyloid (14). Although an exact analog of AD is not
at hand and may not ultimately be attainable, the understanding,
management, and mitigation of AD may be facilitated through the use of
the available animal models. Even though the exact role of senile
plaques remains undefined, massive amyloid deposition is clearly an AD
hallmark and is hypothesized to be seminal in the pathophysiology of
this disease. Because there have been no detailed biochemical studies
of the amyloid present in the APP23 tg mouse, we have undertaken an
integrated chemical and morphological study to examine and compare the
biochemical and biophysical properties of the transgenic mouse amyloid
plaques with those of AD patients.
Preparation of Formic Acid-extracted Brain
Lysates--
The generation of APP23 tg mice has been previously
described (12). These mice express the human APP751 cDNA
with the Swedish double mutation under the control of the
neuron-specific mouse Thy-1 promoter fragment. The cerebral
cortices, hippocampi, and olfactory bulbs, which contained the highest
concentration of amyloid plaques in the transgenic (APP23) mice (12,
15), representing ~165 mg of tissue/animal, were carefully dissected
from five male and five female animals at room temperature. The average
age of the animals was 22.5 months (range of 22.1-22.7 months). The
tissue from each brain was finely minced and immediately homogenized in
3.0 ml of 90% (v/v) glass-distilled formic acid (GDFA) using a glass
Dounce homogenizer at room temperature. Samples of 1.5 ml were loaded
into 2-ml thick-walled polyallomer centrifuge tubes. The acid-insoluble
material was separated by centrifugation in a Sorvall TST 60.4 rotor at
217,000 × g for 1 h at 4 °C. The clear supernatant was carefully collected, avoiding the surface layer of
lipid and the small acid-insoluble material deposited in the bottom of
the tube.
Collection of the Brain Lysate 3-8-kDa Mass Range
Molecules--
Samples (500 µl) of the clear formic acid-extracted
whole brain (brain tissue and its vascular network) lysates were
fractionated by size-exclusion fast protein liquid chromatography on a
1 × 30-cm Superose 12 column (Amersham Pharmacia Biotech,
Uppsala, Sweden) equilibrated and developed with 80% GDFA. The
Superose 12 column fractions were calibrated using a set of proteins of known molecular mass and with the reverse-sequence A Purification of Vascular Amyloid--
To isolate the amyloid
deposited in the walls of the cerebral blood vessels, the complete
cerebral hemispheres from two male and two female APP23 tg mice were
each sectioned into three coronal portions. The brain tissue was gently
stirred for 24 h in 300 ml of 50 mM Tris-HCl (pH 7.5)
containing 2% SDS and 2 mM EDTA at room temperature. The
SDS-insoluble tufts of blood vessels consisting of the extracellular
matrix with attached insoluble vascular amyloid were collected by
filtration (20-µm nylon mesh) and washed with Tris buffer. The
insoluble vascular amyloid was solubilized and extracted with 3 ml of
80% GDFA and centrifuged at 217,000 × g for 1 h
at 4 °C, and the supernatant was submitted in aliquots of 500 µl
to fast protein liquid chromatography as described above for the
fractionation of brain lysate samples.
Purification of A Tryptic Hydrolysis and Separation of A Automatic Amino Acid Analysis--
The tryptic and CNBr-derived
peptides were submitted to acid hydrolysis on a vapor-phase system Work
Station (Waters) in 6 N HCl and 1% (w/v) phenol at
150 °C for 100 min. After removal of the acid by vacuum
centrifugation, the amino acid compositions of the peptide hydrolysates
were determined at 570 and 440 nm using an automatic injector
sampler-HPLC system (Thermo Separation Products, Fremont, CA) and a
post-column ninhydrin reaction system (Pickering Laboratories, Inc.,
Mountain View, CA). The amino acid separations were performed on a
sodium cation-exchange column (4 × 150 mm, 5-µm beads;
Pickering Laboratories, Inc.) using the reagents and programs provided
by the manufacturer.
Protein Sequencing--
HPLC-purified samples were dissolved in
50 µl of 50% (v/v) aqueous acetonitrile containing 0.1%
trifluoroacetic acid. For the major HPLC peaks, ~30 µl of the
sample was dried on a fiberglass peptide disc (Beckman Coulter,
Inc., Fullerton, CA) and used for amino acid sequence determination.
Sequencing was performed on a Porton 2090E gas-phase protein sequencer
(Beckman Coulter, Inc.) equipped with an on-line Hewlett-Packard 1090L
HPLC apparatus. The remainder of the sample was used for mass
spectrometry analyses.
Mass Spectrometry Analyses of the Trypsin-digested A Morphological Characterization of Amyloid Plaques--
Cerebral
hemispheres from transgenic and control mice as well as cerebral
cortices from human AD brains were fixed for 48 h in 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4),
dehydrated by passage through an alcohol and xylene solution series,
and embedded in paraffin wax. Sections of 5 µm were taken on a rotary microtome and stained with hematoxylin and eosin for general structure, thioflavin S for amyloid, and the Campbell-Switzer silver stain for
diffuse amyloid plaques. Stained sections were viewed by
bright-field light microscopy (hematoxylin-eosin;
Campbell-Switzer-stained sections), fluorescence microscopy, and
confocal scanning laser microscopy (thioflavin S-stained sections).
Human and mouse sections were stained under identical conditions, in
the same batch.
APP23 Transgenic Mice Produce More Soluble Amyloid
Plaques--
One of the most striking characteristics of the amyloid
cores present in AD patient brains is their extreme resistance to denaturing agents and detergents such as urea, guanidine hydrochloride, and SDS. During our initial attempt to enrich the amyloid cores from
the APP23 tg mouse cerebral cortices using a 2% SDS solution, we
noticed that in contrast to the human AD tissue samples, the parenchymal amyloid cores and fibrils completely disappeared. Extended
high-speed centrifugation (250,000 × g for 3 h)
of the SDS supernatant failed to sediment any dispersed amyloid fibrils in the animal sample. These observations suggested that the plaque cores present in the APP23 transgenic animals were substantially more
soluble than those in the AD brain. In contrast to the parenchymal deposits, the amyloid deposited around the blood vessels in the APP23
tg mice after SDS treatment remained intact and, as in the case of the
AD brain, firmly attached to the vascular walls. We believe that the
SDS insolubility characteristic of the vascular amyloid may in part be
related to a stronger association between A
Our experiments also revealed that a large proportion of the transgenic
mouse parenchymal A Quantification and Chemical Characterization of the Total A
The minor fraction 4 was identified as A
Finally, the mass spectra of peak 9 also revealed, in addition to its
major component (A Characterization of the SDS-insoluble Vascular
Amyloid--
Separation of the A Morphological Characteristics of APP23 Transgenic Mouse and Human
Amyloid Plaques--
As previously reported (12), senile plaques were
present in the brains of the APP23 tg mice and were readily apparent
with silver and thioflavin S stains in mature animals (14- and
20-month-old animals). Most of the plaques were composed of compact
amyloid, which fluoresced brightly with the thioflavin S stain (Fig.
5A), indicating the presence
of a
The Campbell-Switzer stain revealed additional differences between the
tg mouse and human AD plaques. Mouse compact plaques took up very
little of the stain, appearing light brown in color (Fig. 5,
E and G), unlike human compact plaques, in which
the core and halo regions were both stained intensely black (Fig. 5F). Many mouse compact plaques had a delicate halo of black
fibrillar material, like that seen in surrounding diffuse plaques (Fig. 5E, arrow), whereas others, however, did not
(Fig. 5G, asterisk). Classical human AD plaques
stained with thioflavin S possessed a relatively empty zone between the
intensely stained core and halo (Fig. 5F), whereas no such
empty region was observed in mouse plaques. Diffuse plaques, which were
somewhat apparent with the thioflavin S stain, were, as with human
diffuse plaques, more readily visualized with the Campbell-Switzer
stain (Fig. 5G, arrow). These resembled human
diffuse plaques (Fig. 5H, arrow) in their irregular shape and "cotton wool" appearance, although they lacked the fine granular material commonly observed in the diffuse plaques of
the AD brain.
Amyloid plaque cores isolated from AD patients are entirely
resistant to proteolytic degradation (23, 24), and only a limited
number of powerful chaotropic agents are capable of completely dissolving the human amyloid fibrils. Chemical characterization of the
separated AD amyloid plaque components has revealed the presence of a
complex mixture of A Several transgenic mouse models that reproduce certain aspects of AD
have been engineered (3-12, 37, 38). The APP23 transgenic mice develop
an intense amyloid deposition in the cerebral vasculature and produce
Congo red/thioflavin S birefringent extracellular plaques with
associated microglia in the brain parenchyma (14, 27). Our experiments
have revealed that the condensed amyloid plaque cores deposited in an
age-specific pattern in the transgenic APP23 mice are not precise
reproductions of those observed in AD, but differ in fundamental
chemical and morphological aspects from the human senile plaques. The
majority of the amyloid plaques in the APP23 tg mice consist of compact
amyloid cores that morphologically resemble human plaque cores, but are
much larger and lack the clear zone and halo present in the classical
human plaques. Furthermore, in the APP23 mice, there are relatively few
diffuse amyloid deposits, which are characteristically abundant in the
AD brain.
The increased solubility of the A Chemical analyses of the A It is likely that the structural alterations of the A The values for the human vascular amyloid are more variable since the
A One important factor that may explain the almost complete absence of
structural changes in the A Morphological comparison of tg mouse and human senile plaques revealed
some similarities and some differences. Both the mouse and human
possess compact and diffuse plaques (20-22). In AD, the diffuse
amyloid exhibits a blend of short fibrils and aggregates of a fine
granular material.6
Biochemical analysis of these granular collections revealed the presence of the A Amyloid plaques may be characterized by either the presence or absence
of "dystrophic" neurites. In this respect, it has been reported
that compact plaques in these tg mice are associated with dystrophic
neurites expressing phosphorylated tau epitopes (12), but without
recreation of the paired helical filaments seen in human classical
plaques. Another important difference between the plaques observed in
the APP23 tg mice and the AD brain is that in the tg mice, the plaques
are morphologically homogeneous, whereas in the human AD brains, a
large variety of subtypes are typically observable.
Other (A
) molecules and
the amyloid plaques present in the brains of APP23 transgenic (tg) mice
and human Alzheimer's disease (AD) patients. Despite an apparent
overall structural resemblance to AD pathology, our detailed chemical analyses revealed that although the amyloid plaques characteristic of
AD contain cores that are highly resistant to chemical and physical
disruption, the tg mice produced amyloid cores that were completely
soluble in buffers containing SDS. A
chemical alterations account
for the extreme stability of AD plaque core amyloid. The corresponding
lack of post-translational modifications such as N-terminal
degradation, isomerization, racemization, pyroglutamyl formation,
oxidation, and covalently linked dimers in tg mouse A
provides an
explanation for the differences in solubility between human AD and the
APP23 tg mouse plaques. We hypothesize either that insufficient time is
available for A
structural modifications or that the complex
species-specific environment of the human disease is not precisely
replicated in the tg mice. The appraisal of therapeutic agents or
protocols in these animal models must be judged in the context of the
lack of complete equivalence between the transgenic mouse plaques and
the human AD lesions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(A
) surrounded by dystrophic neurites (1). This association and the
realization that the basis of certain early-onset familial forms of AD
seems to be the enhanced production of one or more A
peptides have
led to the hypothesis that A
is intimately involved in the AD
pathogenic process (2). A promising experimental approach to unraveling the role(s) of A
in AD pathology has been the construction and characterization of transgenic mice that overexpress the amyloid precursor protein (APP) (3-12). Several transgenic mouse lines have
been described that produce A
deposits that accumulate in an
age-dependent fashion and morphologically resemble the
senile plaques characteristic of human AD (3, 6, 8, 12, 37, 38).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
peptide residues 40 to 1. The peptides were loaded into a 500-µl injection loop and separated at a flow rate of 15 ml/h at room temperature, with
the chromatographic eluate absorbance monitored at 280 nm. Fractions of
500 µl were collected into polypropylene tubes every 2 min; those
containing the 3-8-kDa molecules (corresponding to a retention time
interval of 50-66 min) were pooled into a single tube, and 20 µl of
10% (w/v) betaine was added. The formic acid was removed by vacuum
centrifugation (Savant Instruments, Inc., Farmingdale, NY).
Peptides from the 3-8-kDa Range
Pool--
The A
peptides present in the 3-8-kDa size-exclusion
fractions were separated by reverse-phase HPLC on a 4.6 × 150-mm
Source 5RPC column (Amersham Pharmacia Biotech) using a 500-µl
injection loop. Solvent A was 0.1% (v/v) 30% ammonium hydroxide in
water adjusted with GDFA to pH 9.0, and solvent B was 60% (v/v)
acetonitrile and 40% (v/v) solvent A. The chromatography was developed
at room temperature with a linear gradient from 25 to 50% solvent B in 100 min at a flow rate of 1.0 ml/min, with absorbance monitored at 214 nm. The 3-8-kDa fractions obtained from four separate fast protein
liquid chromatography runs of brain cleared lysate samples were pooled
to provide the material for each reverse-phase HPLC separation. To
render the peptides soluble, 20 µl of 2 N NaOH was added
to each of the polypropylene tubes. After pooling, an additional 20 µl of 6 N NaOH was added, followed by 375 µl of base-line solvent (25% solvent A and 75% solvent B) and a final addition of 25 µl of 6 N NaOH. The A
-(1-40) and
A
-(1-42) chromatographic retention times were established using the
corresponding synthetic peptides obtained from California Peptide Inc.
(Napa Valley, CA), which had been further purified in our laboratory by
HPLC prior to their use as size markers.
Peptides--
The A
peptides separated by reverse-phase HPLC were lyophilized, solubilized
in 1 ml of 80% (v/v) GDFA, and dialyzed (Spectrapor No. 6, Mr 1000 cutoff) against two changes of distilled
water and three changes of 100 mM ammonium bicarbonate.
Once equilibrium was attained, the A
peptides were digested with
L-1-tosylamido-2-phenylethyl chloromethyl
ketone-treated trypsin (10 µg/specimen; Worthington) for 16 h at
37 °C. The resulting tryptic peptides were lyophilized, dissolved in
500 µl of 0.1% (v/v) trifluoroacetic acid in water, thoroughly mixed
and centrifuged at 12,000 × g for 10 min to remove any
insoluble material. The insoluble tryptic core, consisting mainly of
the hydrophobic A
C-terminal peptide residues 29-42, was dissolved
in 300 µl of 80% GDFA and, after the addition of a small crystal of
CNBr, incubated for 16 h at room temperature. The soluble tryptic
peptides and the CNBr-cleaved peptides were separated by HPLC at room
temperature on a Spherisorb ODS2 C18 reverse-phase column
(4 × 250 mm, 5-µm beads; LKB, Bromma, Sweden). The
chromatography was developed with a 90-min linear gradient of 0-20%
acetonitrile in 0.1% trifluoroacetic acid, followed by a second linear
gradient of 20-60% acetonitrile in 0.1% trifluoroacetic acid for 30 min using flow rates of 0.7 ml/min. Absorbance was monitored at 214 nm.
Alternatively, the tryptic peptides were separated on a Source 5RPC
column (4.6 × 150 mm, 5-µm beads) using a linear gradient of
0-60% solvent B under the conditions described for the purification
of A
peptides.
Peptides--
Mass spectrometry samples were prepared by mixing 5 µl
of the dissolved HPLC-separated peptides with a saturated solution of
-cyano-4-hydroxycinnamic acid (also dissolved in 50% aqueous acetonitrile containing 0.1% trifluoroacetic acid), and aliquots of
~1 µl were dried on the ends of stainless steel sample pins. For
precise mass determinations, bovine insulin (mass of 5734.5 Da
for the singly protonated molecular ion) was added to the mixture of
sample and matrix and used as an internal standard. Mass spectra were
obtained using a Vestec Lasertec Research mass spectrometer (PE
Biosystems, Framingham, MA) operated in the positive ion mode with an
accelerating voltage of 23 kV. This instrument incorporates a Laser
Science VSL-337ND nitrogen laser that provides 3-ns pulses of 337-nm
light of variable intensity. Data were collected using a TDS 520 digital storage oscilloscope (Tektronix, Beaverton, OR) and analyzed on
a personal computer using LabCalc software (Galactic Industries Corp.,
Salem, NH). Each of the mass spectra reported represents the average of
128 shots.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and the molecules of the
extracellular matrix that required formic acid exposure for complete extraction.
was freely soluble in detergent-free aqueous
buffers. The APP23 tg mouse brains were homogenized at 4 °C in an
aqueous buffer (20 mM Tris-HCl (pH 7.4)) containing a
mixture of protease inhibitors (16), followed by centrifugation (100,000 × g at 1 h). The soluble A
present in
the resulting supernatants was quantified by europium immunoassay
(26). At 22 months of age, the total water-soluble A
and the
total water-insoluble A
represented averages of 56.4 and 750 µg/g
of brain tissue, respectively.
Present in APP23 Transgenic Mouse Brain Tissue--
The increased
water solubility of the APP23 tg mouse amyloid in comparison with its
human AD counterpart suggested that fundamental structural and
compositional differences exist between the amyloid plaques formed in
transgenic mice and humans. Cortical areas, hippocampi, and olfactory
bulbs were carefully dissected from the brain; and to account for the
total amyloid present in the brain parenchyma and vascular walls, the
tissue was acid-lysed and chromatographed in GDFA. Four discrete peaks
were resolved on a size-exclusion column (Fig.
1), of which only the 3-8-kDa range
molecules included A
. These fractions were pooled; their volumes
were reduced by vacuum centrifugation; and they were subjected to
chromatography on a Source 5RPC reverse-phase column. A
polystyrene/divinylbenzene reverse-phase column was employed in these
experiments because, in our experience, A
-(1-42) exhibits better
solubility at alkaline pH (9.0) than at an acid pH (2.0). Of the
several chromatographic conditions investigated, a linear gradient of
25-50% acetonitrile gave the best resolution and eliminated most of
the contaminating molecules either as unbound material or in the >50%
acetonitrile eluate. A total of 10 discrete fractions were recovered
(Fig. 2). The two major fractions had
retention times identical to those of the synthetic A
markers
A
-(1-40) and A
-(1-42). All the fractions were submitted to mass
spectrometry (Table I), which revealed the presence of A
-(1-40) in fraction 7 and A
-(1-42) in fraction 9. Seven steps of automatic amino acid sequencing yielded, in both
cases, the authentic A
N-terminal sequence DAEFRHD. Fractions 1 and
3 were identified as A
-(1-40) with Met35 oxidized to
methionine sulfoxide and A
-(1-38), respectively. Both peptides also
demonstrated the complete A
N-terminal amino acid sequence of
DAEFRHD. The atomic masses of the molecules in the smaller peaks 2 and
5 corresponded to those of A
-(1-37) and A
-(1-39). We suggest
that the A
peptides ending in residues 39, 38, and 37 in the tg mice
represent soluble products of A
degradation. This suggestion is
based on our recent analysis of the soluble A
peptides from the AD
brain, which also show a series of shorter C-terminal
peptides.2 Alternatively, the
A
peptides ending at residues 39, 38, and 37 could represent
by-products of
-secretase-like activity. The loss of C-terminal
residues greatly enhances the solubility of the A
peptides.
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Fig. 1.
Superose 12 size-exclusion fast protein
liquid chromatography of GDFA-extracted APP23 tg mouse brain
lysates. The solid line depicts the UV absorbance
profile of supernatants derived from high-speed centrifugation of
GDFA-extracted brain lysates. The dashed line represents the
peak produced by the reverse-sequence A peptide residues 40 to 1 (4331 Da) employed as a column calibration standard and with a
retention time between 52 and 60 min. The dotted line
depicts the UV absorbance profile produced by GDFA extracts obtained
from human AD amyloid plaque cores. The human-derived amyloid produced
five fractions (fractions 1-5), of which fractions 3-5 were composed
of A
trimers/tetramers, dimers, and monomers, respectively. The
APP23 tg mouse brains yielded four fractions (fractions A-D). The
tubes containing fraction C (3-8-kDa peptides) were pooled and, after
elimination of the acid, submitted to reverse-phase HPLC. The APP23 tg
mouse A
was contained within the positive slope of fraction C. AUFS, absorbance units at full scale.
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Fig. 2.
5RPC reverse-phase HPLC separation of amyloid
peptides from APP23 tg mice. Subjecting the Superose 12 size-exclusion column eluate peak C (see Fig. 1) to reverse-phase
chromatography resulted in 10 discrete factions (fractions 1-10).
These fractions were submitted to mass spectrometry analysis and
peptide sequencing. The identification of each fraction is shown in
Table I. The lower chromatography trace is that of the
calibration standards, synthetic A -(1-40) (peak A) and A
-(1-42)
(peak B), subjected to the same experimental conditions, which had
identical retention times as fractions 7 and 9 from the tg mice.
AUFS, absorbance units at full scale.
Mass spectrometry and amino acid sequencing of APP23 tg mouse
amyloid peptides
-(1-42) with an oxidized
Met35. The smaller peaks 6 and 10 were designated as
formylated forms of A
-(1-38) and A
-(1-42) since their molecular
masses were those of the corresponding peptides increased by 28 mass
units. This was also the situation with a smaller mass spectrometry
signal observed within peak 9, which was identified as formylated
A
-(1-40). The minute amounts of formylated A
are probably
artifactual modifications resulting from the use of formic acid during
the extraction and chromatographic procedures. The minor peak 8 had the
same atomic mass as peak 7 (A
-(1-40)). Based on previous experience
with chromatographic separations of A
, we designated this fraction as A
with D-Asp either at position 7 or 23 (16, 18),
which imparts a slightly delayed retention time upon reverse-phase
chromatography. Under the same chromatographic conditions, the AD A
peptide residues 17-42 or P3 fraction, a component of the diffuse
amyloid deposits, has a retention time of ~70 min. No peaks were
evident in this area in the case of the APP23 tg mouse amyloid.
-(1-42)), a small signal with an atomic mass of
3152.8 Da that may correspond to A
-(11-40) (calculated mass
of 3151.7 Da). This suggestion is supported by the amino acid
sequence data indicating a very small amount of protein, ~3-4% of
the main peptide, starting with the sequence EVHHQ (A
residues
11-15). The finding of this minor peptide is interesting since
in vitro experiments have suggested that the
-secretase, besides generating A
peptides starting at Asp1, is also
capable of cleaving between Tyr10 and Glu11 of
the A
sequence (40, 41). The presence of small quantities of A
starting at residue 11 in the brains of AD patients and in the APP23 tg
mice also supports this contention (31, 43, 44). In addition,
mass spectrometry analysis of non-transfected and
-APP-transfected
mouse neuroblastoma cells has revealed the presence of A
peptides
starting at residue 11 in the former, but not in the latter (42).
Furthermore, ~40 A
-related peptides were detected in the
conditioned medium, probably resulting from the action of amino-,
carboxy-, and endopeptidases (42). To detect any post-translational
A
modifications that were not evident using mass spectrometry, such
as isomerization of Asp at positions 1 and 7, known to occur in the
human A
peptides (16), we digested the Source 5RPC chromatography
peaks 7 (A
-(1-40)) and 9 (A
-(1-42)) with trypsin. The resulting
peptides were separated either by C18 reverse-phase or by
5RPC reverse-phase HPLC (Figs. 3 and
4) and acid-hydrolyzed, and their amino acid sequences were determined. In contrast to the complex tryptic digest pattern obtained from the
human AD A
peptide (which revealed up to 14 distinct peptides due to
degradation of the N-terminal domain; the presence of
L-iso-Asp, D-iso-Asp, L-Asp, and
D-Asp at positions 1 and 7; and cyclization of Glu at
position 3 (16, 19)), the tg mouse A
tryptic digest map pattern was
comparatively simple. The APP23 tg mice (peak 7) yielded only four
tryptic digest peptides (Fig. 3), which corresponded to residues 1-5
(DAEFR), 6-16 (HDSGYEVHHQK), 17-28 (LVFFAEDVGSNK), and 29-40
(GAIIGLMVGGVV) of the A
peptide. The retention times of these
tryptic peptides matched those of the tryptic peptides derived from
synthetic A
-(1-40) employed as calibration standards (Fig. 3). The
C-terminal tryptic peptide of A
-(1-42) produced in the tg mice,
which precipitated as an insoluble core after tryptic digestion, was
further cleaved by CNBr at Met35. This procedure generated
two heptapeptides corresponding to A
residues 29-35
(Gly-Ala-Ile-Ile-Gly-Leu-Hse) and residues 36-42 (Val-Gly-Gly-Val-Val-Ile-Ala). These two peptides were separated by
C18 reverse-phase chromatography as previously described
(Ref. 16 and data not shown). Chromatography of tryptic peptides at pH
9.0 yielded the expected pattern of four peptides (Fig. 4) and did not
reveal the presence of any chemical modifications or N-terminal
degradation.
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Fig. 3.
C18 reverse-phase HPLC profiles
of tryptic peptides derived from the 5RPC reverse-phase HPLC peak 7 as
shown in Fig. 2. Four A tryptic peptides
corresponding to residues 1-5 (peak 1), 6-16 (peak 2), 17-28 (peak
3), and 29-40 (peak 4) were analyzed. The lower chromatography
trace is that of the four tryptic peptides obtained from tryptic
digestion of synthetic A
-(1-40). The retention times of peaks A-D
obtained from synthetic A
coincide with those of peaks 1-4 obtained
from tg mice. All tg mouse tryptic peptide peaks were acid-hydrolyzed,
and their composition was determined by automatic amino acid analysis.
AUFS, absorbance units at full scale.
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Fig. 4.
5RPC reverse-phase HPLC analysis of tryptic
peptides derived from the 5RPC reverse-phase HPLC peak 9 shown in
Fig. 2. The peptides designated as A-D corresponded to
A peptides residues 1-5, 6-16, 17-28, and 29-42, respectively.
The low yield of the C-terminal peptide is due to its hydrophobic
nature and precipitation after tryptic digestion. Tryptic hydrolysis
was also carried out on A
peptide residues 1-38 (peak 3 in Fig. 2).
Separation of the resulting peptides using the 5RPC column revealed
that the C-terminal tryptic peptides (residues 29-38) had a retention
time of ~50 min, which is indicated by the dashed line and
designated as peptide E. AUFS, absorbance units at full
scale.
peptides present in the
cerebrovascular walls by reverse-phase HPLC on a Source 5RPC column
produced only three fractions corresponding to A
-(1-40 ox),
A
-(1-40), and A
-(1-42). Mass spectrometry revealed that the
relative masses of these peptides were 4345.8, 4329.8, and 4517.0 Da, respectively, which is in agreement with their theoretical
expected values.
-pleated sheet conformation. Old animals also displayed small
numbers (<10%) of plaques (Fig. 5G, arrow),
which have been termed "diffuse" deposits in humans (20-22). The
internal structure of the compact plaques was most apparent after
thioflavin S staining, which showed that they were composed of bundles
of filaments radiating outward from a central core (Fig. 5,
A and C). These plaques ranged up to 200 µm in
diameter, averaging ~80-120 µm. In comparison, human AD compact
plaques (Fig. 5, B and D) are classified as
"burned-out" or "naked core" plaques and classical plaques
(20-22). Burned-out plaques consist simply of a central amyloid core
~10-20 µm in diameter (data not shown), whereas classical plaques
have a central core surrounded by an empty region with a halo of
fluorescent material, with overall diameters averaging ~60-80 µm.
Human classical plaques, like the tg mouse plaques, possess wisps of
fluorescent material radiating from a central core region (Fig. 5,
C and D). In the tg mice, these wisps were
clearly composed of filament bundles, whereas in the human, they had no
discernible internal structure.
View larger version (73K):
[in a new window]
Fig. 5.
Photomicrographs of tg mouse (A
and C) and human (E and
G) Alzheimer's disease plaques stained with
thioflavin S and examined by fluorescence microscopy
(A-D) or stained using the Campbell-Switzer silver
method and examined by bright-field microscopy
(E-H). Under conventional fluorescence
microscopy (A and B), mouse compact plaques
(A) appear as spherical structures composed of filamentous
material, whereas human classical plaques (B,
arrow) have a more complex structure (see "Discussion:).
Bars in A and B = 60 µm. Under
confocal scanning laser microscopy (C and D),
mouse compact plaques (C) consist of filaments bundles
radiating from a central core. Human classical plaques (D)
also possess a central core and radiating arms, but filaments are less
apparent. Bar in C = 25 µm; bar
in D = 15 µm. The cores of mouse compact plaques are
relatively unstained by the Campbell-Switzer stain (E,
arrow; and G, asterisk), whereas the
cores of human compact plaques are intensely stained (F,
arrow points to a human classical plaque; observe the
target-like appearance in which the "bull's-eye" is the core).
Both the mouse (G) and human (H) possess diffuse
plaques (arrows), which are similar in appearance.
Bar in E = 120 µ m.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, glycoproteins, and glycolipids, of which the
latter two represent ~20% of the total amyloid core mass (24, 25).
The amyloid core insolubility is partially due to the presence of these
ancillary molecules (28).3 In
addition, the A
peptides isolated from the human brain have numerous
post-translational modifications, are extensively degraded at the N
terminus, and contain a high proportion of cross-linked A
molecules
resulting from intermolecular linkages between tyrosyl residues.4 Of the total
amount of human A
solubilized from the amyloid cores by formic acid,
monomeric, dimeric, and trimeric/tetrameric A
molecules represent an
average of 55, 25, and 20%, of the total amyloid, respectively. All
these factors that contribute to the AD amyloid insolubility and
resistance to proteolytic degradation are apparently absent in the APP
tg mice.
peptides in the APP23 tg mice
relative to that in AD brains is clearly reflected by the amounts of
these peptides extracted by detergent-free aqueous buffer per unit of
tissue. The averaged yield (n = 6, mean age of 22 months) of total water-soluble A
, representing both
A
-(1-40) and A
-(1-42), in the tg mice was 56.4 µg/g of
brain. In contrast, the average (n = 8, mean age of 81 years) water-soluble total A
recovered from AD brains, using an
identical extraction procedure, amounted to 48 ng/g of cortex (17).
This is a >1000-fold increase in the amount of water-soluble A
per
unit weight in the APP23 tg mice over that obtained from the AD brain.
isolated from the APP23 tg mice
demonstrated that most of the A
molecules initiated at
Asp1 in both A
-(1-40) and A
-(1-42). Only a minute
proportion of A
-(11-40) was detected in the tg mice. In the human,
the N-terminal region of brain parenchymal A
is extensively degraded
by aminopeptidases,
-secretase(s), or both. As reported by our
laboratory and other investigators, in the human amyloid plaque cores,
the A
molecules initiate at residues 2, 3, 4, 5, 6, 8, 9, 10, and 17 (13, 16, 18, 19, 29-32). In AD, only a small proportion of A
starts
at position 1 in the form of L-Asp in the amyloid plaques
(~10%), and this fraction is higher in the vascular amyloid
(~65%). In human amyloid plaques, ~50% of the amyloid starts at
position 3 in the form of pyro Glu, whereas in the vascular amyloid,
this form accounts for only 11% (19); no A
molecules with pyro Glu were detected in the APP23 tg mice. In the human A
isolated from neuritic plaques and vascular walls, the N-terminal iso-Asp accounts for ~20 and 6% of the total A
, respectively, whereas none of this
form was detected in the APP23 tg mice. With respect to iso-Asp at
position 7, ~75% of the A
in the human amyloid plaques carries this isomerization. This
-shift is present in lesser quantities in
the vascular amyloid (~20%). Apparently, the APP23 tg mice are free
from this
-shift between the peptide bond of
Asp7-Ser8 that drastically alters the
conformation of the
-carbon backbone of the A
peptide. Finally,
only a reduced proportion (~10%) of the APP23 tg mouse A
appears
to be oxidized at Met35. An important difference between
the tg mice and human experimental subjects is the unavoidable longer
post-mortem delay in sample processing for the latter that may account
for some of the changes present in the amyloid chemistry between the
two species. The presence and extent of potential
time-dependent post-mortem artifacts affecting amyloid
chemistry are currently under investigation.
peptides in
the human neuritic plaque cores account for their remarkable physical
stability as well as for the inability of the phagocytic cells to
remove them from the brain during the course of AD progression. We have
previously demonstrated that N-terminal truncation of the A
molecules results in the net loss of polar residues, thereby increasing
the overall hydrophobicity of the peptides and rendering them
significantly more water-insoluble and resistant to enzymatic degradation (33). This tenet is clearly illustrated by the shorter A
-(17-42) peptide, which, even at low concentrations, avidly precipitates into amorphous aggregates and is abundant in the diffuse
amyloid deposits present in the AD brain and Down's syndrome (13, 36).
It is also well established that isomerization of Asp residues results
in a greater resistance to and even inhibition of proteolytic
degradation in the A
peptides as well as in other molecules (33,
34). Oxidation of Met35 to its sulfoxide form has also been
reported to increase the stability of the A
peptides (33). The
generation of pyroglutamyl at position 3 of A
blocks N-terminal
degradation of these peptides by aminopeptidases and increases the
hydrophobicity of A
(32, 33). Finally, the irreversible
oligomerization of A
due to stable dimers and trimer/tetramers
hinders enzymatic degradation (33). The average ratio among the four
main A
fractions found in the APP23 tg mice, A
-(1-40 ox),
A
-(1-38), A
-(1-40), and A
-(1-42), which in this study
represent the contribution of both vascular and parenchymal A
, was
12:9:50:29. These values contrast with the human neuritic plaque
amyloid cores, in which A
-(n-40) and
A
-(n-42) have an average ratio of 15:85. The relative
ratio of the tg mouse vascular A
-(1-40 ox), A
-(1-40), and
A
-(1-42) peptides was 7:90:3. These data suggest that in the APP23
tg mice, the major contributor to the total amount of A
-(1-40) is
the vascular amyloid and that most of the brain's A
-(1-42)
originates from the cores of parenchymal amyloid.
-(n-40) peptides have a range of 40-90%. The larger the amount of vascular amyloid deposited in AD, the greater the amount
of A
-(n-40) peptide in the vessels, reaching a
peak in those individuals with apoE-
4/
4 genotype (39).
Furthermore, our studies have revealed two patterns of vascular amyloid
deposition in the apoE-
4 AD cases. In the first, there is an
overwhelming number of amyloid deposits affecting the form of spherical
cores intimately attached to the basal lamina of the capillary network, producing an ornate pattern that resembles "pussy willows" (see Fig. 3 (C and D) in Ref. 29). In the second,
there is an abundance of arteriolar and small artery (up to 500 µm in
diameter) amyloid deposits that resemble those observed in the APP23 tg
mice. The dominance of A
-(1-40) over A
-(1-42) in this
strain may reflect the heavy arterial amyloid deposition, an
outstanding characteristic of the APP23 tg mice
(14).5
isolated from the APP23 tg mice is that
the alterations commonly observed in the human brain are thought to be
time-dependent. In the transgenic animals, amyloid is
produced at levels that overwhelm the capacity of any endogenous
clearance mechanisms and that lead to a vastly accelerated depositional
process. In the human brain, the process of amyloid accumulation begins
several decades prior to the onset of clinical symptoms and continues
to occur until the death of the patient. The life span of the APP23 tg
mice is just over 2 years, which may not be sufficient to manifest the
full spectrum of structural A
changes observed in terminal AD (15).
An additional possibility is that the structural modifications observed
in AD do not represent merely a passive and random accumulation of
time-dependent changes in A
structure, but are actually
age- and perhaps species-specific (35), conceivably representing
alterations in the expression and activity of key enzymes. It is
possible that the processing required to create authentic AD plaques
cannot occur in transgenic animals because either the necessary enzyme
homologs are not present or the elevated pace of amyloid deposition
simply precludes the prerequisite maturational reactions.
sequence of residues 17-42 (13), a peptide apparently absent in the tg mice. The mouse compact plaques display a
fibrillar internal structure that is not visible in human compact plaques. These plaque structures are significantly larger in the tg
mice, being on average about twice the diameter of human classical plaques.
-APP tg mouse strains have been constructed, including some in
which there is an accelerated A
deposition resulting from the
coexpression of mutant presenilin-1 and amyloid precursor proteins (37,
38). The precise degree of similarity of the amyloid deposits in these
animals to those in AD awaits experimental investigation. Our studies
indicate that the amyloid fibrils deposited in the brain of the APP23
tg mice are chemically and morphologically distinct from the ones
accumulated in the AD brain. In conclusion, the appraisal of
therapeutic agents or protocols directed toward inhibiting or reversing
plaque formation in these animals must be judged in the context of this
lack of complete equivalence between the transgenic mouse plaques and
the AD lesions.
![]() |
ACKNOWLEDGEMENT |
---|
We are indebted to Dr. Dean Luehrs for constructive discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the State of Arizona Alzheimer's Disease Research Center.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.
To whom correspondence should be addressed: Longtine Center for
Molecular Biology and Genetics, Sun Health Research Inst., 10515 West
Santa Fe Dr., Sun City, AZ 85351. Tel.: 623-876-5465; Fax:
623-876-5698; E-mail: aroher@mail.sunhealth.org.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M007859200
2 A. E. Roher and Y.-M. Kuo, unpublished data.
3 W. J. Goux and A. E. Roher, unpublished data.
4 C. S. Atwood, R. D. Moir, W. D. Jones, X. Huang, R. A. Cherny, Y. S. Kim, A. E. Roher, and A. I. Bush, submitted for publication.
5 Y.-M. Kuo, T. A. Kokjohn, T. G. Beach, L. I. Sue, D. Brune, J. C. Lopez, W. M. Kalback, D. Abramowski, C. Sturchler-Pierrat, M. Staufenbiel, and Alex E. Roher, unpublished data.
6 A. E. Roher, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
A, amyloid-
;
APP, amyloid precursor protein;
tg, transgenic;
GDFA, glass-distilled formic acid;
HPLC, high pressure
liquid chromatography.
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REFERENCES |
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