From the
We have analyzed the effect of internalized amyloid
The major protein component of amyloid deposits associated with
Alzheimer's disease (AD)
Our previous studies have demonstrated
that internalized A
In the pretreatment
experiments, some of the cell cultures exposed to 25 µM A
We examined whether the presence of A
We metabolically
labeled APP with [
The molecular weight of
the amyloidogenic fragments was further characterized by Tris-Tricine
SDS-gel electrophoresis in comparison to a series of carboxyl-terminal
fragments of known structure (Fig. 2). The major amyloidogenic
fragment migrates at a position that is significantly larger than the
authentic
In this report, we have extended our previous studies of
A
Several reports have suggested
that a series of carboxyl-terminal APP fragments may arise from the
endosomal/lysosomal processing of
APP(5, 6, 10, 11, 19) . Under
normal conditions, these fragments are rapidly degraded by lysosomal
cysteine proteases(5, 19) . Sequence analysis of these
APP fragments has indicated that some of these carboxyl-terminal
fragments contain the entire A
Although it is not yet clear what significance the accumulation and
increased stability of the amyloidogenic fragments is for the
mechanisms of amyloid accumulation and AD pathogenesis, there are some
intriguing possibilities that will serve as the basis for further
analysis of this phenomenon. It is possible that the accumulation of
insoluble A
The
increase in the half-life of the amyloidogenic fragments in response to
A
If some of the
fragments that accumulate in response to A
A prediction of this model is that at the normal,
physiological concentrations of A
We thank Dr. Tilman Oltersdorf and Athena
Neurosciences for providing the expression constructs and antibodies.
We also thank Drs. Barbara Cordell and Ziyang Zhong for supplying the
carboxyl-terminal APP fragments of known sequence.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-protein (A
) 1-42 aggregates on the metabolism of the
amyloid precursor protein (APP) in stably transfected 293 cells. The
amount of potentially amyloidogenic fragments of APP immunoprecipitated
by anti-carboxyl-terminal APP and anti-A
antibodies is
dramatically enhanced by the treatment of the cells with
A
1-42, which is resistant to degradation, but not
A
1-28, which does not accumulate in cells. This accumulation
of amyloidogenic carboxyl-terminal fragments is specific, since there
is relatively little effect of A
1-42 on the amount of the
nonamyloidogenic
-secretase carboxyl-terminal fragment. The
amyloidogenic fragments accumulate in the same nonionic
detergent-insoluble fraction of the cell that contains the internalized
A
1-42. Western analysis indicates that a subset of the
amyloidogenic fragments react with antibodies that recognize a
conformation of A
that is specifically associated with aggregated
forms of A
, suggesting that the adoption of this
aggregation-related conformation may be an early event which precedes
the final processing that produces A
. Pulse-chase analysis of the
[
S]Met-labeled 16-kDa amyloidogenic fragment
indicates that it is relatively stable in A
1-42-treated
cells, with a half-life of approximately 50 h. This fragment is
degraded with a half-life of 30 min in control cells treated with
A
1-28. In contrast, the turnover of the nonamyloidogenic
-secretase product is not significantly altered by the presence of
A
1-42. The continuous uptake of A
1-42 from the
medium is not required for the stimulation of amyloidogenic fragment
accumulation, suggesting that the presence of intracellular
A
1-42 aggregates establishes a new pathway for APP
catabolism in cells which leads to the long term stability of the
fragments. If these amyloidogenic fragments of APP ultimately give rise
to A
, then the production of A
may be an autocatalytic,
``runaway'' process in cells containing A
1-42
nuclei. It is conceivable that the accumulation of insoluble APP and
amyloidogenic fragments of APP in response to A
1-42
aggregates may mimic the pathophysiology of dystrophic neurites, where
the accumulation of intracellular APP and APP fragments has been
documented by immunohistochemistry.
(
)is a
39-42-amino acid, self-assembling peptide, known as the amyloid
A
peptide. Although remarkable progress has been made in our
understanding of the proteolytic processing of APP and the secretion of
soluble amyloid A
peptide(1) , the mechanisms for the
accumulation of insoluble amyloid deposits and their role in
Alzheimer's disease pathogenesis remains a matter of speculation.
It is clear that at least two pathways exist for APP processing which
give rise to fragments bearing A
sequences at their amino termini:
processing by
-secretase, which cleaves within the A
sequence
thereby precluding amyloid
accumulation(2, 3, 4) , and
-secretase
processing, which generates carboxyl-terminal APP fragments containing
the entire A
sequence(2, 5, 6, 7) .
Amyloidogenic,
-secretase-processing events may occur within
several intracellular organelles, including the rough endoplasmic
reticulum(8) , trans-Golgi network(9) , and lysosomes
(10-12). Further processing of APP within the transmembrane
domain by
-secretase releases soluble 3- and 4-kDa fragments
containing all or part of the A
sequence(13, 14, 15) . Recent evidence indicates
that the familial AD amino acid substitutions within the transmembrane
domain favor the production of the longer A
1-42 form of
A
(16) which is preferentially localized with diffuse and
senile plaque amyloid deposits in AD brain(17) . This suggests
that A
1-42 is more closely associated with AD pathogenesis
than shorter A
isoforms.
1-42 is largely resistant to degradation
and accumulates as insoluble aggregates in lysosomes. In contrast,
A
1-39 and shorter peptides fail to accumulate, although they
are also internalized by endocytosis(18) . Since lysosomes are a
site of APP processing and catabolism, we examined the effect of
internalized A
1-42 peptide on the proteolytic process of the
APP in APP-overexpressing cells. We found that the intracellular
A
1-42 causes a dramatic increase in the amounts of
amyloidogenic carboxyl-terminal fragments of APP. The accumulation of
these fragments is due to an increase in their stability. These results
suggest that the intracellular A
aggregates may stimulate the
accumulation of more A
by providing a stable nucleus on which
newly synthesized amyloidogenic fragments and A
can accrete,
thereby acquiring resistance to degradation.
Tissue Culture
Two cDNA clones, which transcribe
the full-length human APP751 and APP695 cDNAs under the direction of
the strong cytomegalovirus promoter, were kindly provided by Dr. T.
Oltersdorf of Athena Neurosciences. The constructs were used to
co-transfect the human kidney 293 cell line, along with the selection
marker pSVneo, by CaPO co-precipitation. Cells were then
maintained in DMEM, 10% fetal bovine serum with G418. The stably
transfected cells were then screened for APP expression by Western blot
analysis.
Antibody Production and Purification
Rabbits were
immunized with high performance liquid chromatography-purified
synthetic peptides A1-28 or A
1-42 containing an
extra cysteine residue at the carboxyl terminus conjugated to ovalbumin
lysine residues using the cross-linker, N-succinimidyl
3-(2-pyridyldithio)propionate (Pierce) and emulsified in Freud's
complete adjuvant. Subsequent injections utilized unconjugated
synthetic A
1-42 emulsified in Freud's incomplete
adjuvant. After at least three immunizations, serum samples were
collected and affinity-purified. To immobilize the immunizing peptide,
10 mg of the cysteine-containing A
1-28 or A
1-42
were dissolved in 5 ml of 20 mM TES buffer (Sigma) and allowed
to react with 8 ml of Bio-Rad Affi-Gel 401 for 16 h at room
temperature. Preparation and washing of the gel was done according to
the manufacturer's recommendations. Serum samples (20-30
ml) were diluted 1:1 in PBS, and the IgG fraction was eluted from a
protein G-Sepharose column using 0.2 M glycine, pH 2.7. The
IgG fraction was neutralized, dialyzed in PBS, recirculated over the
A
1-42 affinity resin, and eluted with 0.2 M glycine, pH 2.7. The purified antibody was neutralized and
dialyzed against PBS, and aliquots were stored at -80 °C.
Once thawed, antibody samples were kept at 4 °C for use. Typically,
30 ml of serum yielded 2 mg of affinity-purified antibody. Monoclonal
antibody 13G8, which recognizes the carboxyl terminus of APP, and
anti-BX5 polyclonal antibody, which recognizes an epitope in the
extracellular domain of APP, were generous gifts of Athena
Neurosciences.
Metabolic Labeling and
Immunoprecipitation
Transfected cell cultures (1
10
cells in a 10-cm plate) were preincubated with
methionine-deficient DMEM for 2 h prior to labeling. The cells were
then incubated in 2 ml of methionine-deficient DMEM, containing 25
µM amyloid peptide and 1% bovine serum albumin, and
labeled with 100 µCi/ml of
[
S]methionine/cysteine (1000 Ci/mmol;
Tran
S-label, ICN) for 4-16 h. At the end of the
labeling period, the conditioned medium was collected, and cells were
washed twice with cold PBS and lysed either in RIPA (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% Nonidet P-40, 0.5%
deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 5
mM EDTA, 2 µg/ml leupeptin, 0.2 unit/ml soybean trypsin
inhibitor, 1 µg/ml aprotinin) or Nonidet P-40 lysis buffer (50
mM Tris, pH 8.0, 150 mM NaCl, 1.0% Nonidet P-40, 1
mM phenylmethylsulfonyl fluoride, 5 mM EDTA, 2
µg/ml leupeptin, 0.2 unit/ml soybean trypsin inhibitor, 1 µg/ml
aprotinin). The cell lysate was then clarified at 10,000
g for 10 min, and the supernatant was transferred to a fresh tube.
The pellet fraction was then resuspended in 88% formic acid,
centrifuged at 10,000
g for 10 min, and lyophilized.
After lyophilization, the dry sample was resolublized with 2
RIPA, sonicated until clarified, diluted to 1
RIPA, and
centrifuged at 10,000
g for 10 min. The supernatant of
these samples was then subjected to immunoprecipitation analysis. The
immunoprecipitation of soluble APP with anti-BX5 antibody from
conditioned medium was carried out by the method described by
Oltersdorf et al. (3). The immunoprecipitated products were
then subjected to either Tris-Tricine SDS (45) or Tris-glycine (46) PAGE analysis, the gel was treated with ENHANCE (DuPont
NEN), and the dry gel was exposed to x-ray film.
1-42 for 6 h were treated with trypsin and resuspended
in fresh medium lacking A
1-42 prior to metabolic labeling.
Cells treated with A
peptide for 6-12 h were incubated in 2
ml of methionine-free DMEM containing 1% bovine serum albumin for 2 h
and then pulse-labeled with 250 mCi/ml [
S]Met
for 30 min. The [
S]Met-labeled cells were then
washed twice with PBS and chased in the methionine-deficient DMEM
supplemented with 10 µg/ml methionine for 2-12 h.
Immunoprecipitation of APP and its products was then performed as
described above.
Immunoblotting
Cells treated with A peptide
were lysed in 1 ml of RIPA as described above and centrifuged at 10,000
g for 15 min. The RIPA-insoluble pellet was then
solubilized in 88% formic acid, and the supernatant was then collected
by centrifugation at 10,000
g for 10 min. The formic
acid-soluble cell extract was then lyophilized and redissolved in 4
SDS sample buffer with sonication. About 30 µg of protein
were subjected to SDS-PAGE and blotted onto nitrocellulose.
Nitrocellulose filters were blocked with 5% dried milk and incubated
with primary antibody that was specific to either A
peptide or APP
COOH-terminal fragments. The bound primary antibody was then detected
by a horseradish peroxide-coupled secondary antibody and an ECL
detection system (Amersham Corp.).
1-42 affects
the catabolism of APP and APP fragments in stably transfected 293 cell
lines. We chose this cell culture model because the nonamyloidogenic
-secretase and amyloidogenic
- and
-secretase pathways
for APP processing have been demonstrated in this
system(2, 3, 11, 13, 19) . The
ability to compare the products of nontransfected cells is an important
control for immunoprecipitation and Western blot identification of
fragments, since the endogenous levels of APP expression in
nontransfected cells is relatively low(3) . We first confirmed
that A
1-42 is internalized by APP751-transfected 293 cells
and accumulates as nonionic detergent-insoluble aggregates that are
resistant to degradation as described previously in human
fibroblasts(18) . Five percent of the total
I-labeled A
1-42 in the medium accumulates
intracellularly over a 6-h incubation period in comparison to 0.02% of
the total A
1-28 as determined by the amount of
cell-associated A
that is resistant to removal by trypsin
treatment. After lysis of the cells in the nonionic
detergent-containing RIPA, two-thirds of this internalized,
I-labeled A
1-42 is sedimentable at 10,000
g. These results closely parallel those previously
described for human fibroblasts(18) .
S]Met in cell cultures treated
with A
1-42 or nonaccumulating A
1-28 and compared
the amount of
S-labeled APP or amyloidogenic fragments of
APP associated with the cell by immunoprecipitation with 13G8
monoclonal antibody directed against the carboxyl terminus of APP.
Since most of the intracellular A
1-42 is contained in the
low speed sedimentable fraction after nonionic detergent lysis, we
examined both the insoluble and soluble fractions for APP fragments.
The amounts of APP and amyloidogenic APP fragments are greatly
increased in the insoluble fraction of A
1-42-treated cells (Fig. 1A). In particular, the accumulation of a band
migrating at an apparent mass of 16 kDa is dramatically stimulated by
A
1-42. Although the amount of this band is much lower in
control cells, it can be visualized at longer exposures (see below).
The stimulatory effect is specific for A
1-42, since the
amounts of APP and APP fragments in cells treated with nonaccumulating
A
1-28 are indistinguishable from that in cells incubated in
the absence of peptide. The response to A
1-42 treatment is
also specific for potentially amyloidogenic fragments of APP, since the
amounts of the nonamyloidogenic 12-kDa
-secretase product are not
significantly affected by the presence of A
1-42. Similar
increases in the amounts of APP and amyloidogenic fragments of APP were
observed in cells transfected with APP695 (data not shown). Control
experiments with nontransfected cells indicate that the
immunoprecipitated bands arise from the transfected APP751 gene and are
not an artifact of immunoprecipitation. In contrast to the results
observed in the insoluble fraction of the cells, no significant effect
of A
1-42 on the amounts of APP and APP fragments was
observed in the RIPA-soluble fraction of cells (Fig. 1B). The stimulation of the accumulation of
fragments of APP does not appear to be due to an increase in the rate
of APP synthesis or a decrease in the
-secretase processing of
APP, since the amounts of soluble APP immunoprecipitated from the
culture medium are the same in A
1-42-treated and control
cells (Fig. 1C).
Figure 1:
A1-42 stimulates the
accumulation of amyloidogenic fragments of APP. A,
[
S]Methionine-labeled APP and carboxyl-terminal
fragments from APP were immunoprecipitated with the monoclonal
antibody, 13G8, from the insoluble fraction of A
1-42-treated
and control cell cultures. The amounts of APP and amyloidogenic
fragments of APP are greatly increased in cells treated with
A
1-42 (lane 6) in comparison to controls treated
with A
1-28 (lane 5) or cells labeled in the absence
of A
peptide (lane 4). In particular, the amount of a
16-kDa amyloidogenic fragment (indicated by the arrowhead) is
dramatically increased. In contrast, the amount of the nonamyloidogenic
12-kDa
-secretase band is not significantly altered by
A
1-42. The amount of immunoprecipitable fragments is greatly
reduced in nontransfected cells which have low levels of endogenous APP
expression, indicating that the labeled bands are derived from the
transfected APP gene and are not an artifact of immunoprecipitation. B, A
1-42 has no significant effect on the amounts
of APP or amyloidogenic APP carboxyl-terminal fragments in the soluble
fraction of the cells. APP and its carboxyl-terminal fragments were
immunoprecipitated from the RIPA-soluble cell extracts of cells
incubated with A
1-42 for 6 h (695
cells-A
1-42; 751 cells-A
1-42) or
control cells (695 cells only; 751 cells only) with
the monoclonal antibody 13G8. No significant and reproducible changes
in the amounts of APP or amyloidogenic fragments were observed (compare lanes 1 and 2; 3 and 4). A small
(2-fold) increase in the amount of the
-secretase product
(indicated by the arrowhead) is observed in
A
1-42-treated APP751-transfected cells, but this small
increase has not proven to be reproducible in subsequent experiments. C, the secretion of soluble APP is not affected by the
treatment of cells with A
1-42. Conditioned medium from cells
treated with A
1-42 for 6 h (751 cells,
A
1-42) or untreated control cells (751 cells, no
peptide and 293 cells) were immunoprecipitated with
antisera directed against APP residues 444-592 of APP695
(polyclonal anti-Bx5). No difference in the amount of
immunoprecipitated APP is observed in the medium from
A
1-42-treated and control APP751 cells. D,
immunoprecipitation of APP COOH-terminal fragments with antisera
directed against residues 1-28 of A
. APP751-transfected 293
cells were metabolically labeled with [
S]Met in
the presence (+) or absence (-) of 25 µM A
1-42 for 6 h. The APP COOH-terminal fragments were
then immunoprecipitated by anti-A
1-28 antibody and resolved
on a 15% Tris Tricine gel. The levels of a 14.4-kDa APP COOH-terminal
fragment (arrowhead) are greatly increased in
A
1-42-treated cells (lane 2) in comparison to
control cultures (lane 1). Bands with a higher electrophoretic
mobility also accumulate in response to A
treatment. The
A
1-42-induced APP COOH-terminal fragments can be eliminated
by preadsorbing the anti-A
1-28 antibody with 10 µg/ml of
excess A
peptide before immunoprecipitation (lanes 3 and 4).
The accumulation of amyloidogenic
fragments of APP in the insoluble fraction of cells treated with
A1-42 was also observed by immunoprecipitation with
antibodies directedagainst residues 1-28 of A
, providing
further evidence that these fragments contain A
epitopes (Fig. 1D). The amount of APP and amyloidogenic APP
fragments immunoprecipitated by affinity-purified anti-A
1-28
antisera is observed to increase dramatically in the cells incubated
with A
1-42. The amount of amyloidogenic fragments is greatly
reduced in control, nontransfected cells, indicating that the labeled
bands are products of the transfected APP gene (data not shown). The
immunoprecipitation of the amyloidogenic fragments is blocked by
preadsorption of the antibody with excess A
1-28 peptide. On
Tris-Tricine gels (Fig. 1D), the apparent molecular
weight of the APP fragments is slightly lower than that observed on
Tris-glycine gels (Fig. 1A) and the carboxyl-terminal
fragments are further resolved into a series of closely spaced bands as
has been previously described(6) .
-secretase products from transfected CHO cells (lane
1),
(
)or the carboxyl-terminal 100 residues
of APP (20) expressed in a reticulocyte lysate (lane
4), which have an apparent molecular mass of approximately 12 kDa.
These results indicate that the fragments which accumulate in response
to A
1-42 are sufficiently large to contain the entire A
sequence and suggest that the amino terminus of the major fragment may
extend approximately 20 amino acids amino-terminal to the beginning of
the A
domain.
Figure 2:
Characterization of the sizes of the
amyloidogenic fragments by Tris-Tricine gel electrophoresis. The
electrophoretic mobility of the amyloidogenic carboxyl-terminal
fragments of APP were compared to APP fragments of known structure. The
amyloidogenic fragments were immunoprecipitated with 13G8 monoclonal
antibody from APP-751-transfected cells and resolved on 15%
Tris-Tricine gels as described previously. The major carboxyl-terminal
fragment of APP from A1-42-treated cells migrates with an
apparent molecular mass of 14 kDa on Tris-Tricine gels (lane
2) which is substantially larger than the smallest amyloidogenic
fragment from transfected CHO cells (lane 1) that was verified
by protein sequencing. It also migrates more slowly than an in
vitro translation product of the carboxyl-terminal 100 residues of
APP expressed in a rabbit reticulocyte lysate (lane
4).
The accumulation of the amyloidogenic fragments
of APP in A1-42-treated cells is also observed by Western
analysis (Fig. 3A). The 16-kDa band and several
additional higher molecular mass bands are stained by affinity-purified
anti-A
1-42 antibodies in A
1-42-treated
transfected cells (lanes 2 and 3), but not in
transfected cells incubated in the absence of A
(lane 1)
nor in nontransfected control cells treated with A
1-42 (lane 5). These results indicate that the immunoreactive bands
arise from the transfected APP751 gene and not the added
A
1-42 and confirm that the accumulation of these bands in
the preceding immunoprecipitation experiments is not an artifact. The
reactivity of these bands with this antibody also reveals that some of
the amyloidogenic fragments display a conformation that is resistant to
denaturation in SDS and found only in A
aggregates. The
specificity of this antibody for an epitope found in SDS-resistant
A
aggregates is demonstrated in Fig. 3B, which
compares the staining of synthetic A
1-42 and
A
1-40 standards. Previously published work has demonstrated
that A
1-42 forms aggregates that are not disrupted by
heating at 100 °C in SDS sample buffer, while aggregates formed by
A
1-40 are disrupted by this
treatment(21, 22) . Although bands corresponding to
4-kDa A
monomer are prominently revealed by Amido Black staining
of the nitrocellulose membrane for both A
1-40 and
A
1-42 (lanes 4-6), only the bands
corresponding to aggregated forms of A
1-42 (at approximately
18 kDa and at the top of the gel) strongly react with the
anti-A
1-42 antibodies (lane 3). The 4-kDa band
corresponding to the A
1-40 monomer is not stained by this
antibody, and the 4-kDa band corresponding to A
1-42 reacts
only weakly (lanes 1-3). The fact that the amyloidogenic
fragments are recognized by the same antibody suggests that they
display the same SDS-resistant epitope found in A
aggregates.
Figure 3:
The
amyloidogenic fragments of APP that accumulate in response to
A1-42 treatment display a conformation-dependent epitope
associated with aggregated forms of A
. A, Western
analysis of A
1-42-treated and control cells with an
affinity-purified antibody which reacts with a conformation of A
found specifically in A
aggregates. This panel shows the
fluorogram of a blot transferred from a 15% Tris-glycine gel. The
prominent 16-kDa amyloidogenic and minor bands at molecular masses of
30 and 28 kDa are labeled by the antibody (lane 2). No bands
are detected in control 293 cells (lane 4) or
APP751-transfected cells incubated in the absence of A
1-42 (lane 1) nor in nontransfected 293 cells incubated with
A
1-42 (lane 5). The steady-state amounts of these
fragments does not decrease rapidly after the
A
1-42-containing medium is removed and replaced with fresh
medium lacking A
and incubated for an additional 6 h (lane
3), suggesting that the fragments are relatively stable. B, the specificity of the antibody for a conformation of
A
associated with A
aggregates is demonstrated by Western
analysis of synthetic A
peptides. At concentrations above 25
µM, A
1-42 forms higher molecular weight
aggregates that are not disrupted by heating at 100 °C in SDS
sample buffer (21, 22), while the aggregates formed by A
1-40
and shorter A
peptides are disrupted by this treatment. Lanes
1-3 are anti-A
1-42-stained, and lanes
4-6 are Amido Black-stained nitrocellulose strips after the
electrophoretic transfer of peptide from A
1-40 (lanes 1 and 4), A
1-41 (lanes 2 and 5) or A
1-42 (lanes 3 and 6). The
aggregated forms of A
1-42 migrating with an apparent
molecular mass of 18 kDa and material that remains at the top of the
gel are intensely stained by the antibody, even though there is little
Amido Black-staining peptide at these positions. The 4-kDa band
corresponding to the monomeric A
reacts weakly or not detectably
with the antibody, even though this band is prominently stained by
Amido Black.
Since the amount of soluble APP secretion is not significantly
altered by the presence of A1-42 peptide, the accumulation
of the insoluble amyloidogenic APP fragments may be due to a decrease
in their rate of turnover. Western analysis suggests that the 16-kDa
amyloidogenic fragment is relatively stable after the removal of
A
1-42 from the medium (Fig. 3A, lane
3). The intensity of the immunoreactivity of the 16-kDa band is
only slightly reduced when the cells are removed from the
A
1-42-containing medium and incubated in peptide-free medium
for an additional 6 h (lane 3) as compared to cells incubated
continuously with A
1-42 for 12 h (lane 2). This
suggests that the steady-state amount of the fragment does not change
rapidly when the cells are no longer exposed to A
1-42. To
quantify the stability of the 16-kDa fragment, we determined its
turnover rate by pulse-chase analysis. As shown in Fig. 4A, the 16-kDa fragment turns over at a much slower
rate in cells treated with A
1-42 as compared to the control
cells exposed to A
1-28. Although the amount of this 16-kDa
fragment is greatly reduced in control cells, the turnover of this
fragment can be quantified by longer exposures of the PhosphorImager
plate. In the presence of A
1-42, the half-life is
approximately 50 h, whereas in the presence of A
1-28 the
half-life is approximately 30 min. The increased stability of the
amyloidogenic APP fragments does not appear to be due to a generalized
inhibition of degradative enzymes. The half-life of the
nonamyloidogenic
-secretase carboxyl-terminal fragment of APP is
not detectably stabilized by A
1-42 treatment, suggesting
that the stability is specific for amyloidogenic fragments (Fig. 4B). This is consistent with previous results that
demonstrate that intracellular A
1-42 aggregates do not
interfere with the degradation of control peptides and proteins
internalized from the culture medium(18) .
Figure 4:
The turnover of the 16-kDa amyloidogenic
fragment is specifically stabilized by A1-42. A,
pulse-chase analysis of the turnover of the 16-kDa carboxyl-terminal
fragment. The 16-kDa fragment is much more stable in
A
1-42-treated cells (
--
) in
comparison to the lifetime of the fragment in control cells treated
with A
1-28 (
--
). The half-life of
the 16-kDa fragment is approximately 50 h in A
1-42-treated
cells and approximately 30 min in control cells. Although the amount of
the 16-kDa fragment in control cells is much lower than that in
A
1-42-treated cells, this fragment can be detected and
quantified accurately using a PhosphorImager. B, pulse-chase
analysis of the turnover of the 12-kDa nonamyloidogenic
-secretase
product. The lifetime of the nonamyloidogenic fragment is that same in
A
1-42-treated cells (⊡--⊡) and cells
treated with A
1-28 (
--
). This
indicates that A
1-42 treatment does not result in a
generalized inhibition in the degradation of membrane proteins, but
rather suggests that the effect is specific for amyloidogenic fragments
of APP.
To probe the
mechanism for the accumulation of amyloidogenic fragments of APP in
response to A1-42 treatment, we asked whether the continued
internalization of A
1-42 from the medium is required for the
stimulation of amyloidogenic fragment accumulation. APP751-transfected
cells were incubated in the presence of 25 µM A
1-42 for 6 h. One set of cultures was immediately
labeled with [
S]Met in the presence of A
and prepared for immunoprecipitation, whereas another set of cultures
was trypsin-treated to remove surface-adsorbed A
(18) ,
cultured for an additional 6 h in the absence of A
, and then
labeled and prepared for immunoprecipitation. Both cultures labeled in
the continuous presence of A
and cultures pretreated with A
before labeling show an increase in amyloidogenic carboxyl-terminal
fragments of APP ranging from 8 to 14 kDa (Fig. 5) on a Tris
Tricine gel. These results demonstrate that the continued uptake of
A
from the culture medium is not required to stimulate the
accumulation of amyloidogenic fragments and suggest that, once stable
A
1-42 aggregates have nucleated within the cell, the
pathways for APP catabolism are altered to favor the accumulation of
amyloidogenic fragments and potentially more A
.
Figure 5:
The
accumulation of amyloidogenic APP fragments does not require the
continued uptake of A1-42 from the medium.
APP751-transfected 293 cells were incubated with A
1-42 for 6
h, after which half of the cultures were treated with trypsin to remove
any surface-adsorbed A
and resuspended in fresh medium lacking
A
. The A
-treated cultures and control cells not exposed to
peptide were then labeled for 6 h with [
S]Met,
and samples were immunoprecipitated with monoclonal antibody 13G8. The
amounts of amyloidogenic fragments immunoprecipitated is not
significantly different in the cell cultures labeled in the continuous
presence of A
1-42 (lane 2) and the cultures
pretreated with A
1-42 and labeled in the absence of A
in the medium (lane 3).
internalization and turnover to examine the effects of
internalized A
1-42 aggregates on the catabolism of APP and
its proteolytic processing products. Our results suggest that
intracellular A
1-42 aggregates alter the pathways which
normally degrade APP and its amyloidogenic fragments to stabilize them
and favor their accumulation. The accumulation of amyloidogenic
fragments is not due to an increase in the rate of APP synthesis or an
inhibition of the
-secretase processing pathway, since the amount
of soluble APP secreted into the medium is not altered by
A
1-42 treatment. Instead, the mechanism for the accumulation
of the amyloidogenic fragments appears to be derived from a specific
enhancement in their stability. The half-life of the 16-kDa
amyloidogenic fragment is approximately 100-fold longer in cells
treated with A
1-42 than in control cells treated with
nonaccumulating A
1-28.
peptide sequence and are
potentially amyloidogenic(7) . A similar series of
carboxyl-terminal fragments are detected in both AD and aged control
brains(5) , and some reports indicate that there may be a direct
correlation between the extent of neuronal degeneration and
accumulation of APP carboxyl-terminal
fragments(23, 24) . These results indicate that APP
fragments of the same size that we have observed accumulating in
A
1-42-treated cultured cells are also produced by brain
cells in vivo and accumulate in AD and aged brain tissue.
, APP, and amyloidogenic APP fragments may mimic the
pathophysiology of dystrophic
neurites(25, 26, 27, 28, 29, 30, 31, 32, 33) and
vascular smooth muscle cells in amyloid
angiopathy(34, 35) , where A
and carboxyl-terminal
fragments of APP have been demonstrated to accumulate intracellularly
by immunohistochemical staining. The amounts of potentially
amyloidogenic, carboxyl-terminal fragments of APP and 4-kDa A
were
also found to be significantly enriched in lysates of leptomeningeal
vessels from AD cases with amyloid angiopathy but not
controls(35) . The increased stability and accumulation of
amyloidogenic, carboxyl-terminal fragments of APP has also been
reported in lymphoblastoid cells from patients with familial forms of
AD(36, 37) . The accumulation of carboxyl-terminal
fragments of APP may also contribute directly to pathogenesis. In some
cell culture and transgenic animal models, overexpression of
carboxyl-terminal fragments bearing the entire A
sequence has been
reported to result in cell death and
neurotoxicity(20) -(41) . Determining whether these
possibilities are valid or not will require further analysis.
1-42 treatment is not due to a generalized inhibition of
the activity of the degradative machinery of the cell. Our results also
indicate that the accumulation is very specific for amyloidogenic
fragments of APP, since the amount of the nonamyloidogenic
-secretase product is not substantially altered by the presence of
A
1-42, and its rate of turnover is similarly unaffected. The
simplest model to account for these results is to postulate that the
amyloidogenic fragments interact with the degradation-resistant A
aggregates and that this interaction allows the fragments to evade the
normal degradation pathways. A prediction of this model is that the
rate of amyloid accumulation would depend on the flux of APP and
amyloidogenic fragments down the catabolic pathway and the balance
between their rates of degradation and accretion onto existing A
aggregates. The fact that the amyloidogenic fragments of APP and the
A
aggregates end up in the same insoluble fraction of the cell
support the notion of their interaction, but experiments designed to
demonstrate a direct interaction between A
and amyloidogenic
fragments of APP in vitro have so far been equivocal. The
finding that the amyloidogenic fragments display a
conformation-dependent epitope found in A
aggregates provides a
conceptual basis for their potential interaction. If the fragments have
the same conformation of A
that is required for amyloid assembly,
they may be capable of interacting with A
aggregates prior to
completion of their proteolytic processing to A
.
1-42 treatment are
further processed to A
, then as a consequence of the nucleation of
stable A
aggregates intracellularly, amyloid accumulation would be
a self-stimulating, ``runaway'' process. A major caveat to
this interpretation is that it must await the unambiguous demonstration
that these fragments ultimately give rise to more A
. This model
for amyloid accumulation is mechanistically related to models proposed
for the replication of the scrapie prion (42, 43), and the finding that
A
1-42 stimulates the accumulation of long-lived fragments
which are potential precursors to A
is consistent with a key
feature of this model, which postulates that the scrapie prion
catalyzes its own production from its precursor protein. Another
prediction of this model is that the scrapie precursor protein
undergoes a conformation change leading to the acquisition of
resistance to degradation. The observation that aggregation-specific
epitopes are displayed by amyloidogenic fragments of APP is also
consistent with this prediction. Our working model for A
accumulation differs in some details from the model originally proposed
for scrapie prion replication. In this model, it is the aggregates of
A
, rather than the monomer which interacts with the precursor
protein or its fragments. It is not clear whether aggregation induces a
conformation change in the fragments or whether the conformation change
precedes aggregation and merely allows the fragment to interact with
the A
aggregates. It seems simpler to propose that the resistance
to degradation arises from the interaction of the fragments with the
stable A
aggregates rather than from the conformation change per se.
1-42, the formation of
A
aggregates would be a rare event, and the turnover of APP
fragments would proceed normally. The predicted stochastic nature of
stable A
aggregate formation (44) at the low, physiological
concentrations of A
1-42 can explain a peculiar feature of
A
accumulation and AD pathogenesis. Amyloid deposits are focal
lesions and even within the regions of the brain which contain large
numbers of amyloid deposits, a large number of neurons remain
apparently unaffected. The neurons at risk for AD pathogenesis may
represent ones which have had the misfortune of containing a stable
A
aggregate over their lifetime. The remnant of these cells may
form the focal nucleus of an amyloid deposit and initiate a cascade of
events, including the growth and maturation of the deposits by the
addition of soluble, extracellular A
to eventually yield senile
plaque. This hypothesis remains to be tested by further
experimentation.
, amyloid
-protein; APP,
amyloid precursor protein; PBS, phosphate-buffered saline; PAGE,
polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
DMEM, Dulbecco's modified Eagle's medium; TES,
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic
acid; RIPA, radioimmune precipitation buffer; CHO, Chinese hamster
ovary. We use the term ``amyloidogenic'' to refer to
fragments of APP that are sufficiently large to contain the entire
A
sequence. These are actually potentially amyloidogenic and it
remains to be established that they actually give rise to A
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.