* Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104; and Cephalon, Incorporated, West Chester, Pennsylvania 19380
Previous studies have demonstrated that
NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type -amyloid precursor protein (APP) to
amyloid
peptide in an intracellular compartment.
These studies indicate that other proteolytic fragments
generated by intracellular processing must also be
present in these cells. Here we show that the NH2-terminal fragment of APP generated by
-secretase cleavage (APP
) is indeed produced from the endogenous
full length APP (APPFL). Pulse-chase studies demonstrated a precursor-product relationship between
APPFL and APP
as well as intracellular and secreted
APP
fragments. In addition, trypsin digestion of intact NT2N cells at 4°C did not abolish APP
recovered
from the cell lysates. Furthermore, the production of
intracellular APP
from wild-type APP appears to be a
unique characteristic of postmitotic neurons, since intracellular APP
was not detected in several non-neuronal cell lines. Significantly, production of APP
occurred even when APP was retained in the ER/
intermediate compartment by inhibition with brefeldin
A, incubation at 15°C, or by expression of exogenous
APP bearing the dilysine ER retrieval motif.
AMYLOID
Newly synthesized APP matures in the endoplasmic
reticulum and the Golgi apparatus, acquiring N- and O-linked
carbohydrates, tyrosine sulfates (Weidemann et al., 1989 Although the identities of the putative Recently, we showed that both A Cell Culture
NT2 cells derived from a human embryonal carcinoma cell line (Ntera
2/c1.D1) were grown and passaged twice weekly in Opti-Mem (Life Technologies, Inc., Gaithersburg, MD) supplemented with 5% FBS and penicillin/streptomycin (P/S) as described previously (Pleasure et al., 1992 Metabolic Labeling, Gel Electrophoresis,
Immunoblotting, and Quantitation
Cultured NT2N neurons were starved in methionine-free DME HG (Life
Technologies, Inc.) for 30 min before incubation in fresh, methionine-free
DME HG containing 0.5 mCi/ml of [35S]methionine (sp act 1,000 Ci/
mmol; NEN-Du Pont, Boston, MA). For steady-state labeling studies,
NT2N neurons were labeled with [35S]methionine continuously for 16 h.
For pulse-chase studies, cells were labeled with [35S]methionine for 1 h,
washed twice with methionine-containing DME, and then chased in the
same medium for 0 to 24 h. APPFL, APP Sample Preparation and Serial Immunoprecipitations
Cell lysates were prepared as described elsewhere (Golde et al., 1992 Trypsin Treatment of NT2N Neurons
NT2N neurons were metabolically labeled with 0.5 mCi/ml [35S]methionine for 16 h, as described above. After rinsing the cultures twice with
PBS, the NT2N neurons were incubated on ice for 20 min with PBS, with 10 µg/ml of trypsin in PBS alone (Life Technologies, Inc.), or with 10 µg/ml
trypsin and 0.1% Triton X-100 in PBS. After this treatment, trypsin was
inactivated by the addition of 100 µg/ml soybean trypsin inhibitor. The
cells were then washed with PBS, scraped into cell lysis buffer, and processed for immunoprecipitation, as described above.
BFA Treatment of NT2N Neurons and Deglycosylation
of Immunoprecipitated APP NT2N neurons were pretreated with 20 µg/ml of BFA for 1 h before the
addition of 0.5 mCi/ml of [35S]methionine to the cultures for 16 h in the
absence or presence of BFA. The cell lysates and media were processed
for immunoprecipitation as described above. For deglycosylation of
APP Antibodies for Immunoprecipitation
and Immunoblotting
The antibodies used in this study and their epitope specificities are summarized in Fig. 1. Briefly, Karen is a goat polyclonal antisera raised to the
large, secreted NH2-terminal fragment of APP, and antibody 53 is a rabbit
polyclonal antisera raised to a synthetic peptide corresponding to the
amino acid sequence SEVKM. Antibody 53 binds specifically to the free
COOH terminus of APP Preparation of SFV-bearing pSFV-1(APP695) and
pSFV-1(APP695 The dilysine motif was introduced into APP695 by standard PCR site-
directed mutagenesis of pSFV-1(APP695) using primers 5 NT2N Neurons Exhibit Intracellular
Our previous studies have demonstrated that NT2N cells
produce intracellular A
The detection of intracellular APP To determine if the recovery of APP
Intracellular APP To determine if other cell types are capable of producing
intracellular APP
NT2N Neurons Produce Intracellular APP The experiments shown in Figs. 2-4 demonstrated that
intracellular APP
We next employed a pulse-chase paradigm to study more
rigorously the temporal relationship between intracellular
and secreted APP
Intracellular Since APP
We sought next to determine if incomplete maturation
of APP is indeed the cause of the shift in electrophoretic
mobility of the APP
In addition to N-linked glycosylation, however, APP undergoes a variety of posttranslational modifications, including the addition of O-linked carbohydrate chains.
Therefore, we removed both N- and O-linked carbohydrate chains from immunoprecipitated APP To further verify that
A third approach was adopted to confirm that To determine whether or not APP
APP serves as a substrate for a variety of proteolytic processing pathways, only some of which result in the production of A Several lines of evidence presented here demonstrate
that APP The detection of APP Our data also suggest that the The effect of the Swedish mutation on APP processing
is interesting. Overexpression of APPsw in transfected,
non-neuronal cells results in a 5-10-fold increase in A In view of the foregoing, three potential The possibility of A (A
)1 peptides are the building blocks of
the amyloid fibrils found in neuritic plaques and
vascular deposits that accumulate in the brains of
patients with Alzheimer's disease (AD; Selkoe, 1994
). A
is derived from proteolytic processing of one or more isoforms of the amyloid precursor protein (APP; Kang et al.,
1987
). APP isoforms are alternatively spliced type I transmembrane glycoproteins that are encoded by a single gene on human chromosome 21 (Kang et al., 1987
; St. George-Hyslop et al., 1987
). The 39-43-amino acid-long A
sequence begins in the ectodomain of APP and extends into
the transmembrane region (see Fig. 1). Of the three major
A
-containing isoforms encoded by the APP gene (i.e.,
APP695, APP751, and APP770; Kang et al., 1987
; Kitaguchi et al., 1988
; Ponte et al., 1988
; Tanzi et al., 1988
), APP695 is expressed almost exclusively by neurons of the
central and peripheral nervous systems (Golde et al., 1990
;
Kang and Müller-Hill, 1990
; Arai et al., 1991
).
Fig. 1.
Proteolytic processing of APPFL. The diagram depicts APP fragments generated by both the - and
-secretase pathways. A
large, secreted ectodomain called APP
is generated by the putative
-secretase(s) that cleaves APPFL within the A
domain. A second
cleavage by the
-secretase(s) releases a subfragment of A
known as p3. Alternative cleavage by the
-secretase(s) generates a similarly large ectodomain fragment known as APP
. After the subsequent
-secretase cleavage, A
is released. This schematic also shows
the epitope location of the antibodies used in this study to identify the different proteolytic fragments.
[View Larger Version of this Image (8K GIF file)]
;
Oltersdorf et al., 1990
), and phosphates (Oltersdorf et al.,
1990
; Suzuki et al., 1992
; Knops et al., 1993
). Several pathways of APP metabolism have been described in cultured
cells, and evidence suggests that the relative importance of
each pathway depends on the cell type. For example,
non-neuronal cells preferentially process APP by the
-secretase pathway, which cleaves APP within the A
sequence, thereby precluding the formation of A
(Esch et
al., 1990
; Sisodia et al., 1990
). The putative
-secretase enzyme(s) is active at or near the cell surface, causing the
NH2-terminal fragment (APP
) to be quickly secreted. In
contrast, neuronal cells process a much larger portion of
APP by the
-secretase pathway(s), which generate intact
A
by the combined activity of two enzyme classes. The
-secretase(s) cleaves APP at the NH2 terminus of the A
domain releasing a distinct NH2-terminal fragment (APP
).
In addition, the
-secretase(s) cleaves APP at alternative
sites of the COOH terminus, generating species of A
that
are either 40 (A
40) or 42 amino acids long (A
42; Seubert
et al., 1993
; Suzuki et al., 1994
; Turner et al., 1996
).
-,
-, and
-secretases remain speculative, and the precise subcellular localization of their activity is poorly understood, in
vitro studies have suggested the existence of at least two
-secretase pathways. In the endosomal/lysosomal pathway, APP targeted to the cell surface is endocytosed
and delivered to endosomes and lysosomes where
- and
-cleavages can occur (Golde et al., 1992
; Haass et al., 1992a
; Nordstedt et al., 1993
; Koo and Squazzo, 1994
; Lai
et al., 1995
; Perez et al., 1996
). The alternative
-secretory
pathway is predicted to generate A
in Golgi-derived vesicles, most likely secretory vesicles, before secretion
(Haass et al., 1995a
; Higaki et al., 1995
; Perez et al., 1996
;
Thinakaran et al., 1996b
). Whether these pathways operate in the same or different cell types is not known, nor is
the biological importance of each pathway for the production of A
in vivo understood.
40 and A
42 are produced intracellularly from endogenous wild-type APP695
by cultured postmitotic central nervous system (CNS) neuronal cells (NT2N) that are induced to differentiate from a
human teratocarcinoma cell line (NT2) by treatment with
retinoic acid (Pleasure et al., 1992
; Pleasure and Lee, 1993
;
Wertkin et al., 1993
; Turner et al., 1996
). To date, the
human-derived NT2N neuron is the only cell line documented to generate intracellular A
40 and A
42 before
their eventual release into the medium (Turner et al.,
1996
). Because neurons are the cell type most adversely
affected by AD, the NT2N neurons represent a unique
system for the study of intracellular
-secretase pathways
in a human neuronal model. An essential first step in the
analysis of such pathways is the identification of the proteolytic fragments that are the products of these cleavages.
We report here that in addition to A
40 and A
42, the NH2-terminal fragment generated by
cleavage (i.e., APP
) is
produced intracellularly in NT2N neurons before secretion.
More significantly, we demonstrate that novel
-secretase
activity occurs in the ER/intermediate compartment (IC)
of neuronal cells using inhibition with Brefeldin A (BFA),
incubation at 15°C, and expression of exogenous APP
bearing the dilysine ER-retrieval motif.
Materials and Methods
;
Pleasure and Lee, 1993
). To begin differentiation, 2.5 × 106 cells were
seeded in a 75-cm2 (T75) flask and fed with DME HG (Life Technologies,
Inc.) containing 10 µM retinoic acid, 10% FBS, and P/S twice weekly for 5 wk. The cells in a single T75 flask were then replated at a lower density in
2 × 225 cm2 (T225) flasks for 10 d (Replate 1 cells). Greater than 99%
pure NT2N neurons were then obtained by enzymatic treatment and mechanical dislodegment of Replate 1 cells and replated at a density of 6 × 106
cells/10-cm dish previously coated with polylysine and Matrigel (Pleasure
et al., 1992
). The NT2N neurons were maintained in medium consisting of
one part conditioned medium and one part DME HG containing 10%
FBS and P/S. For experiments involving the incubation of NT2N neurons
at 15°C for 16 h, regular medium containing DME HG and 10% FBS was
replaced by DME HG containing 25 mM Hepes, 10% FBS, and P/S. Cultures of NT2N neurons were used for experiments when they were between 3 to 4 wk old. CHO695 cells, a gift from Dr. S. Sisodia (Johns Hopkins University School of Medicine, Baltimore, MD), were grown and
passaged three times per week in
-MEM (Life Technologies, Inc.) supplemented with 10% FBS and P/S. M17 cells were grown and passaged
once per week in Opti-Mem (Life Technologies, Inc.) containing 10% iron-enriched calf serum and P/S.
, and APP
were separated on
7.5% Laemmli SDS-PAGE gels, and A
and p3 were separated on 10/
16.5% step-gradient Tris-tricine gels. These gels were either stained with
Coomassie brilliant blue R (Pierce, Rockford, IL) and dried or transferred
to nitrocellulose membranes and dried before exposure on PhosphorImager plates (Molecular Dynamics, Sunnyvale, CA) for 3-5 d. The nitrocellulose replicas containing the immunoprecipitates were further probed
with different antibodies, as described previously (Wertkin et al., 1993
).
Quantitation of bands in the autoradiogram was performed using the ImageQuant software (Molecular Dynamics) as described previously
(Turner et al., 1996
). Radiolabeled proteins in SDS-PAGE gels and nitrocellulose replicas were also analyzed by standard autoradiographic methods. All experiments were repeated between three to six times.
).
Protein concentration was determined by the bicinchoninic acid procedure (Pierce). Media were centrifuged at 100,000 g for 1 h at 4°C before
immunoprecipitation. Both cell lysates and media were precleared with
protein A-Sepharose (Pharmacia Fine Chemicals, Piscataway, NJ) in RIPA
for 1 h at 4°C. After recentrifugation at 15,000 g for 1 min, the supernatants were rocked overnight at 4°C with fresh protein A-Sepharose and
the appropriate primary antibody. After collecting the immunoprecipitates by recentrifugation at 15,000 g for 1 min, the supernatants were used
in a second round of immunoprecipitation with fresh protein A-Sepharose
and a different primary antibody.
, the immunoprecipitates containing APP
were washed twice in sodium phosphate buffer (20 mmol/liter, pH 7.2) and boiled for 2 min in 10 µl
of 1% SDS. The samples were then boiled for an additional 2 min after
adding 90 µl of the sodium phosphate buffer with sodium azide (10 mmol/ liter), EDTA (50 mmol/liter), and n-Octylglucoside (0.5% wt/vol). After
the denaturation step as described, deglycosylation was initiated by the
addition of 2 mU neuraminidase (Arthrobacter; Boehringer Mannheim,
Indianapolis, IN), 2.5 mU O-Glycosidase (Boehringer Mannheim), and
0.4 U N-Glycosidase F (Boehringer Mannheim). The samples were then
incubated at 37°C for 18 h, and deglycosylated APP
was run on 7.5%
SDS-PAGE gels as described above. For endoglycosidase H (Endo H)
sensitivity test, cell lysates and media were immunoprecipitated with
Karen as described. The immunoprecipitates were then recovered in 100 µl
60 mM phosphate buffer, pH 5.7, with 1% SDS. The samples were then
split in half (50 µl each) and incubated with 4 µl Endo H (Boehringer
Mannheim) or vehicle at 37°C for 18 h. The samples were then run on
7.5% SDS-PAGE gels as described above.
(Howland et al., 1995
). Antibody 369W is a
rabbit polyclonal antiserum raised to a synthetic peptide corresponding to
the last 45 amino acid residues at the COOH terminus of APP and was
generously donated by Dr. Sam Gandy (Cornell University School of
Medicine, New York, NY). Also used in this study were three mAbs to A
that are specific for residues 1-17 (6E10; Kim et al., 1988
, residues 1-10
(Ban50; Suzuki et al., 1994
), and residues 18-25 (4G8; Kim et al., 1988
).
KK)
-CGAAAACCACCGTGGAGCTCC TT-3
and 5
-TTAACCCGGGCTAGTTCTGCTTCTTCTCAAAGAACTTGT-3
. The mutation-containing PCR
fragment was isolated by digestion with BsmI and XmaI and then ligated
into pSFV(APP695) to yield pSFV(APP695
KK). All pSFV-1 constructs,
including a pSFV helper plasmid with SFV structural genes, were linearized by digestion with SpeI and then used as a template for RNA synthesis
with SP6 RNA polymerase. Coelectroporation of RNA from the expression and helper plasmids into BHK cells yielded an infectious, replication-defective virus that was harvested 24 h later (Liljestrom and Garoff, 1991
). Accurate determination of viral stock titers was made as described elsewhere (Cook et al., 1996
). For all infection experiments, ~1 × 106 NT2N
neurons per 35-mm dish were infected in serum-free medium at a multiplicity of infection (MOI) of 7-10. When called for, 20 µg/ml BFA was
added after the completion of the infection step.
Results
-Secretase Activity
(Wertkin et al., 1993
; Turner et al.,
1996
). To determine if intracellular APP
(Fig. 1) can also
be recovered from these cells, samples of cell lysate were
immunoprecipitated with Karen (an antiserum raised to
the NH2-terminal region of APP). Then, the presence of
APP
in the immunoprecipitate was determined by immunoblot analysis using 53 (a polyclonal antibody specific for
the free COOH terminus of APP
). We found that 53 detects a single band of ~95 kD (Fig. 2 a). That this 95-kD
APP fragment is indeed APP
, cleaved at the
-secretase
site, was further substantiated by (a) the inability of 369W,
an antibody specific for the COOH terminus of APP, to
recognize this fragment; (b) the inability of 6E10, an antibody specific for the first 10 amino acid residues of A
, to
detect this fragment; (c) the binding of Karen, an antibody
that recognizes all APP species, to this fragment; (d) the
fact that this intracellular APP fragment is ~11-12 kD
smaller than APPFL (Fig. 2 a); and (e) the detection of the
same 95-kD APP fragment using a different antibody
specific for APP
(i.e., 192; Seubert et al., 1992
; and data
not shown). To determine if APP
is secreted, media from
NT2N neurons were again immunoprecipitated with Karen
and subsequently immunoblotted with various antibodies
(Fig. 2 a). We found that APP
was readily detected in the
media of NT2N neurons and that it comigrated with APP
recovered from the cell lysates. However, as expected,
APP
migrated slightly faster than the product of
-secretase cleavage (APP
), which was also recovered from the
media.
Fig. 2.
NT2N neurons produce intracellular APP and A
. To
demonstrate the presence of APP
, samples of cell lysate and
medium were collected from NT2N cultures and processed for
immunoprecipitation (IP) with Karen, a polyclonal antibody that
recognizes epitopes within the large ectodomain of APP. The
presence of APP
, APP
, APP
/
, and APPFL was detected by
immunoblotting (IB) with the corresponding antibodies (A). To
show that A
but not p3 is produced intracellularly, NT2N neurons were radiolabeled with [35S]methionine for 16 h. The cell lysate and the medium were then processed for immunoprecipitation with 4G8, a mAb that binds to both A
and p3, or Ban50, a
mAb that recognizes only A
(B). Immunoprecipitates of A
and p3 were separated by electrophoresis in 10/16.5% step-gradient Tris-tricine gels. M, mature APPFL; I, immature APPFL.
[View Larger Version of this Image (33K GIF file)]
and A
in NT2N neurons is consistent with our view that both
- and
-secretase
activities occur in an intracellular compartment. The absence of intracellular APP
, however, suggests that the
majority or all of the
-secretase activity occurs at a different site. To further confirm that the
-secretase pathway,
but not the
-secretase pathway, occurs inside these cells,
we examined the cell lysate of NT2N neurons for the products of these respective pathways: A
, which is generated by
- and
-secretase cleavages; and p3, a product of
- and
-secretase cleavages. To do this, we immunoprecipitated
the cell lysates of metabolically labeled NT2N neurons
with mAbs that can distinguish between these peptides:
4G8 recognizes both A
and p3; Ban50, however, binds
only to A
and not p3 (Fig. 2 b). Our data clearly demonstrate that A
, but not p3, is produced intracellularly. The
p3 fragment was not detected in cell lysates even after prolonged exposure of the film. By contrast, both A
and p3
were readily recovered from the media. This observation
supports previous findings that the
-secretase pathway
occurs at or near the plasma membrane (Haass et al.,
1992a
, 1995b
; Sisodia, 1992
).
from the cell lysates reflects its intracellular origin or its association with
the cell surface, we treated cultures of NT2N neurons with
trypsin at 4°C. Under such conditions, cell surface-associated but not intracellular APP
should be proteolyzed.
Fig. 3 shows that a similar amount of APP
was recovered
from NT2N neurons regardless of trypsin treatment (Fig.
3, compare lanes 1 and 2). By contrast, when the NT2N
neurons were treated with trypsin and 0.1% Triton X-100, intracellular APP
was completely eliminated (Fig. 3, lane
3). This experiment provides evidence that the APP
recovered from the NT2N cell lysate is indeed produced in
an intracellular compartment.
Fig. 3.
APP is produced intracellularly in NT2N neurons.
Culture dishes containing >99% pure NT2N cells were metabolically labeled with [35S]methionine for 16 h. Cells were rinsed
twice with PBS and then 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 with the anti-APP
antibody 53, as described in Materials and Methods.
[View Larger Version of this Image (32K GIF file)]
Derived from Wild-Type APP Is
Detected Only in Cells with a CNS Phenotype
, the following cell lines were included
in this study for comparison: (a) retinoic acid-naive NT2
cells, the undifferentiated precursors of the NT2N neurons
that express high levels of the APP751 and APP770 isoforms; (b) Chinese hamster ovary (CHO) cells stably transfected with APP695; and (c) human M17 neuroblastoma cells. Approximately 800 µg of total protein collected in
the cell lysates of each cell type was first immunoprecipitated with Karen and then immunoblotted with either antibody 53 to detect APP
(Fig. 4 a) or Karen to detect all
forms of APP (Fig.4 b). We found that while all four cell
types synthesized similar amounts of APP, the NT2N neuron was the only cell type capable of producing detectable
levels of intracellular APP
(Fig. 4 a). However, both NT2N neurons and stably transfected CHO cells expressing APP695, but neither NT2 cells nor the M17 neuroblastoma cells, secreted APP
, raising the possibility that secretion of APP
may be isoform specific. While our data
does not preclude low levels of intracellular
-secretase
activity or faster rate of APP
secretion in these cell lines,
the evidence clearly indicates that the fraction of APP processed by
-secretase(s) as well as the subcellular site(s) of
this activity may be strongly cell-type dependent.
Fig. 4.
Intracellular APP is observed only in NT2N neurons.
Samples of cell lysate and medium collected from cultures of NT2N,
NT2, M17, and CHO cells stably expressing APP695 (CHO695)
were processed for immunoprecipitation with the antibody Karen.
The immunoprecipitates were separated by SDS-PAGE gels and
transferred onto nitrocellulose replicas. APP
present in the cell
lysates and the media were detected by immunoblotting with the
anti-APP
antibody 53 (A). After stripping the nitrocellulose
replica in A with 0.1% SDS, the blot was reprobed with Karen to
detect all APP ectodomain species (B).
[View Larger Version of this Image (26K GIF file)]
Before Secretion
can be detected in NT2N neurons. These
data suggest that APP
may be generated inside the cell
before secretion. To demonstrate unequivocally that a precursor-product relationship exists between intracellular and
secreted APP
, we adopted the following approaches. In
our first approach, NT2N neurons were washed with fresh
medium, and then the amount of intracellular as well as secreted APP
and APP
were measured over an 8-h period. This was accomplished by immunoprecipitation of
cell lysates and media with Karen followed by immunoblotting with either antibody 53 (for APP
) or 6E10 (for
APP
). As shown in Fig. 5, secreted APP
was first detected in 3 to 5 h, and its accumulation in the medium continued over the 8-h incubation period. By contrast, APP
was detected in 1 h, suggesting that APP
is produced at a
faster rate than APP
. As seen with APP
, APP
accumulated in the conditioned media over time. Finally, our
data also show that intracellular APP
is produced constitutively, since a steady state level of APP
is recovered
from NT2N cell lysates prepared from parallel cultures
over a period of 8 h (Fig. 5). These findings are consistent
with the idea of APP
being generated inside NT2N neurons before secretion.
Fig. 5.
NT2N neurons produce intracellular APP before secretion. Cultures of NT2N neurons were washed and fresh medium was replenished before measuring the amount of intracellular and secreted APP
over an 8-h period. Cell lysate and
medium collected at the times indicated were immunoprecipitated with Karen. The immunoprecipitates were separated by
SDS-PAGE and then transferred onto nitrocellulose membranes.
APP
was identified in immunoblots using the antibody 53. APP
was detected using the antibody 6E10. APPFL and APP
/
were
recognized by Karen.
[View Larger Version of this Image (44K GIF file)]
. To this end, NT2N cultures were
pulsed with [35S]methionine for 1 h and then chased for
different lengths of time (Fig. 6). We found that after 1 h
of chase time, full length APP (APPFL) immunoprecipitated from the cell lysate began to decline, while the intracellular level of APP
continued to increase until 4 h, after
which it also declined (Fig. 6, a and c). This lag in maximum production of intracellular, radiolabeled APP
supports the idea that APP
is produced intracellularly from
APPFL by
-secretase cleavage. Finally, the 1-h delay in
the secretion of APP
into the medium as well as the accumulation of this fragment with increasing chase time supports a temporal relationship between APP
that is produced intracellularly and APP
that is secreted into the medium (Fig. 6, b and d). Therefore, we conclude that
APP
is produced in an intracellular compartment in
NT2N neurons before secretion.
Fig. 6.
Pulse-chase labeling demonstrates that intracellular APP is produced in
an intracellular compartment
before secretion in NT2N
neurons. NT2N neurons
were pulse labeled with
[35S]methionine for 1 h and
chased for 0, 1, 4, 8, and 24 h.
Radiolabeled cell lysates (A)
or media (B) were immunoprecipitated sequentially with antibody 53 (for APP
)
followed by Karen (for APPFL
in the cell lysates and APP
/
in the media). Radiolabeled
immunoprecipitates were used
to expose PhosphorImager
plates (72 h) or X-ray film (3 wk) for visualization. C and
D summarize the quantitation
of experiments shown in A
and B. Counts from three different experiments were normalized to percentage of maximum and plotted as shown (mean ± standard error).
[View Larger Version of this Image (33K GIF file)]
-Cleavage in NT2N Neurons Occurs in a
pre-Golgi Compartment
is produced in an intracellular compartment
in NT2N neurons, we sought to identify the subcellular
site(s) of
-secretase cleavage. Therefore, NT2N neurons
were metabolically labeled with [35S]methionine in the presence or absence of 20 µg/ml BFA (Fig. 7). BFA is a pharmacological agent that causes a redistribution of the Golgi
into the ER (Doms et al., 1989
; Lippincott-Schwartz, 1989;
Pelham, 1991
). In the absence of BFA, APPFL, APP
, and
A
were recovered from the cell lysates, while APP
,
APP
, and A
were detected in the media of NT2N neurons (Fig. 7, a-c, lanes 1 and 3). Surprisingly, in the presence of BFA, not only APPFL but also APP
and A
continued to be recovered from NT2N cell lysates (Fig. 7, a-c,
lane 2). The effectiveness of BFA was verified by the fact
that the secretion of APP
, APP
, and A
into the medium was completely abolished in its presence (Fig. 7, a-c, lane 4). Furthermore, we found that APP
recovered from
BFA-treated cells (Fig. 7 b, lane 2) migrate with an accelerated electrophoretic mobility compared to APP
from
nontreated cells (Fig. 7 b, lane 1), suggesting that this fragment may have been derived from immature APP. Indeed,
the faster mobility of mature APPFL in the presence of
BFA (Fig. 7 a, compare M of lanes 1 and 2) indicates that
this agent blocks APP from acquiring at least some of the
posttranslational modifications. Thus, A
may be generated from immature as well as mature forms of APP.
Fig. 7.
Intracellular and
cleavages occur in a pre-Golgi
compartment in NT2N neurons. Cultures of NT2N cells were first
preincubated with 20 µg/ml BFA for 1 h before radiolabeling with
[35S]methionine for 16 h in the continuous presence of 20 µg/ml
BFA. Control cultures were processed similarly, except that BFA
was absent in the medium. Radiolabeled proteins from BFA-treated and untreated cell lysates and media were immunoprecipitated with Karen (for APPFL in the cell lysates and APP
/
in
the media as shown in A), with antibody 53 (for APP
in B), and
with the mAb 6E10 (for A
in C). Note that APP
and A
were
recovered in the cell lysate but not in the medium of BFA-treated
cells. (M, mature APPFL; I, immature APPFL.
[View Larger Version of this Image (43K GIF file)]
fragment generated in the presence
of BFA. Therefore, NT2N cells were metabolically labeled
with [35S]methionine in the presence or absence of BFA,
and APP
immunoprecipitated from the cell lysate was incubated with N-glycosidase F (Nglyc F), an enzyme that
removes N-linked carbohydrate chains. As shown, APP
from BFA-treated NT2N neurons (Fig. 8 a, lane 1) migrated more quickly than APP
recovered from untreated
cells (Fig. 8 a, lane 2). After digestion with Nglyc F, APP
demonstrated a mobility downshift in SDS-PAGE (Fig. 8
a, compare lanes 2 and 4). However, APP
from BFA-treated cells (Fig. 8 a, lane 3) still migrated faster than
APP
from nontreated cells (Fig. 8 a, lane 4) despite enzymatic removal of all N-linked carbohydrate chains. Thus, the
increased electrophoretic mobility of APP
in the presence of BFA cannot be accounted for solely by differences
in N-linked carbohydrate processing.
Fig. 8.
APP generated in the presence of BFA is partially
glycosylated. Cultures of NT2N neurons were metabolically labeled as in Fig. 7 in the presence or absence of 20 µg/ml BFA.
The cell lysates were then immunoprecipitated with the antibody
53. (A) Samples in lanes 3 and 4 were treated with Nglyc F for 16 h
to remove N-linked sugars, whereas immunoprecipitates in lanes
1 and 2 were treated with the vehicle. (B) Samples in lanes 2 and
4 were deglycosylated with a combination of Nglyc F, neuraminidase, and O-glycosidase for 16 h to remove both N- and O-linked
chains (lanes 2 and 4); lanes 1 and 3 represent samples that were
mock digested.
[View Larger Version of this Image (41K GIF file)]
by simultaneous digestion with Nglyc F, O-glycosidase, and neuraminidase. As shown, fully deglycosylated APP
(Fig. 8 b, lane 2) comigrated with APP
recovered from BFA-treated NT2N neurons (Fig. 8 b, lane 3). Furthermore,
combined BFA inhibition and deglycosylation (Fig. 8 b,
lane 4) did not induce a greater mobility shift than either
of these treatments alone (Fig. 8 b, lanes 2 and 3). Taken together, these results suggest that APP
generated from
BFA-treated NT2N neurons may represent
-secretase
processing of immature (nonglycosylated) APPFL in a pre-Golgi compartment.
-secretase cleavage indeed occurs early in the biosynthetic pathway of NT2N neurons,
we employed an alternative nonpharmacological method
to block protein transport from the ER to the Golgi. Incubation of cultured cells at 15°C has been shown to inhibit
newly synthesized proteins from exiting the intermediate
compartment (Saraste and Kuismanen, 1984
; Saraste et al.,
1986
; Schweizer et al., 1990
). To this end, NT2N cells were
incubated at 15°C for 16 h. Fig. 9 a shows that only the immature form of APPFL was present after a 16-h incubation
at 15°C, as indicated by its sensitivity to Endo H digestion,
suggesting that it is not transported to the Golgi apparatus
under these conditions (Fig. 9 a, lanes 3 and 4). By contrast, incubation of the NT2N cells at 37°C yielded both
immature and fully processed APPFL (Fig. 9 a, lanes 1 and
2). As expected, the immature APPFL was Endo H sensitive, while the mature forms of APPFL, having acquired
posttranslational modifications after exiting the ER, were
Endo H resistant. In addition, secreted forms of APP were not detected in cells maintained at 15°C, further substantiating the effectiveness of the temperature block. Significantly, continuous production of intracellular APP
was
observed at 15°C, despite the fact that the secretion of
APP ectodomain is completely abolished (Fig. 9 b). Taken
together, these data support the ER/IC of NT2N neurons
as a
-cleavage site.
Fig. 9.
APP is generated
in the ER/IC of NT2N neurons. Approximately 6 × 106
NT2N neurons were incubated at either 15° or 37°C
for 16 h. The cell lysates and
media were harvested and
immunoprecipitated with Karen. (A) The immunoprecipitates were then split, and half
of the samples was treated
with Endo H for 18 h, while
the other half was mock digested. Subsequent to this
step, the immunoprecipitates were separated by SDS-PAGE, transferred onto nitrocellulose replicas, and
probed with the antibody
Karen. The following observations serve to verify the effectiveness of the temperature block: (a) immature
forms of APPFL (I and I
) in
the cell lysate retain Endo H
sensitivity at 15°C; (b) mature glycosylated forms of
APPFL (M) in the cell lysate are not detected at 15°C; and (c) secreted fragments are not detected in the conditioned medium at 15°C. (B) Immunoprecipitates were separated by SDS-PAGE, transferred onto nitrocellulose replicas, and probed with antibody 53. APP
continued to be produced intracellularly despite the effective temperature block. However, secreted APP
was not detected in the medium at 15°C. Note that splitting intracellular APP
samples recovered at 15°C for Endo H digestion decreased the yield to below the
level of detection by this assay (data not shown). M, mature APPFL; I, immature APPFL; I
, immature APPFL demonstrating a mobility
shift due to Endo H sensitivity.
[View Larger Version of this Image (33K GIF file)]
-secretase cleavage indeed occurs in a pre-Golgi compartment of
NT2N neurons. To accomplish this, we compared the processing of wild-type APP695 and APP695 bearing an ER-retrieval motif (APP695
KK; Jackson et al., 1990
, 1993
) in
the NT2N cells. We used recombinant Semliki Forest virus
(SFV) vectors to express APP695
KK, in which the third
and fourth amino acids from the COOH terminus of APP are changed to lysines (i.e., APP695
KK). Our previous
studies have shown that despite high levels of SFV-mediated APP expression, SFV-infected NT2N cells display a
high degree of fidelity in processing APP (Wertkin et al.,
1993
; Turner et al., 1996
; Cook et al., 1997
). Furthermore,
we have found that cytopathic effects of SFV infection in
NT2N cells as measured by LDH release do not develop
until >48 h after infection (data not shown). Importantly,
all if not a significant majority of APP695
KK colocalize
with calnexin, the ER marker, by immunofluorescence
upon expression in NT2N neurons (Cook et al., 1997
).
can be produced
from APP695
KK, wild-type APP695 and APP695
KK were
separately expressed in NT2N neurons by infection with
SFV vectors bearing these constructs. After infection, duplicate wells containing wild-type APP695-infected cells were
also treated with 20 µg/ml BFA. The [35S]methionine-
labeled cell lysates and the media were then sequentially immunoprecipitated with the antibodies 53 and Karen.
Only the immature form of APPFL was detected from cells
expressing APP695
KK (Fig. 10 b, compare lanes 1 and 3).
Significantly, intracellular production and secretion of
APP
was not affected by genetic targeting of APP to the
ER (Fig. 10 a, lanes 3 and 6). Furthermore, we found that
unlike inhibition with BFA that eliminates transport of all proteins from the ER to the Golgi, specific retrieval of full length APP695
KK to the ER allowed the APP
fragment
generated in the ER/IC to be transported to the Golgi
complex for modification before secretion (Fig. 10 a, compare lanes 2 and 3 and lanes 5 and 6). This suggests that
once the ER retention motif is cleaved from the APP
fragment, it can then be transported to the Golgi complex
for further maturation and subsequent secretion.
Fig. 10.
APP is generated from APPFL that is concentrated in the ER. NT2N
cultures of ~1 × 106 cells
were infected with recombinant SFV containing either wild-type APP695 or
APP695
KK constructs. The
dilysine motif concentrates APPFL to the ER by an efficient retrieval mechanism.
Duplicate cultures infected
with wild-type APP695 were
treated with 20 µg/ml BFA
for comparison. Under these
conditions, the cells were
metabolically labeled with
[35S]methionine for 16 h. Radiolabeled cell lysates and
media were then immunoprecipitated with antibody 53 (for APP
, A) and Karen (for APPFL and APP
/
, B).
Radiolabeled immunoprecipitates were used to expose PhosphorImager plates (72 h) for visualization of bands. Unlike APP
produced under BFA inhibition,
APP
derived from APP695
KK was modified and secreted into the medium.
[View Larger Version of this Image (31K GIF file)]
Discussion
(Selkoe, 1994
). However, A
is the major component of senile plaques in the AD brain. Moreover,
mutations in the APP gene associated with Familial Alzheimer's disease alter APP processing and A
production
in vitro (Citron et al., 1992
; Cai et al., 1993
; Suzuki et al.,
1994
). Thus, it will be important to determine the proteolytic events that lead to A
production and to identify
the proteases responsible for each step as well as the sites
of their action. In addition, it will be important to consider
the cell type in which these processes occur. Non-neuronal
cells favor the nonamyloidogenic
-secretase pathway. By
contrast, neuronal cells exhibit increased
-secretase activity (Busciglio et al., 1993
; Wertkin et al., 1993
). To better
understand APP processing in neurons, we have used the
NT2N system for this study. We have previously shown that NT2N neurons express the isoform of APP expressed
almost exclusively in the CNS (i.e., APP695) and that they
constitutively produce intracellular and secreted A
. In
this study, we have identified and characterized some of
the intracellular
-secretase activities that cleave on the
NH2 terminus side of A
by using specific antibodies to
APP
and to other proteolytic fragments. More significantly, however, we have used three independent approaches to document novel
- and
-secretase activities that occur
in a pre-Golgi compartment.
is derived from APPFL within the cell before secretion. First, APP
was recovered from NT2N cell lysates
even after intact NT2N neurons were treated with trypsin.
Such treatment would eliminate cell surface-associated
APP
but not intracellular APP
. Indeed, the loss of
APP
after trypsin treatment of detergent-permeabilized NT2N neurons further confirms the intracellular origin of
APP
in NT2N neurons. Second, the continuous presence
of steady state levels of APP
in NT2N neurons, together
with a delay in the detection of APP
in freshly replenished medium, suggested that APP
is generated intracellularly before secretion. Third, pulse-chase experiments
demonstrated that the turnover of intracellular APP
lags
behind the turnover of newly synthesized APPFL, thereby
confirming that APP
is generated from APPFL inside
NT2N neurons before secretion.
in the cell lysate of NT2N neurons, together with the presence of A
40 and A
42 (Turner
et al., 1996
), firmly established that an intracellular
-secretase pathway(s) must exist in these cells. At present, no
other cell line has been reported to produce detectable levels of intracellular APP
from endogenous or over-expressed
wild-type APP (Seubert et al., 1993
; Haass et al., 1995a
;
Thinakaran et al., 1996b
). Only human kidney 293 cells
stably transfected with APPsw cDNA yield the related
APP
sw fragment from the cell lysates (Haass et al., 1995a
; Martin et al., 1995
). In these non-neuronal cells, however,
treatment with BFA completely eliminates APP
sw and
A
production (Haass et al., 1995a
; Martin et al., 1995
; Essalmani et al., 1996
). In contrast, NT2N neurons continue
to produce APP
and A
during treatment with BFA, implying that the subcellular site(s) of the
-secretase pathway is cell-type specific. Furthermore, this lack of inhibition of APP
and A
production by BFA in NT2N cells
suggests that at least one of the
-secretase pathways is localized to the ER/IC. Two additional independent means of
testing this hypothesis (i.e., the use of 15°C temperature
block and expression of APP bearing the dilysine ER retrieval signal) yielded consistent results.
-secretase pathway, but
not the
-secretase pathway, occurs inside NT2N neurons.
This view is based on the absence of APP
and p3 fragments in NT2N cell lysates. Of course, this observation
alone cannot rule out the possibility of their presence below the level of detection by our assay. Nevertheless, these
results imply that at least in this regard, NT2N neurons are
similar to almost all other cell lines in which the enzymes
of the
-secretase pathway are active at or near the cell
surface. The uniqueness of intracellular processing in postmitotic neuronal cells such as the NT2N neurons lies in the fact that unlike non-neuronal cells, the amyloidogenic
-secretase pathway(s) is preferred. Accordingly, the level
of A
secretion is much higher than that of p3 in postmitotic NT2N neurons.
secretion (Citron et al., 1992
; Cai et al., 1993
). Concomitant
with this change, intracellular APP
sw is also detected in
non-neuronal cells stably transfected with APPsw (Haass
et al., 1995a
; Thinakaran et al., 1996b
). Transfection of wild-type APP695 in non-neuronal cells, however, fails to produce intracellular APP
and results in the secretion of
more p3 than A
(Thinakaran et al., 1996b
). Thus, it appears that the introduction of the Swedish mutation shifts APP processing away from the
-secretase pathway to the
-secretase pathway. However, unlike NT2N neurons that
may use multiple
-secretase pathways to produce both intracellular A
and APP
, APPsw expressing non-neuronal cells use primarily the endosomal/lysosomal pathway or the Golgi-derived vesicles to generate intracellular
A
and APP
sw, since treatment of these cells with BFA
completely inhibits APP
sw and A
production (Haass et
al., 1995a
; Martin et al., 1995
).
-secretase
pathways have been identified to date. Of these three, the
endosomal/lysosomal pathway, which processes APP targeted to the cell surface after its reinternalization into endosomes and lysosomes, is the most ubiquitous. Both primary cultures of neuronal and non-neuronal cells, as well
as multiple cell lines, use this pathway to produce A
.
However, the contribution of endosomal/lysosomal processing to the overall production of A
is relatively minor
since non-neuronal cells transfected with wild-type APP
produce mostly p3 and very little A
(Haass et al., 1992a
,
b
; Koo and Squazzo, 1994
; Lai et al., 1995
; Thinakaran et al.,
1996b
). In contrast, an alternative
-secretase pathway
that produces A
in Golgi-derived vesicles is the most important for the production of A
in cells transfected with
APPsw. Consistent with this view, transfection of an
APPsw construct lacking the cytoplasmic tail, which eliminates reinternalization of cell surface APPsw, does not reduce the secretion of A
(Haass et al., 1995a
; Essalmani et
al., 1996
). It is likely that the neuron-like NT2N cells also
use this
-secretase pathway since neuronal cells (including
hippocampal neurons and NT2N neurons) produce much
higher levels of A
than p3. Finally, the third
-secretase pathway localized to the ER/IC appears to be preferentially used by postmitotic neuronal cells, since intracellular
APP
was not detected in several non-neuronal cell lines
when treated with BFA.
generation in the ER of NT2N
neurons identifies these cells as a unique system in which
to test the hypothesis that amyloidogenic processing of
APP within that compartment plays an important role in
the pathogenesis of AD. There is now strong evidence that
mutations in both the APP gene and the recently identified presenilin genes cause AD by altering APP processing in ways that lead to the production of more amyloidogenic
form of A
(i.e., A
42; Scheuner et al., 1996
). Recently, in
both non-neuronal and neuronal cells (including the
NT2N neurons used in this study), the presenilin proteins
have been localized to the ER (Cook et al., 1996
; Kovacs
et al., 1996
; Thinakaran et al., 1996a
). Thus, the identification of amyloidogenic processing that may occur within
the ER of neurons raises the formal possibility that direct or indirect interaction may occur between the presenilins
and APP. Furthermore, the mutations in the presenilin
genes may alter this interaction in a manner that leads to
increased production of A
42. Therefore, it will be particularly interesting to examine the effects of both Familial
Alzheimer's disease-linked mutations occurring in the
APP as well as the presenilin genes on the processing of
APP in the ER.
Received for publication 14 February 1997 and in revised form 22 May 1997.
Please address all correspondence to Dr. Virginia M.-Y. Lee, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Third Floor Maloney, HUP, Philadelphia, PA 19104-4283. Tel.: (215) 662-6427; Fax: (215) 349-5909; E-mail: vmylee{at}mail.med.upenn.eduWe thank Drs. T.E. Golde and J.Q. Trojanowski for critical review of the manuscript. We also thank Drs. D. Schenk, S. Gandy, and N. Suzuki for providing us with the antibody 192, antibody 369W, and the mAb Ban50, respectively. C.D. Page is thanked for providing some of the NT2N cells used in this study.
This work was supported by National Institutes of Health NIA grant AG-11542.
A, amyloid
;
AD, Alzheimer's disease;
APP, amyloid precursor protein;
BFA, brefeldin A;
CNS, central
nervous system;
Endo H, endoglycosidase H;
IC, intermediate compartment;
Nglyc F, N-glycosidase F;
SFV, Semliki Forest virus.
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