(Received for publication, September 21, 1995; and in revised form, February 5, 1996)
From the
Previous studies have demonstrated the presence of amyloid
(A
) in neurons (NT2N) derived from a human embryonal carcinoma
cell line (NT2) by steady state metabolic radiolabeling and
immunoprecipitation. We show here that A
is present
intracellularly since trypsin digestion of intact NT2N cells at 4
°C did not eliminate the A
recovered in cell lysates. To
determine whether both A
and A
are
produced intracellularly, quantitative sandwich enzyme-linked
immunosorbent assay (ELISA) was performed using COOH-terminal
end-specific anti-A
monoclonal antibodies. Sandwich ELISA detected
intracellular A
and A
in NT2N cell
lysates at a ratio of 3:1, whereas secreted A
and
A
were recovered in medium conditioned by NT2N cells
at a ratio of approximately 20:1. Metabolic steady state and
pulse-chase labeling studies demonstrated a 2-h delay in the detection
of cell-associated A
/A
in the
medium, suggesting that A
is generated at a slow rate
intracellularly prior to its secretion. Finally, as NT2N cells mature
over time in culture, the secretion of A
and
A
increases more than 5-fold over 7 weeks. This
increase in the secretion of A
/A
in
NT2N cells as a function of time may recapitulate a similar phenomenon
in the aging brain.
The amyloid (A
) (
)peptide deposited as
insoluble amyloid in plaques and vascular deposits is one of the most
striking neuropathological features of the Alzheimer's disease
(AD) brain, and these plaques may play a critical role in the
pathogenesis of AD. A
, purified from AD blood vessels (1) and brain parenchyma (2) and sequenced as 39- to
43-amino acid peptides of 4 kDa. A
was shown to be an insoluble
peptide with a
-pleated sheet secondary conformation. The amyloid
precursor protein (APP), a transmembrane glycoprotein, was subsequently
cloned (3) and localized to human chromosome 21(4) .
Alternative mRNA splicing generates primarily 695- (APP
),
751- (APP
), and 770- (APP
) amino acid
isoforms of APP(5, 6, 7) . Some kindred of
early onset familial AD (8, 9, 10, 11, 12) and
hereditary cerebral hemorrhage with amyloidosis of the Dutch type (13, 14, 15) are linked with APP missense
mutations which localize within or adjacent to the A
sequence
contained within the carboxyl-terminal region of APP. Thus, it is
likely that these mutations influence APP processing and A
generation, at least in familial AD.
Several proteolytic pathways
for APP metabolism have been described. An -secretase cleaves APP
within the A
region at or near the plasma membrane and thus
precludes amyloid deposition(16, 17) . A
-secretase cleaves APP at the amino terminus of A
and a
-secretase cleaves APP at the carboxyl terminus of A
,
releasing A
from APP that is either 40 amino acids
(A
) or 42 amino acids (A
)
long(18) . An endosomal/lysosomal pathway generates a number of
intracellular carboxyl-terminal APP fragments, the larger of which
contain the entire A
peptide sequence and thus are also
potentially
amyloidogenic(19, 20, 21, 22) .
A
is found in conditioned medium from mixed brain-cell primary
cultures and in normal cerebrospinal fluid(23, 24) ,
suggesting that it is produced and secreted constitutively. A
peptides are also secreted by a number of nontransfected and
APP-transfected cell
lines(22, 24, 25, 26) . Transfection
of cells with the APP
tandem mutation linked to
early onset familial AD results in a 6-fold increase in the secretion
of A
(27, 28) , and transfection of cells with the
APP
mutation increases the production of
A
(29) . These observations support the
hypothesis that overproduction of A
or increased production of
A
leads to familial AD. However, the exact mechanism
that leads to the production and secretion of A
and
A
and the identity of the
-,
-, and
-secretase remains elusive. This is in part due to the fact that
APP metabolism is cell type-specific, and overexpression of APP may
affect its metabolism. For example, A
was detected associated with
cell lysate as well as in the conditioned medium from cultured
postmitotic CNS neuronal cells (NT2N) produced by the terminal
differentiation of a teratocarcinoma cell line (NT2) with retinoic
acid(26, 30) . By contrast, cell associated A
has
not been demonstrated convincingly in the less differentiated NT2
cells, and many APP-transfected or nontransfected cell lines although
A
has been recovered from the conditioned medium of a variety of
cultured cells(22, 24, 25, 26) .
However, it was recently reported that A
is found in a human
neuroblastoma cell line (SY5Y) (31) .
To date, the cell
types responsible for the production of A that forms amyloid
deposits in the AD brain are not known. Since A
deposits are found
exclusively in the brain, and APP
is found predominantly
in neurons, it is reasonable to hypothesize that A
produced by
human CNS neurons from APP
is the likely source of
amyloid in senile plaques. Since NT2N cells resemble human CNS
neurons(32, 33) , express primarily the APP
isoform and produce A
, they may provide a unique system to
test this hypothesis. In the present study, we demonstrated that A
is indeed produced intracellularly before secretion. We also showed
increase secretion of A
and A
in
NT2N cells as a function of time in culture.
Figure 1:
A is produced intracellularly in
NT2N cells. Culture dishes containing >99% NT2N cells (Replate 3)
were metabolically labeled with 500 µCi/ml
[
S]methionine for 16 h. Cells were rinsed twice
with PBS and incubated on ice for 20 min with PBS alone (lane
1), with 10 µg/ml trypsin (lane 2), or with 10
µg/ml trypsin and 0.1% Triton X-100 (lane 3). The cells
were processed for immunoprecipitation and gel electrophoresis as
described under ``Experimental Procedures.'' All lanes were
immunoprecipitated with the anti-A
antibody
6E10.
Figure 2:
NT2N
cell-associated A precedes its recovery from conditioned medium.
Replate 3 NT2N cells were metabolically labeled with 200 µCi/ml
[
S]methionine for 1, 3, 8, or 24 h. Proteins
from cell lysates (A) or conditioned medium (B) were
immunoprecipitated sequentially with Karen (for APP) or 6E10 (for
A
). Radiolabeled immunoprecipitates were subsequently separated on
7.5% Laemmli SDS-PAGE gels (APP) or 16.5% Tris-Tricine gels (A
),
stained, dried, and exposed to PhosphorImager plates (72 h) for
quantitation and to autoradiographic film (14-21 days). Molecular
mass markers are in kilodaltons. Panels C and D are
quantitation of intracellular APP and A
, and secreted APPs and
A
, respectively. Counts from five experiments for cell lysates (A) and 10 experiments for medium (B) were converted
to percent of maximal counts at 24 h. Because results from experiments
using a number of polyclonal anti-A
(e.g. 2332, SGY2134)
antibodies in Replate 2 and Replate 3 NT2N cells were similar, the data
were pooled for statistical analysis (mean ± standard
error).
To demonstrate unequivocally that A is produced
intracellularly before secretion, the NT2N cells were pulsed with
[
S]methionine for 3 or 4 h and then chased for
different lengths of time. Notably, it was necessary to pulse label the
NT2N cells for a longer period of time, since at shorter labeling
times, intracellular A
was at too low a level to be detected.
Nevertheless, it became evident that the levels of intracellular APP
and A
decreased with increasing chase times and that only a small
amount of intracellular APP and A
was detectable after a 24 h
chase time (Fig. 3, A and C). By contrast, the
levels of A
and APPs secreted into the medium increased with
increasing chase time (Fig. 3, B and D). These
studies demonstrate definitively that A
is produced
intracellularly before secretion.
Figure 3:
Newly synthesized A is rapidly
secreted into the medium in NT2N cells. NT2N cells (Replate 3) were
pulsed with 400 µCi/ml [
S]methionine for 16
h and chased for 0, 1, 3, 8, and 24 h. Radiolabeled proteins from cell
lysates (A) or conditioned medium (B) were
immunoprecipitated sequentially with Karen (for APP) followed by 6E10
(for A
). Immunoprecipitated APP was separated on 7.5% Laemmli
gels, and A
was separated on 16.5% Tris-Tricine gels which were
exposed to PhosphorImager plates (72 h) or exposed to x-ray film for 2
weeks for quantitation. Molecular mass markers are in kilodaltons. Panels C and D are quantitation of experiments shown
in Panels A and B. Counts from six experiments were
converted to percent of maximal counts at 24 (A) or 8 (B) hours.
Figure 4:
Soluble A levels increase with NT2N
cell maturation. Six ml of serum-free DMEM were conditioned for 24 h by
10-cm dishes of 7.5
10
NT2N cells (Replate 2).
Medium was harvested weekly for 13 weeks, stored at -70 °C,
and analyzed by a sandwich ELISA using BAN-50/BA-27 and BAN-50/BC-05
for specific quantitation of A
(A) or
A
(B), respectively. The means ±
standard error (n = 4-8 dishes) are indicated,
and the data are representative of three separate
experiments.
Figure 5:
The proportion of secreted
A and A
remains constant with NT2N
cell maturation. NT2N cell-conditioned medium was analyzed by ELISA as
described in the legend to Fig. 4, and the proportions of
A
and A
are expressed as a percent
of total A
.
In this study, we show for the first time that both
A and A
are produced
intracellularly in NT2N cells. Although cell-associated A
has been
detected in NT2N cells and human neuroblastoma SY5Y cells(31) ,
no other cell line has been shown to produce detectable levels of
intracellular A
and A
. The presence
of A
in the neuron-like NT2N cells and its subsequent
secretion suggests that human CNS neurons may contribute to the
formation of amyloid plaques, since recent studies have shown that
A
is the major A
species in amyloid plaque cores (35, 36) .
Several lines of evidence were presented
here to demonstrate that both A and A
are produced intracellularly prior to secretion. First, A
was recovered from NT2N cell lysates even after intact NT2N cells were
treated with trypsin. Such treatment would eliminate cell surface
associated A
but not intracellular A
. Indeed, the loss of
A
following trypsin treatment of detergent permeabilized NT2N
cells further confirmed the intracellular location of A
in NT2N
cells. Second, the steady state metabolic labeling experiments showed
that A
was detected intracellularly before it was recovered in the
medium. In fact, there was a lag time of about 2 h before the detection
of secreted radiolabeled A
and APPs from the medium. Third, the
pulse-chase experiments showed that newly synthesized APP and freshly
produced A
from cell lysates could be chased into the medium after
a lag time of about one hour. Finally, our ELISA data showed that both
A
and A
peptides were present at
much higher levels in NT2N cell-conditioned medium than in NT2N cell
lysate suggesting that A
and A
that
were produced intracellularly eventually accumulated in the medium.
Previously we showed that, after a 15-min pulse, a 95-kDa band
corresponding to APP695 was detected in the NT2N cells(26) .
Here, we showed that prolonged labeling resulted in the appearance of
diffused bands from about 95-110 kDa. This higher molecular mass
material most likely represents glycosylated APP695, although the
presence of small amounts of APP751 and APP770 cannot be ruled out.
Further, prolonged pulse labeling was required for the detection of
intracellular A before chase since the level of intracellular
A
was quite low even after a 3-4-h pulse. Thus, it was not
possible to determine the exact time course of the reduction of the
intracellular pool of A
even though we were able to monitor and
quantitate the accumulation of secreted A
over time after pulse
labeling.
Our data show that as much as 30% of the total A
produced intracellularly in the NT2N cells is A
.
Currently, it is unclear whether or not A
and
A
are produced in the same or different intracellular
compartments, or whether intracellular A
is the
precursor of intracellular A
. The detection of both
A
and A
in the intracellular
compartment of the NT2N cells will provide a useful system to dissect
the pathways leading to the generation of these two forms of A
.
Our observation that 30% of the total intracellular A
is
A
while only about 5-6% of the total A
released into the medium is A
suggests a hitherto
unanticipated complexity in A
metabolism. For example, some of the
A
could be degraded intracellularly before secretion,
thereby reducing the amount of A
recovered in the
medium. Alternatively, multiple pathways could be involved in the
production of secreted A
especially since the
production of A
has been shown to involve both secretory and
lysosomal
pathways(16, 17, 18, 19, 20, 21, 22) .
One such pathway involves the rapid reinternalization of membrane
inserted APP into early endosomes followed by excision and release of
the A
peptide(21) . Since intracellular A
has not
been detected in cultured cells that undergo APP reinternalization, and
since A
is the major A
species recovered from
the medium(29) , we speculate that this secretory pathway may
lead to the production of A
but not
A
. Thus, by selectively increasing the production of
secreted A
, the amount of A
recovered in the medium would increase. This increase would alter
the ratio of A
and A
such that
A
to A
would be higher in the
medium than in the cell lysate. We also noted a 5-7-fold increase
in A
and A
secretion as the NT2N
cells matured in culture. This increase in A
and
A
secretion was not due to a concomitant increase in
APP synthesis or intracellular A
and A
since the amount of intracellular A
and
A
is increased by only about 30% even after 6 weeks
in culture. Thus, it appears that APP processing is altered as NT2N
cells age in culture such that the production of secreted A
and A
is dramatically increased. It is unclear
at the present time whether this is due to increased
-secretase or
reduced
-secretase activities. Future studies will directly
address this important issue since the aging of NT2N cells in culture
may recapitulate key aspects of the aging brain by increasing the
production of A
peptides leading to amyloid deposition. Finally,
our ability to detect intracellular A
and
A
suggests that the NT2N cells may be a unique system
to identify the
- and
-secretases that produce A
and A
in neurons.