Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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Abstract |
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The amyloid- peptide (A
) is produced at
several sites within cultured human NT2N neurons with
A
1-42 specifically generated in the endoplasmic reticulum/intermediate compartment. Since A
is found as
insoluble deposits in senile plaques of the AD brain, and the A
peptide can polymerize into insoluble
fibrils in vitro, we examined the possibility that A
1-40,
and particularly the more highly amyloidogenic A
1-42, accumulate in an insoluble pool within NT2N neurons. Remarkably, we found that formic acid extraction of the NT2N cells solubilized a pool of previously undetectable A
that accounted for over half of the total intracellular A
. A
1-42 was more abundant than A
1-40
in this pool, and most of the insoluble A
1-42 was generated in the endoplasmic reticulum/intermediate compartment pathway. High levels of insoluble A
were
also detected in several nonneuronal cell lines engineered to overexpress the amyloid-
precursor protein.
This insoluble intracellular pool of A
was exceptionally stable, and accumulated in NT2N neurons in a
time-dependent manner, increasing 12-fold over a 7-wk
period in culture. These novel findings suggest that A
amyloidogenesis may be initiated within living neurons
rather than in the extracellular space. Thus, the data
presented here require a reexamination of the prevailing view about the pathogenesis of A
deposition in the
AD brain.
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Introduction |
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ALZHEIMER'S disease (AD)1 is characterized by accumulation of fibrillar amyloid- peptides (A
) in
senile plaques. That the accumulation of A
is essential for the pathogenesis of AD is supported by genetic
studies showing that mutations in the amyloid-
precursor
protein (APP) (which gives rise to A
through proteolytic processing) are linked to a subset of familial AD (FAD)
cases with autosomal penetrance, and alter A
production
(reviewed in Selkoe, 1997
). For example, the double mutation found in a Swedish FAD kindred leads to overproduction of A
, while other mutations alter the relative levels of the two major forms of A
, resulting in an increased
A
1-42/1-40 ratio (Citron et al., 1992
; Scheuner et al.,
1996
). Previous studies have shown that A
1-42 is more insoluble than the more abundant A
1-40, and that it is
the most prevalent A
species found in senile plaques
(Iwatsubo et al., 1994
). Other FAD mutations that account
for the majority of early-onset FAD cases have been
linked to the Presenilin 1 (PS1) and Presenilin 2 (PS2)
genes (Levy-Lahad et al., 1995
; Sherrington et al., 1995
). Mutations in these genes, like some of those in the APP
gene, also increase the A
1-42/1-40 ratio (Borchelt et al.,
1996; Duff et al., 1996
; Scheuner et al., 1996
).
Since genetic studies have established a role for A in
the pathogenesis of AD, it is essential to understand how
A
is produced from APP. For example, it has been
shown that APP is cleaved by
-secretase(s) to generate
the NH2 terminus of A
, and by
-secretase(s) to generate
the COOH terminus of A
(Haass et al., 1992
; Shoji et al.,
1992
). These cleavages may occur in a variety of subcellular locations, including the endoplasmic reticulum/intermediate compartment (ER/IC; Chyung et al., 1997
; Cook
et al., 1997
; Hartmann et al., 1997
; Xu et al., 1997
), the
trans-Golgi network (TGN; Xu et al., 1997
), and the endosomal/lysosomal system (Koo and Squazzo, 1994
). Whereas
A
produced by these pathways may be secreted (as has been shown for TGN-generated A
) or may remain intracellular (as has been shown for A
generated by the ER/
IC pathway), the relative roles of intracellular and secreted A
in the pathogenesis of AD remain to be determined.
While numerous studies have documented that nonneuronal cells engineered to express APP secrete both A1-40
and A
1-42, intracellular A
is not commonly seen in
these cells (Forman et al., 1997
; Xu et al., 1997
). However,
intracellular A
can be detected readily in human NT2N
neurons after metabolic labeling, and its production precedes that of secreted A
(Wertkin et al., 1993
; Turner et al., 1996
). Analysis of intracellular A
by ELISA indicates
that intracellular and secreted A
are composed of different ratios of A
1-42/1-40, with A
1-40 being more prevalent in secreted material (Turner et al., 1996
). In addition
to being produced by mechanisms with different time
courses, and being composed of different proportions of
A
1-40 and A
1-42, intracellular and secreted A
can be
produced by different pathways in NT2N neurons. Recent
studies have shown that A
1-42, but not A
1-40, is produced by an ER/IC pathway, and that this pathway does
not contribute to the secreted pool of A
(Cook et al.,
1997
). Finally, secretion of A
by NT2N neurons increases
with time in culture (Turner et al., 1996
). An age-dependent increase in A
secretion by neurons in vivo may play
a role in the deposition of A
into senile plaques in the extracellular space of the brain during normal aging and in
AD, as well as in the cortex and hippocampus of transgenic mice that overexpress mutant forms of APP (Games
et al., 1995
; Hsiao et al., 1996
).
In addition to forming insoluble extracellular plaques,
A may also accumulate intracellularly in an aggregated
insoluble pool. For example, exogenous A
1-42 added to
culture medium can be taken up by cells, after which it can
be solubilized only by formic acid extraction (Knauer et al.,
1992
; Yang et al., 1995
). Thus, these findings raise the possibility that endogenously produced intracellular A
may
aggregate within neurons as well. Because formic acid is
required to solubilize A
from senile plaques, we sought
to detect the presence of insoluble A
within NT2N neurons and other cell lines by formic acid extraction, and
found that a significant fraction of the total intracellular
A
, particularly A
1-42, was retained as an insoluble pool
within these cells. Further, this insoluble pool of A
increased 12-fold in postmitotic NT2N neurons over a period of 7 wk in culture. Since the prevailing view of amyloidogenesis in AD is that plaque formation is initiated in
the extracellular space by secreted A
, our findings challenge this assumption by implicating the intracellular compartment as a site where A
may accumulate in an insoluble form.
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Materials and Methods |
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Cell Culture
NT2 cells derived from a human embryonal carcinoma cell line (Ntera2/
cl.D1) were grown and passaged as described previously (Pleasure et al.,
1992; Pleasure and Lee, 1993
). Cells were differentiated by two weekly retinoic acid treatments (10 µM) for 5 wk, and were replated (replate 2 cells) in the presence of mitotic inhibitors to yield nearly pure NT2N neurons (Pleasure et al., 1992
). To obtain 99% pure neurons (replate 3 cells),
replate 2 cells were removed enzymatically and mechanically, and were
replated in 10-cm dishes (Pleasure et al., 1992
). Cultures of Replate 2 or
Replate 3 NT2N cells were used for experiments when they were 3-4 wk
old unless otherwise indicated. CHO Pro5 cells were grown and passaged three times per week in Alpha-MEM (Life Technologies, Inc., Gaithersburg, MD) containing 10% FBS and penicillin/streptomycin. Baby hamster kidney (BHK-21) cells were grown and passaged three times per week in Glascow MEM (Life Technologies, Inc.) supplemented with 10%
tryptose phosphate, 5% FBS, and 0.02 M Hepes. CHO-695 cells were obtained from Dr. S.S. Sisodia, and were grown and passaged as described
above for CHO Pro5 cells with the addition of 0.2 mg/ml of G418 to the
culture medium.
Preparation of Semliki Forest Virus and Infection of Cultured Cells
Semliki Forest Virus (SFV) expressing wild-type APP695 (SFV-APPwt)
or an APP mutant in which the third and fourth amino acids from the carboxyl terminus of APP have been changed to lysines (SFV-APPKK)
were prepared and titered as previously described (Chyung et al., 1997
;
Cook et al., 1997
). CHO-Pro5, BHK-21, NT2, and NT2N cells were infected in serum-free medium at a multiplicity of infection of ~10. After 1 h,
complete growth medium was replaced and infection was allowed to proceed for 12 h.
Metabolic Labeling, Immunoprecipitation, and Gel Electrophoresis
Cultured NT2N cells were methionine-deprived by incubation in methionine-free DMEM (Life Technologies, Inc.) for 30 min before adding
[S35]methionine (500 µCi/ml in methione-free DMEM + 5% dialyzed
FBS; DuPont-NEN, Boston, MA) for a 12-h labeling period. Cells were washed twice in PBS and lysed in 600 µl RIPA buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% NP40, 5 mM EDTA in TBS, pH 8.0) with a cocktail of protease inhibitors (1 µg/ml each of Pepstatin A, Leupeptin, TPCK,
TLCK, STI, and 0.5 mM PMSF). After brief sonication, cell lysates were
centrifuged at 40,000 g for 20 min at 4°C, and the supernatant was subjected to immunoprecipitation with 6E10 (a monoclonal antibody specific
for A1-17; Kim et al., 1988
) as previously described (Turner et al., 1996
).
The remaining pellets were resuspended in 100 µl 70% formic acid and
sonicated until clear. For direct extraction into formic acid, cells were
scraped in 1 ml PBS, pelleted by centrifugation, and lysed in 100 µl of 70%
formic acid with sonication. Formic acid from both directly extracted and
sequentially extracted samples was removed by vacuum centrifugation for
40 min, and the resulting dry pellet was resuspended in 100 µl of 60% acetonitrile. RIPA buffer (1.9 ml) was added to each of the samples before
they were subjected to immunoprecipitation with 6E10. Immunoprecipitated A
was resolved on a 10/16.5% step gradient Tris-Tricine gel, fixed
in 60% methanol, dried, and placed on PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) plates for 72 h.
Trypsin Treatment of CHO Cells
CHO Pro5 cells were infected with SFV-APPwt for 12 h, rinsed twice in
PBS, and incubated on ice for 20 min in either PBS alone, 10 µg/ml of
trypsin (Life Technologies, Inc.) in PBS, or 10 µg/ml trypsin plus 0.1%
Triton X-100 in an adaptation of a previously described technique (Turner
et al., 1996; Chyung et al., 1997
). Trypsin was then inactivated by adding
100 µg/ml soybean trypsin inhibitor. The treated cells were then washed
with ice-cold PBS, scraped into PBS buffer, centrifuged at 2,000 g for 2 min, resuspended in 100 µl formic acid, sonicated, and centrifuged at
40,000 g for 20 min at 4°C. The supernatant was neutralized with 1.9 ml of
1 M Tris base and diluted 1:3 in H2O for quantification of A
1-40 and
A
1-42 by sandwich-ELISA.
Lysis of Cells and Sandwich ELISA
For serial extraction in RIPA and formic acid, cells were washed twice in
PBS and then lysed in 600 µl RIPA buffer and centrifuged for 20 min at
40,000 g at 4°C. Supernatant was subjected directly to sandwich ELISA,
and the pellet was resuspended in 100 µl 70% formic acid with sonication
until clear. Formic acid samples were then neutralized by adding 1.9 ml
1 M Tris base and diluted 1:3 in H2O before quantifying A by sandwich-ELISA.
For direct extraction into formic acid, cells were scraped in PBS after
washing twice with PBS. Cells were pelleted by centrifugation at 2,000 g
for 2 min, and were then lysed in 100 µl formic acid. Insoluble material
was pelleted by centrifugation at 40,000 g at 4°C for 20 min, and the supernatant was neutralized by adding 1.9 ml 1 M Tris base and diluted 1:3 in
H2O before quantification of A by sandwich-ELISA.
For extraction into PBS, cells were scraped in PBS after washing twice
with PBS. Cells were lysed by sonication, and insoluble material was pelleted by centrifugation at 40,000 g at 4°C for 20 min, and A in the soluble
fraction was quantitated by sandwich-ELISA.
Sandwich-ELISA was performed as described previously using mAbs
specific for different species of A (Suzuki et al., 1994
; Turner et al.,
1996
). BAN-50 (a mAb specific for the first 10 amino acids of A
) was
used as a capturing antibody, and horseradish peroxidase-conjugated BA-27
(a mAb specific for A
1-40) and horseradish peroxidase-conjugated BC-05
(a mAb specific for A
1-42) were used as secondary antibodies. To calibrate the sensitivity of the ELISA for detecting A
after formic acid extraction and neutralization, synthetic A
1-40 and A
1-42 peptides
(Bachem Bioscience Inc., King of Prussia, PA) used to generate the standard curves were treated with formic acid and neutralized in the same
manner as the cell lysates. Under these conditions, the sandwich ELISA
had a detection limit of <1 femtomole of synthetic A
per sample. The
BAN50, BA-27, and BC-05 mAbs were prepared and characterized as described previously (Suzuki et al., 1994
).
Cycloheximide Treatment
For experiments involving cycloheximide treatments, NT2N cells were incubated in media containing 150 µg/ml cycloheximide for various time points up to 24 h. Cells were harvested and extracted sequentially in RIPA and formic acid as described above. Samples were then subjected to sandwich ELISA analysis.
Western Blot Analysis of APP Levels
RIPA-extracted cell lysates (15 µg as determined by BCA assay) were resolved on a 7.5% Tris-glycine acrylamide gel and transferred to nitrocellulose for immunoblotting with Karen (a goat anti-APP antibody) at a 1:1,000
dilution (Turner et al., 1996; Chyung et al., 1997
). After application of a
rabbit anti-goat IgG linker, [125I]Protein A was applied, and radiolabeled
APP was quantitated by PhosphorImager analysis.
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Results |
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Neurons Contain Insoluble Amyloid Peptide
To evaluate the possibility that A exists in multiple intracellular pools with different solubility characteristics,
NT2N neurons were sequentially extracted in aqueous
buffer (PBS), detergent buffer (RIPA), and then 70% formic acid. The levels of A
1-40 and A
1-42 present in each
fraction were quantified by sandwich-ELISA. Previous
studies have shown that nonionic detergents liberate intracellular A
, but not A
deposited in senile plaques or
fibrillar A
formed in vitro (Selkoe et al., 1986
; Burdick et al.,
1992
; Harigaya et al., 1995
; Turner et al., 1996
). However,
more rigorous solubilization methods using 70% formic
acid liberate A
from these insoluble aggregates. Sonication of cells in PBS in the absence of detergent failed to release any soluble A
(data not shown). By contrast, significant levels of A
1-40 and A
1-42 were solubilized by
RIPA buffer. Nonetheless, RIPA buffer released only a
fraction of the total intracellular A
since subsequent extraction of the detergent-insoluble material with 70% formic acid revealed a much larger pool of both A
species
(Fig. 1 A). Since increased production of A
1-42 relative
to A
1-40 has been associated with AD (Borchelt et al.,
1996; Duff et al., 1996
; Scheuner et al., 1996
), we examined
the ratios of these A
species in the detergent-soluble and
-insoluble pools in NT2N neurons. The ratio of A
1-42/1-40
in the RIPA soluble pool was 1.0 ± 0.1 (Fig. 1 B), consistent with previous studies in a variety of experimental systems (Cook et al., 1997
; Forman et al., 1997
). However,
A
1-42 was more abundant in the detergent-insoluble
pool, with an A
1-42/1-40 ratio of 2.7 ± 0.3 (Fig. 1 B).
This finding is consistent with the reduced solubility of
A
1-42 relative to A
1-40 in vitro, and the predominance
of A
1-42 in insoluble deposits in the AD brain (Jarrett et
al., 1993a
; Iwatsubo et al., 1994
).
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The identification of a large and previously undetected
pool of insoluble A in NT2N neurons prompted us to establish precise conditions for reproducible recovery of the
maximum amount of formic acid-extractable A
. Sonication was found to be necessary for efficient A
extraction,
and a volume of 100 µl formic acid was found to extract
A
optimally from cell lysates containing ~1 mg of total
protein. However, longer incubation times in formic acid
(up to 24 h) or high incubation temperatures (up to 37°C) did not increase A
recovery (data not shown). To confirm that formic acid-extracted A
was present in intracellular compartments and not attached to the cells or culture
dish, cells were treated with trypsin in the presence or
absence of 0.1% Triton X-100. We found that formic acid-
solubilized intracellular A
was resistant to trypsin digestion in the absence of detergent, but sensitive to trypsin digestion after solubilization by Triton X-100 (data not
shown). This finding indicates that the formic acid-soluble pool of A
is located intracellularly, and is accessible to
trypsin only when cell membranes are first permeabilized
by detergent. Finally, we found that cells extracted directly
into formic acid yielded amounts of A
similar to the sum
of RIPA-soluble and RIPA-insoluble A
(Fig. 1 A). From
these studies, we concluded that neurons contain at least
two major pools of intracellular A
: a detergent soluble pool, and a larger formic acid soluble pool that is enriched
in A
1-42.
Insoluble A is Present in a Range of APP-Expressing
Cell Types
To determine if insoluble intracellular A is present in cell
types other than neurons, NT2, CHO Pro5, and BHK-21
cells were sequentially extracted with RIPA followed by
formic acid, and A
levels were measured by sandwich-ELISA (Fig. 2). To evaluate the consequences of increased APP production on the generation of soluble and
insoluble intracellular A
, each cell type was also infected
with a recombinant SFV vector that led to the expression of high levels of APP695. Additionally, A
levels in stably
transfected CHO cells expressing APP695 (CHO-695)
were examined (Fig. 2 A). Steady-state APP levels present
in each cell type were determined by Western blotting in
order to correlate the levels of intracellular A
with APP
(Fig. 2 B).
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In contrast to NT2N neurons, retinoic acid naïve NT2
cells did not produce significant amounts of A, despite
expressing nearly equivalent levels of APP (Fig. 2, A and
B). This observation is consistent with previous experiments that have demonstrated that NT2 cells do not efficiently process APP by the
-secretase pathway, and thus
generate only low levels of A
(Wertkin et al., 1993
; Forman et al., 1997
). Furthermore, the engineered expression of APP695 in NT2 cells at levels similar to those found in
NT2N neurons resulted in only a modest increase in intracellular A
levels (Fig. 2, A and B), indicating that the
lack of intracellular A
in NT2 cells relative to NT2N neurons was not due to differential expression of APP isoforms in the two cell types (APP751/770 in NT2 cells vs.
APP 695 in NT2N cells), but to differential processing of
APP. In addition, the fact that only low levels of A
were detected by sandwich-ELISA in this cell line further confirms that this assay is highly specific for A
, and does not
significantly cross-react with other cellular proteins, including full-length APP, other A
-containing carboxy-terminal fragments, or non-A
APP-derived fragments.
CHO Pro5 and BHK-21 cells expressed barely detectable levels of APP, and they did not produce detectable
levels of soluble or insoluble A, further confirming the
specificity of the A
ELISA. CHO-695 cells, however, did
produce intracellular A
, 22 ± 3% of which was insoluble
(Fig. 2 A). Likewise, infection of CHO Pro5 cells and
BHK-21 cells with SFV-APPwt led to a markedly increased production of APP as well as intracellular A
, of
which up to 74 ± 5% was insoluble. This dramatic increase
in A
production over a relatively short period of time
could favor A
aggregation, resulting in a decrease in A
solubility. Indeed, CHO cells stably expressing APP contained a much lower proportion of insoluble A
than did
SFV-APP-infected CHO cells (Fig. 2 A).
These findings indicate that in addition to cell type-specific factors, the level of APP expression also governs deposition of insoluble A. In cells that efficiently use the
-secretase pathway to generate A
, increased APP expression generally resulted in increased levels of both soluble and insoluble A
. However, while CHO-695 cells and
NT2N neurons both expressed similar levels of APP and
produced similar levels of soluble A
, NT2N neurons accumulated significantly higher levels of insoluble A
(Fig. 2; compare tracks labeled NT2N vs. CHO-695). This difference may be due to the higher metabolic rate of CHO-695 cells, which may result in increased turnover of A
,
thus hindering aggregation. Alternatively, A
aggregation
in CHO-695 cells may be impeded by continual dilution
due to cell division. In postmitotic neurons, A
may accumulate intracellularly over time, and thus favor the formation of insoluble aggregates.
Taken together, these results indicate that while NT2N
neurons accumulate intracellular insoluble A as a consequence of endogenous APP production, other cell types
also exhibit this property when they overexpress APP. It is
interesting to note that increased expression of APP in
NT2N neurons as a consequence of SFV-APPwt infection
did not result in increased levels of intracellular A
1-42,
consistent with some of our previous work indicating that
-secretase cleavage in the ER/IC pathway is rate-limiting
(Cook et al., 1997
). By contrast, increased expression of
APP in NT2N neurons resulted in increased levels of intracellular A
1-40. That this increase was due solely to increased levels of soluble A
1-40 is consistent with this
form of A
being produced late in the secretory pathway,
and being recovered from cells before secretion.
A Can be Immunoprecipitated from an Insoluble Pool
To further confirm that the material recovered by extraction with formic acid and measured by sandwich ELISA
was indeed A, SFV-APPwt-infected NT2N and CHO
cells were metabolically labeled with [35S]methionine for
12 h, and A
was immunoprecipitated using 6E10. As
shown in Fig. 3, a band of ~4 kD was immunoprecipitated
by an A
-specific antibody in the RIPA-soluble cell lysate.
Additional A
was immunoprecipitated from the RIPA-insoluble (formic acid-extracted) cellular fraction, thus
confirming that a pool of A
remained insoluble in RIPA
buffer, and could be extracted by formic acid (Fig. 3).
However, the yield of A
after formic acid extraction was
lower than that predicted by A
sandwich ELISA. To determine if formic acid extraction compromised the recovery of A
by immunoprecipitation, [35S]methionine-labeled
SFV-APPwt-infected cells were extracted directly into
formic acid. Direct extraction of cells into formic acid would
be expected to yield amounts of A
equal to the sum of
A
extracted in the RIPA-soluble and -insoluble pools.
However, lower levels of A
than expected were recovered by this method (Fig. 3; compare lane 3 with lanes 1 and 2, and lane 6 with lanes 4 and 5). Thus, immunoprecipitation of formic acid-extracted cells was not quantitative,
and resulted in only partial recovery of A
. This low
recovery of A
may have been due to incomplete resolubilization of A
in acetonitrile after lyophilization, or reaggregation of A
during immunoprecipitation. To evaluate the contribution of each of these factors to the
incomplete recovery of A
by immunoprecipitation, we
measured A
levels in the formic acid-extracted cell lysate before and after lyophilization and immunoprecipitation by sandwich-ELISA. We found that ~43% of formic
acid-extracted A
could be resolubilized in acetonitrile
after lyophilization, and ~45% of this resolubilized A
could be captured by immunoprecipitation with the antibody 6E10 (data not shown). Nevertheless, despite the
shortcomings of the immunoprecipitation protocol as compared with the A
sandwich-ELISA, these data confirm
that the formic acid-extracted pool does indeed contain A
.
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Differential Production of Insoluble A1-40 and
A
1-42 in Subcellular Compartments
While it has been shown that secreted A is mainly produced in the TGN, intracellular A
1-42, but not A
1-40,
is produced in the ER/IC (Cook et al., 1997
). To determine if A
1-42 produced in the ER/IC enters the insoluble pool, NT2N neurons and CHO Pro5 cells were infected
with SFV-APPwt or SFV-APP
KK (an APP mutant containing the dilysine ER retrieval sequence). Infection of
both cell types with SFV-APP
KK gave similar results: almost a complete abrogation of A
1-40 production, with
no diminution of A
1-42 production relative to SFV-APPwt
infected cells (Fig. 4, A and B). Importantly, the levels of
insoluble A
1-42 were the same in SFV-APPwt and SFV-APP
KK-infected cells. These results demonstrate that
A
1-42 produced in the ER/IC pathway represents the
bulk of the insoluble A
1-42 inside cells. By contrast, insoluble A
1-40 is produced by a post-ER/IC pathway. Finally, these results also prove that insoluble A
can accumulate in the absence of secretion, and they provide
additional evidence that the A
solubilized by formic acid
is intracellular.
|
Time-dependent Accumulation of Insoluble A
Our previous studies have shown that secretion of A1-40
and A
1-42 by the NT2N neurons increases with time in
culture without an increase in APP synthesis (Turner et al.,
1996
). However, a time-dependent increase in intracellular A
was not detected. Conversely, we found that retention of APP in the ER/IC resulted in continued production
of A
1-42, but without either secretion or intracellular accumulation (Cook et al., 1997
). Our observation here that
intracellular A
(particularly the A
1-42 species produced in the ER/IC) forms an insoluble pool provided a
possible explanation for both of these earlier findings. To
test the hypothesis that insoluble A
can accumulate intracellularly over time, NT2N neurons were analyzed at
various time points after replating by sequential extraction
in RIPA and formic acid, followed by sandwich-ELISA for A
quantitation. We found a dramatic increase (12-fold over 7 wk in culture) in the levels of formic acid-
extractable intracellular A
1-40 and A
1-42 in NT2N
cells concomitant with increased time in culture (Fig. 5, A
and B). In addition to an increase in the absolute amount
of insoluble A
with longer times in culture, an increase in
the fraction of insoluble A
was also observed. For example, at 4 wk, ~58% of A
was insoluble, while at 7 wk ~78% of A
was insoluble (Fig. 5, A and B). This result
suggests that the equilibrium of soluble to insoluble A
may be shifted to favor insoluble A
in NT2N cells that
were cultured longer (i.e., older neurons).
|
The Intracellular Accumulation of A Over Time
in Culture is Due to the Slow Turnover of Insoluble A
The time-dependent accumulation of insoluble intracellular A in neurons could be due to several factors, including the slow turnover of insoluble A
. To examine this
possibility, we treated NT2N cells with cycloheximide to
prevent protein synthesis, and measured endogenous levels of A
in the soluble and insoluble pools over time in
culture. This approach was needed (rather than a standard
pulse-chase analysis) because immunoprecipitation of A
after formic acid extraction was not quantitative (Fig. 3). Fig. 6 shows that over the 24-h cycloheximide treatment,
soluble A
1-40 and A
1-42 decreased by ~63% and ~ 77%,
respectively. Assuming a constant rate of degradation, we
calculated half-lives of ~18 h and ~12 h for the decay of
intracellular soluble A
1-40 and A
1-42, respectively. By
contrast, insoluble A
1-40 and A
1-42 levels did not decrease significantly over 24 h. The slow turnover of the insoluble pool of A
precluded an accurate estimate of the
half-life of this pool. In addition, this analysis is complicated by two factors. First, although no new APP will be
synthesized in the presence of cycloheximide, existing
pools of APP continue to be processed to generate A
.
However, the half-life of APP in NT2N neurons is ~3 h.
Thus, de novo production of A
from existing pools of
APP is unlikely to contribute significantly to intracellular A
pools, especially at later time points. Second, soluble
A
1-40 and A
1-42 may enter the insoluble pool over
time, again making accurate estimates of turnover rates
difficult. Nevertheless, our results show that intracellular
insoluble A
is very long-lived, and that this long life is
likely to play an important role in the time-dependent accumulation of insoluble A
we observed in NT2N cells
over weeks in culture.
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![]() |
Discussion |
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The presence of insoluble aggregates of A in senile
plaques is a well-characterized feature of AD (Selkoe,
1997
). A
is composed of two major species that terminate
at residues 40 and 42 of the intact A
sequence. Both species can be recovered from the CSF of normal and AD individuals, with A
1-40 being approximately 10-fold more
abundant than A
1-42 (Citron et al., 1992
). However, A
1-42 is the major A
species present in senile plaques,
with A
1-40 being only a minor constituent (Iwatsubo et
al., 1994
). That alterations in APP processing can lead to
development of AD has been shown by several FAD-associated APP mutations that, when expressed in vitro or in
transgenic animals, lead to either an overall increase in A
production or an increase in the amount of A
1-42 relative to A
1-40 (Borchelt et al., 1996; Duff et al., 1996
).
The differential production of A
1-40 and A
1-42 as a
consequence of AD-associated APP mutations as well as
the preferential deposition of A
1-42 in senile plaques
raises important questions as to the intracellular sites of
A
1-42 generation, the origin of A
that is recovered
from senile plaques, and the factors that control its deposition.
Both A1-40 and A
1-42 are constitutively produced
and secreted from cells in vitro and in vivo as judged by
their recovery from conditioned medium and CSF (Shoji
et al., 1992
; Tamaoka et al., 1996
). Since FAD-associated
APP mutations lead to increased secretion of A
, and senile plaques are extracellular lesions, it is possible that secreted A
is ultimately deposited in senile plaques, even
though the factors controlling its deposition are obscure.
However, we have recently discovered that retention of APP in the ER/IC induced by a variety of methods leads
to continued production of intracellular A
1-42, but not
A
1-40 (Cook et al., 1997
). While A
1-42 is constitutively
produced by this novel pathway in NT2N neurons, other
cell types can also process APP to generate A
in the ER/
IC after overexpression of APP (Wild-Bode et al., 1997
).
A
1-42 has also been shown to be localized to the ER/IC by immunoelectron microscopy and by cell fractionation
(Hartmann et al., 1997
; Wild-Bode et al., 1997
). Interestingly, this compartment also is the site where PS1 and PS2
are localized (Cook et al., 1996
; Kovacs et al., 1996
). Since
mutations in PS1 and PS2 account for the majority of
early-onset FAD cases, and FAD-associated PS1 and PS2
mutations have been shown to result in an increased ratio
of A
1-42/1-40 (Borchelt et al., 1996; Duff et al., 1996
; Scheuner et al., 1996
), colocalization of the presenilins
with a major site of constitutive A
1-42 production raises
the possibility that alterations in A
production by the
ER/IC pathway may play an important role in AD pathogenesis.
While retention of APP in the ER/IC resulted in continued and selective production of A1-42, we were unable
to document either secretion of this material or its intracellular accumulation (Cook et al., 1997
). Taken at face
value, this result indicates that the production and turnover of A
1-42 by the ER/IC pathway are in equilibrium.
However, given the propensity of A
1-42 to aggregate in
vivo and in vitro, we asked whether A
1-42 also aggregated intracellularly. Since formic acid has been shown to
effectively solubilize aggregated A
present in senile
plaques, we solubilized cell lysates in formic acid. Using
this approach, we found that a considerable fraction of total intracellular A
1-42, and to a lesser extent A
1-40,
could be solubilized by formic acid, but not by a variety of
detergents. Currently, we do not know whether or not the
formic acid-extractable A
self-aggregates or coaggregates with other proteins. Our observation that none of
the cell-associated A
(including that targeted for secretion) can be extracted with aqueous buffer suggests that it
may be bound to other cellular proteins. On the other
hand, in vitro studies of A
aggregation suggest that A
is
prone to self-aggregation. Future ultrastructural studies on
the accumulated intracelullar A
will help to resolve this
issue. Intracellular insoluble A
was recovered in a number of different APP-expressing cell lines. Overexpression of
APP generally resulted in increased production of insoluble
A
. However, insoluble A
was produced most efficiently
in NT2N neurons. Thus, while aggregation of intracellular
A
is not cell type-specific, the subcellular environment in
neurons appears to favor this process.
Identification of a novel form of intracellular A that
has previously escaped detection could explain our failure
to detect secretion or intracellular accumulation of A
1-42
produced by the ER/IC pathway (Cook et al., 1997
). To
test this possibility, we expressed APP bearing an ER/IC
retrieval signal in the cytoplasmic domain in NT2N neurons and in CHO cells. Production of intracellular soluble
and insoluble A
1-40 was almost completely inhibited after expression of this construct, while levels of soluble and
insoluble intracellular A
1-42 were unchanged by ER retention. Thus, almost all of the formic acid-soluble A
1-42
can be derived from the ER/IC pathway. By extension, insoluble A
1-40 must be produced by a post-ER/IC compartment. Production of insoluble A
1-40 and A
1-42 in
different subcellular compartments may help explain the
predominance of A
1-42 in the intracellular pool. A
1-40
is produced late in the biosynthetic pathway, and may
spend relatively little time in the cell before secretion,
thereby minimizing the opportunity for aggregation. By
contrast, the bulk of the intracellular A
1-42 is produced
by the ER/IC pathway. This fact represents an environment distinct from that in which A
1-40 is produced, and
one that does not result in A
1-42 secretion. The long-lived nature of A
1-42, its continued production at an intracellular site from which it cannot be secreted, and the
fact that it is intrinsically less soluble than A
1-40 all may
contribute to its propensity to enter a stable, intracellular
pool of insoluble material. It will be important to define
further the factors that govern A
deposition in this insoluble pool, and to more carefully study its physical state.
During the course of our experiments, we found that recovery of insoluble A from NT2N neurons was somewhat variable. However, we found that this result was due
to a time-dependent accumulation of A
. Specifically, we
found that A
levels increased by 12-fold as the NT2N
neurons aged over 7 wk in culture. While insoluble A
1-40
and A
1-42 accumulated at similar rates, more detailed kinetic studies are needed to determine if production of
insoluble A
1-40 and A
1-42 is contemporaneous, or if
generation of insoluble A
1-42 seeds subsequent polymerization of A
1-40, as has been reported in vitro. In any
event, time-dependent accumulation of insoluble A
could be due to increased production, decreased turnover, or stable accumulation of A
at a relatively constant rate.
We found that intracellular insoluble A
was exceptionally stable. Thus, even slow addition of A
to the insoluble
pool over weeks in culture could result in steady accumulation of A
seen in the insoluble pool over time. This observation may have implications for AD pathogenesis,
where it is thought that accumulation of A
occurs slowly
over decades. Since AD is an age-dependent disease, the
data presented here suggest that gradual accumulation of
intracellular A
may be a factor in the slow onset and progression of AD. It will be important to determine if accumulation of intracellular insoluble A
is simply the result
of the stability of this form of A
, or if other time-dependent factors (such as altered APP processing or neurotoxic
insults) contribute to this process.
Although intracellular -amyloid fibrils have been observed in the AD brain (Kim et al., 1988
) as well as in a
transgenic mouse model of AD (Masliah et al., 1996
), it is
unclear whether A
fibrils can form within neurons from
endogenously produced A
. The experiments presented
here demonstrate that significant levels of A
are insoluble within neurons. The observation that A
can accumulate with time in a relatively stable insoluble pool may explain how A
deposition in senile plaques can begin
despite relatively low levels of secreted and CSF-soluble
A
1-42. Concentrated intracellular A
1-42 could rapidly
nucleate fibril formation, and intracellularly produced
A
1-42 and A
1-40 could add to these fibrils over time,
thus serving as a nidus for a developing senile plaque.
![]() |
Footnotes |
---|
Received for publication 20 January 1998 and in revised form 31 March 1998.
We gratefully thank Dr. N. Suzuki and Tekeda Pharmaceutical for providing the monoclonal antibodies for the AThis work was supported by grants from the National Institute on Aging. D.M. Skovronsky is the recipient of a Medical Scientist Training Program Predoctoral Fellowship from the National Institutes of Health, and R.W. Doms is the recipient of a Paul Beeson Faculty Scholar Award.
![]() |
Abbreviations used in this paper |
---|
A, amyloid-
peptide;
AD, Alzheimer's disease;
APP, amyloid-
precursor protein;
BHK, baby hamster
kidney;
ER/IC, endoplasmic reticulum/intermediate compartment;
FAD, Familial Alzheimer's disease;
SFV, Semliki Forest Virus;
SFV-APPwt, SFV expressing wild-type APP695;
TGN, trans-Golgi network.
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