From the Center for Neurodegenerative Disease Research,
Department of Pathology and Laboratory Medicine, and
Department of Microbiology, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania 19104
Received for publication, October 2, 2002, and in revised form, December 4, 2002
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ABSTRACT |
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Insoluble pools of the
amyloid- Alzheimer's disease (AD)1 is characterized by the
accumulation of aggregated A Whereas some truncations may be due to the partial degradation of
full-length A Untransfected non-neuronal cells and murine cells are not well suited
toward the study of BACE-derived N-terminally truncated A NT2N neurons are a post-mitotic, terminally differentiated human
neuronal cell culture model derived from retinoic acid treatment of the
human embryonal carcinoma cell line NTera2/c1.D1 (NT2 Cell Culture--
Undifferentiated NTera2/c2.D1 cells were
maintained as described previously (26, 27) in Opti-MEM (Invitrogen)
containing 5% fetal bovine serum (FBS), 100 units/ml penicillin, and
10 µg/ml streptomycin sulfate. Cells were differentiated by twice
weekly 10 µM retinoic acid treatments for 5 weeks and
then replated (replate 2 neurons) in DMEM with high glucose, 5% FBS,
and mitotic inhibitors (10 µM uridine, 10 µM 5-fluoro-2'deoxyuridine, 1 µM cytosine
arabinoside, Sigma) to obtain nearly pure NT2N neurons (26). Greater
than 99% pure neurons (replate 3 neurons) (27) were isolated by
mechanical separation of neurons after brief trypsinization of mixed
cultures and replated into 6-well plates coated with Matrigel and
poly-D-lysine.
Western Blot Analysis--
Cell lysates were collected in RIPA
buffer (0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 5 mM EDTA in TBS, pH 8.0) in the presence of protease
inhibitors (1 µg/ml each of pepstatin A, leupeptin,
L-1-tosylamido-2-phenylethyl chloromethyl ketone, 1-chloro-3-tosylamido-7-amino-2-heptanone, soybean trypsin
inhibitor, and 0.5 mM phenylmethylsulfonyl fluoride) and
briefly sonicated. Protease inhibitors were also added to conditioned
media samples. Samples were centrifuged at 100,000 × g
for 20 min at 4 °C, electrophoresed on 7.5% Tris-glycine acrylamide
gels, and transferred to nitrocellulose. When indicated, samples were
immunoprecipitated with Karen, a goat polyclonal antibody raised
against sAPP, prior to electrophoresis. APP and total sAPP were probed
with Karen. sAPP Metabolic Labeling and Immunoprecipitation--
Cells were
incubated in methionine-free DMEM (Invitrogen) for 30 min, labeled with
[35S]methionine (250 µCi/ml in methionine-free DMEM
supplemented with 5% dialyzed FBS; PerkinElmer Life Sciences) for 90 min, and chased for 1 h. Cells were treated with 10 µM PMA in Me2SO (Sigma) during the chase
period and/or with 10 µM TAPI in Me2SO
(Peptides International, Louisville, KY) 30 min prior to and throughout the chase period. Protease inhibitors were added to conditioned media,
and sAPP was immunoprecipitated with Ban50 prior to electrophoresis on
7.5% Tris-glycine acrylamide gels. For C-terminal fragment analysis,
cells were labeled for 2 h with [35S]methionine in
the presence of 200 µM MG132 (Peptides International), rinsed, and lysed in 1000 µl of RIPA buffer containing protease inhibitors for immunoprecipitation. Lysates were briefly sonicated, and
both lysates and media were cleared by centrifugation at 100,000 × g for 20 min at 4 °C. C-terminal fragments were
immunoprecipitated with 2493, a rabbit polyclonal antibody recognizing
the C-terminal region of APP, and resolved on 10/16.5% step gradient
Tris-Tricine gels. Gels were fixed in 50% methanol, 5% glycerol,
dried, and exposed to PhosphorImager plates for visualization. Finally,
to detect both secreted and intracellular A Northern Analysis--
Total RNA was extracted from NT2 Stable Transduction of NT2 Immunoprecipitation/Mass Spectrometry--
Media conditioned for
10 days were collected in the presence of protease inhibitors and
cleared by centrifugation at 100,000 × g for 20 min at
4 °C. Media were immunoprecipitated with 4G8, and immunoprecipitated
material was eluted with a saturated solution of
Sandwich ELISA Analysis--
Secreted A sAPP and A TAPI-insensitive sAPP Production in NT2N Neurons--
The
increased production of sAPP secretion by NT2N neurons indicated that
BACE and/or
Because Ban50 recognizes both sAPP BACE Expression in NT2
In agreement with the Northern analysis, BACE protein was undetectable
in NT2
To assay BACE cleavage at Asp-1, NT2 Secretion of Full-length and N-terminally Truncated
A Intracellular Generation of Truncated A
To demonstrate further the presence of intracellular N-terminally
truncated A Insoluble amyloid deposits from AD brains are heterogeneous in
morphology and composition. Although the seeding and maturation of
senile plaques in vivo is not well understood, increased
Until recently, the mechanism whereby A Additional A Although secretion of A Multiple subcellular sites are responsible for the production of
different A Increased BACE expression has been implicated in the pathogenesis of AD
(29). Interestingly, BACE expression had a more profound effect on A peptide (A
) in brains of Alzheimer's disease patients
exhibit considerable N- and C-terminal heterogeneity. Mounting evidence
suggests that both C-terminal extensions and N-terminal truncations
help precipitate amyloid plaque formation. Although mechanisms
underlying the increased generation of C-terminally extended peptides
have been extensively studied, relatively little is known about the
cellular mechanisms underlying production of N-terminally truncated
A
. Thus, we used human NT2N neurons to investigate the production of
A
11-40/42 from amyloid-
precursor protein (APP) by
-site
APP-cleaving enzyme (BACE). When comparing undifferentiated human
embryonal carcinoma NT2
cells and differentiated NT2N neurons, the
secretion of sAPP and A
correlated with BACE expression. To study
the effects of BACE expression on endogenous APP metabolism in human
cells, we overexpressed BACE in undifferentiated NT2
cells and NT2N neurons. Whereas NT2N neurons produced both full-length and truncated A
as a result of normal processing of endogenous APP, BACE
overexpression increased the secretion of A
1-40/42 and
A
11-40/42 in both NT2
cells and NT2N neurons. Furthermore, BACE
overexpression resulted in increased intracellular A
1-40/42 and
A
11-40/42. Therefore, we conclude that A
11-40/42 is generated
prior to deposition in senile plaques and that N-terminally truncated
A
peptides may contribute to the downstream effects of amyloid
accumulation in Alzheimer's disease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptides
in senile plaques and vascular deposits. A
has classically been
described as a 4-kDa peptide, derived from proteolytic processing of
APP, varying in length from 40 to 42 amino acids due to C-terminal
heterogeneity. The generation of the longer A
1-42 peptide is
specifically increased by several mutations linked to familial
Alzheimer's disease (1). Furthermore, A
1-42 is more fibrillogenic
than A
1-40 in vitro (2), consistent with the finding
that both diffuse and senile plaques are composed of primarily A
peptides that terminate at position 42 (3). However, several N-terminal
truncations are also found in A
peptides derived from AD brains,
demonstrated as early as the first biochemical isolation of A
peptides from senile plaques (4). The relative importance of
N-terminally truncated A
peptides in the pathogenesis of AD is
unknown. Interestingly, some individuals with sporadic AD (5), familial
AD (5, 6), and Down's syndrome (7) preferentially accumulate
N-terminally truncated A
species. Also, overexpression of APP
harboring disease-associated mutations within the A
domain
(i.e. A692G and E693G) results in increased secretion of
A
11-40/42 (8, 9).
after secretion of peptides into the extracellular milieu, A
peptides beginning with glutamine at position 11 are derived from the membrane-bound
-site APP-cleaving enzyme (BACE) (10-12). To generate full-length A
, BACE cleaves APP between the methionine and aspartate at position 1 (Asp-1) of the N terminus of
A
(
-cleavage), resulting in the secretion of a large N-terminal ectodomain, sAPP
, and the retention of a 99-amino acid C-terminal fragment, C99. To generate N-terminally truncated A
, BACE cleaves APP between tyrosine and glutamate at position 11 (Glu-11) within the
A
domain (
'-cleavage), resulting in the secretion of a slightly larger N-terminal ectodomain, sAPP
', and the retention of an 89-amino acid C-terminal fragment, C89. C89 can also be produced by
proteolysis of C99 by BACE at Glu-11 (11). Alternatively, APP may also
be cleaved at position 16 by
-secretase (13, 14), resulting in
secretion of the N-terminal sAPP
along with the retention of an
83-amino acid C-terminal fragment, C83. Membrane-bound C-terminal
fragments are subjected to further proteolysis within the transmembrane
domain by
-secretase, with cleavage typically occurring at either
position 40 or 42 within the A
region. Whereas all of the components
of
-secretase have not been identified, the presenilin proteins are
necessary for secretion of A
peptides and have been postulated to
contain the active site of
-secretase (1). However, multiple
-secretases may exist as presenilin is not needed for A
1-42
production early in the secretory pathway (15).
. Many
non-neuronal cells preferentially use the
-secretase pathway at the
expense of
-cleavage of APP (13, 14), although the secretion of
full-length and N-terminally truncated A
can be increased upon
overexpression of either APP or BACE (11, 12, 16, 17). Furthermore, due
to low BACE activity and low APP expression, intracellular A
is
difficult to detect in non-neuronal cells (16, 18). In contrast,
neuronal cells cleave a larger proportion of APP by BACE (19-21),
although overexpression of human APP in rodent cells does not lead to
the generation of A
11-40/42 from human APP (22), consistent with
evidence that
'-cleavage is species-specific (23). Furthermore,
rodent neuronal cells preferentially cleave endogenous APP at position
11, whereas the presence of
'-cleavage in human neuronal cells has
not been adequately addressed. Therefore, rodent cell culture models
may not accurately reflect the proteolytic processing of APP in human neurons.
) (24-27).
Their high APP expression and
-secretase activity make them amenable
to biochemical analysis of both neuronal specific and human-specific
characteristics of APP processing (18, 21). We have shown that NT2N
neurons generate A
1-40/42 intracellularly prior to secretion and
that endogenous secretion of A
1-40/42 from NT2N neurons increases
with age in culture (19, 20). Furthermore, a detergent-insoluble pool
of intracellular A
accumulates with time in NT2N neurons (28). In
this study, we found that sAPP and A
secretion increase upon
neuronal differentiation of NT2N neurons, correlating with BACE
expression. Given a recent report (29) that BACE protein expression and
activity are increased in AD, we sought to refine further our
understanding of APP metabolism in NT2N neurons by examining the effect
of exogenous expression of BACE in undifferentiated NT2
cells and
differentiated NT2N neurons. We found that the increase in A
production due to BACE overexpression was more pronounced in NT2N
neurons than in non-neuronal NT2
cells. Furthermore, we found that
A
11-40/42 is produced endogenously by NT2N neurons and that BACE
overexpression increases the secretion of both full-length and
truncated A
peptides. We further demonstrate that A
11-40/42 is
generated intracellularly, indicating that A
11-40/42 is produced
prior to deposition in senile plaques. The effect of BACE expression on
the generation of both full-length and N-terminally truncated A
underscores the role of BACE activity in the generation of A
peptides and indicates that N-terminally truncated A
peptides may
contribute to the pathogenesis of AD.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and sAPP
' were probed with Ban50, a mouse
monoclonal antibody recognizing A
residues 1-10, or with NAB228, a
mouse monoclonal antibody recognizing A
residues
1-11.2 sAPP
was
specifically probed with C5A4/2, a rabbit polyclonal antibody raised
against a synthetic peptide (CSEVKM) corresponding to the C terminus of
sAPP
(11). sAPP
' was specifically probed with C10A4, a rabbit
polyclonal antibody raised against a synthetic peptide (CHDSGY)
corresponding to the C terminus of sAPP
'. The specificity of C5A4
and C10A4 was determined by their lack of immunoreactivity with
full-length APP or C-terminal APP fragments, and by blocking
experiments in which only peptides with the corresponding free C
terminus are able to block immunoreactivity (data not shown). Immunoblots were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences) after application of species-specific horseradish peroxidase-conjugated anti-IgG antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). However, for quantification of APP and sAPP, Karen
immunoblots were labeled with 125I-protein A (PerkinElmer
Life Sciences) after application of a rabbit anti-goat IgG linker.
Radiolabeled APP and sAPP were quantified using PhosphorImager analysis
(Amersham Biosciences). For detection of BACE, crude membrane fractions
were prepared by first collecting cells in hypotonic buffer (10 mM NaCl, 10 mM Tris, 1 mM EDTA, pH
7, protease inhibitors) followed by centrifugation at 100,000 × g for 20 min at 4 °C. After an additional wash with
hypotonic buffer, membrane proteins were extracted by sonication of the pellet in 1 M NaCl, 40 mM Tris, 4 mM EDTA, protease inhibitors, 0.5% Triton X-100, pH 7. The
fraction was cleared by another round of centrifugation, and the
BACE-containing supernatants were electrophoresed as above and
immunoblotted with a rabbit polyclonal anti-BACE (CT) antibody (ProSci,
Poway, CA).
, two 10-cm dishes of replate 2 NT2N neurons were infected with recombinant Semliki Forest
virus encoding wild type APP695, prepared as described previously (18,
30). Cells were infected in serum-free medium for 1 h, cultured in
complete growth medium for 14 h, and then labeled with
[35S]methionine (500 µCi/ml) for 8 h. Conditioned
media and RIPA cell lysates were collected, cleared by centrifugation,
and immunoprecipitated with 4G8 (Senetek, Maryland Heights, MO) prior
to electrophoresis on 10/16.5% step gradient Tris-Tricine gels.
cells
and NT2N neurons with the Trizol Reagent (Invitrogen) as per
manufacturer's protocol. RNA concentrations were determined by optical
density readings, and equal amounts of RNA were separated on 1%
agarose-formaldehyde gels. RNA was transferred to a nitrocellulose
membrane (Amersham Biosciences) and hybridized with a radiolabeled
probe generated by either PstI digestion or
AccI-HincII double digestion of a BACE cDNA.
The blot was washed and exposed to a PhosphorImager plate for
visualization. Glyceraldehyde-3-phosphate dehydrogenase levels were
obtained by using a glyceraldehyde-3-phosphate dehydrogenase probe
purchased from Ambion (Austin, TX). 28 S and 18 S ribosomes were
visualized by staining a duplicate gel with ethidium bromide.
Cells--
NT2
cells were stably
transduced with a vesicular stomatitis virus surface glycoprotein
(VSV-G) pseudotyped self-inactivating lentiviral
vector.3 To generate the
virus, QBI 293A cells were plated on poly-D-lysine-coated 10-cm dishes. Cells were transfected with pMD.G (containing the VSV-G
envelope glycoprotein), pCMV
R8.2 (containing viral structural, enzymatic, and accessory genes), and SIN-EFp-GFP/SIN-EFp-BACE (containing minimal human immunodeficiency virus-based viral sequences, the elongation factor 1-
promoter, and either a GFP or BACE
cDNA) using standard CaPO4 techniques. Media
conditioned with viral particles were harvested over 3 days,
centrifuged at 1000 rpm for 5 min, and passaged through a 0.45-µm
filter to remove any cellular debris. Viral supernatants were added to
NT2
cells, and transduced NT2
cultures were subcloned by limited
dilution into 96-well plates. Uniform expression was verified either by direct fluorescence for GFP-expressing cells or indirect
immunofluorescence with BaceN1, a rabbit polyclonal antibody raised
against the N terminus of BACE (32), for BACE-expressing cells.
-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid, 50%
acetonitrile. Data were collected on an ABI/Perspective (Framingham, MA) Voyager DE-PRO MALDI-TOF instrument in the positive-ion mode at the
Protein Microchemistry/Mass Spectrometry Facility of the Wistar
Institute (Philadelphia). Samples were spotted to a 100-well plate
using
-cyano-4-cinnamic acid matrix (Sigma) at 10 mg/ml. Reflector
mode with the accelerating potential at 20 kV was used. External
calibration was performed on all samples.
was detected using
ELISA protocols described previously (20, 33). Briefly, Ban50
(anti-A
1-10) or BNT77 (anti-A
11-28) were used as capturing
antibodies. After application of culture media samples, horseradish
peroxidase-conjugated BA-27 and BC-05 were used to report A
species
ending at position 40 and 42, respectively. For quantification of A
levels, synthetic A
1-40 and A
1-42 purchased from Bachem
Bioscience Inc. (King of Prussia, PA) were serially diluted in
unconditioned cell culture media to generate standard curves.
Monoclonal antibodies, Ban50, BNT77, BA-27, and BC-05 were prepared as
described previously (33-35). To detect intracellular A
, cells were
washed thoroughly with phosphate-buffered saline and scraped into RIPA
buffer containing protease inhibitors. Lysates were sonicated and spun
at 100,000 × g for 20 min at 4 °C. A
from RIPA
lysates was captured with either JRF/cA
40/10 or JRF/cA
42/26, monoclonal antibodies specific for A
40 and A
42, respectively (36), supplied by Dr. M. Mercken (Janssen Research Foundation, Beerse,
Belgium). To detect full-length A
, captured peptides were reported
with horseradish peroxidase-conjugated JRF/A
N/25, a monoclonal
antibody directed against A
1-7. To detect full-length and
N-terminally truncated A
, captured peptides were reported with
horseradish peroxidase-conjugated m266, a monoclonal antibody recognizing residues 13-28 of A
(37).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Production Correlates with Neuronal
Differentiation--
APP processing differs between non-neuronal and
neuronal cells, underscoring the importance of using neuronal systems
to study APP metabolism and A
generation (18-20, 28). Prior to
studying BACE activity and
'-cleavage in NT2N neurons, we quantified
the differences in the expression and proteolytic processing of APP between NT2
cells and NT2N neurons. Whereas both NT2
cells and NT2N
neurons express high levels of APP, the two cell types express different isoforms of APP (Fig.
1A). We have shown previously (19) that NT2
cells predominantly express the 751- and 770-amino acid
isoforms of APP (APP751/770) that appear as a doublet corresponding to
immature (~110 kDa) and mature N- and
O-glycosylated APP (~125 kDa). NT2N neurons predominantly
express the shorter 695-amino acid isoform of APP (APP695), the
majority of which is immature APP695 (~95 kDa) with relatively less
mature APP695 (~110 kDa). Despite the difference in isoform
expression, densitometric quantification indicated that when normalized
for total protein content, NT2
cells expressed APP at 98% ± 5 (S.E.) compared with NT2N neurons. However, despite equivalent total
APP expression, we found that proteolytic processing of APP was more
efficient in NT2N neurons compared with NT2
cells. To measure total
sAPP, derived from both
- and
-secretase cleavage of APP, media
conditioned by NT2
cells or NT2N neurons for 24 h were
immunoprecipitated with a polyclonal antibody raised against the
N-terminal domain of APP. We found that NT2N neurons secreted more
total sAPP relative to NT2
cells (Fig. 1B). Densitometric
quantification of total sAPP indicated that NT2N neurons secreted over
5-fold more sAPP than NT2
cells (Fig. 1C). Differentiation
had an even larger effect on A
secretion, as NT2N neurons secreted
over 20-fold more A
than NT2
cells (Fig. 1D),
determined by sandwich ELISA for A
1-40. Because differences in APP
expression cannot explain the increase in A
secretion, differences
in either
- or
-secretase activity are likely to be responsible
for the more efficient proteolysis of APP in NT2N neurons.
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Fig. 1.
Dissociation between APP expression and
sAPP/A secretion. A,
protein-corrected RIPA lysates from NT2
cells and replate 3 NT2N
neurons were separated on a 7.5% Tris-glycine gel and immunoblotted
with Karen for full-length intracellular APP. The bands represent
immature APP751/770 (*, left lane), mature APP751/770
(arrowhead, left lane), immature APP695 (*,
right lane). and mature APP695 (arrowhead, right
lane). B, after normalization to intracellular
full-length APP expression levels, total sAPP was purified from media
conditioned from either NT2
cells or replate 3 NT2N neurons for
24 h by immunoprecipitation with Karen, separated on a 7.5%
Tris-glycine gel, and immunoblotted with Karen. C, total
sAPP was quantified by Karen immunoblots of conditioned media, analyzed
by PhosphorImager, corrected for intracellular APP expression, and
shown as mean values relative to NT2
cells ± S.E. One-way
analysis of variance revealed p < 0.0001; post hoc
analysis showed p < 0.01 (*) compared with NT2
cells. D, A
1-40 levels were quantified by Ban50/BA-27
ELISA, normalized to intracellular APP expression levels, and shown as
mean values relative to NT2
cells ± S.E. One-way analysis of
variance revealed p value of 0.0015. Post hoc analysis
showed p < 0.01 (*) compared with NT2
cells.
-secretase activity might be elevated in NT2N neurons
upon neuronal differentiation of the NT2
cells. To distinguish
between these possibilities, we first addressed the presence of
-secretase by testing the pharmacologic responses of NT2
cells and
NT2N neurons upon
-secretase inhibition.
-Secretase has been
attributed to members of a family of proteases that contain a disintegrin and a
metalloprotease domain (ADAM), including ADAM10 and tumor
necrosis factor-
converting enzyme.
-Cleavage is enhanced by
phorbol ester-induced stimulation of tumor necrosis factor-
converting enzyme via protein kinase C and can be inhibited by metalloprotease inhibitors. Therefore, we treated metabolically labeled
NT2
cells and NT2N neurons with either phorbol 12-myristate 13-acetate (PMA) or the specific metalloprotease inhibitor, TAPI. sAPP
from media samples was immunoprecipitated using Ban50, a monoclonal
antibody that recognizes the first 10 amino acids of A
, thereby
recognizing both sAPP
and sAPP
'. In NT2
cells, PMA treatment
increased sAPP secretion by 2.15 ± 0.29-fold, whereas TAPI
treatment inhibited sAPP production to 0.54 ± 0.04-fold of untreated NT2
cells (Fig. 2,
A and B). The magnitude of sAPP inhibition in
NT2
cells was comparable with that reported for other non-neuronal
cells (38). Thus we concluded that
-secretase activity is present in
NT2
cells, and a large proportion of the base-line sAPP secreted by
NT2
cells is derived from
-secretase. Whereas NT2N neurons also
exhibited PMA-induced up-regulation of
-secretase activity
(1.80 ± 0.37), TAPI had no effect on sAPP production (0.95 ± 0.14; Fig. 2, A and B). Furthermore, in both NT2
cells and NT2N neurons, TAPI was able to prevent PMA-induced sAPP
production, demonstrating that TAPI is able to inhibit
PMA-induced
-secretase in both non-neuronal and neuronal cells.
However, the inability of TAPI to decrease sAPP generation by NT2N
neurons below base-line levels indicated that a relatively small
proportion of APP is normally proteolyzed by
-secretase in NT2N
neurons.
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Fig. 2.
TAPI-insensitive secretion of sAPP in NT2N
neurons. A, NT2 cells and replate 3 NT2N neurons were
metabolically labeled for 90 min and chased for 60 min. Cells were
treated with 10 µM PMA during the chase period and/or 10 µM TAPI 30 min prior to and throughout the chase period.
Conditioned media were immunoprecipitated with Ban50 and separated on
7.5% Tris-glycine gels. A representative gel out of three separate
experiments is shown. B, sAPP
/sAPP
' levels were
quantified by PhosphorImager and shown as mean values ± S.E.
normalized to control, untreated cells. C, total sAPP was
immunoprecipitated with Karen from media conditioned for 24 h by
NT2
cells and replate 3 NT2N neurons, corrected for cell lysate
concentration or intracellular APP expression. Samples were then
separated on a 7.5% Tris-glycine gel and immunoblotted with Ban50
(top panel), NAB228 (2nd panel), C5A4/2
(3rd panel), or C10A4 (bottom panel). The
specific sAPP fragments recognized by these antibodies are
labeled.
and sAPP
', the ability of
Ban50 to immunoprecipitate TAPI-insensitive sAPP from NT2N neurons
suggested that NT2N neurons may produce sAPP
' endogenously. Therefore, to demonstrate more directly the presence of BACE-derived sAPP fragments, we used a panel of antibodies that recognize different sAPP species to analyze media conditioned for 24 h by NT2
cells and NT2N neurons. To better compare the relative abundance of sAPP
species, media samples were corrected for either lysate protein concentration or intracellular full-length APP levels prior to analysis. Despite preferential utilization of
-cleavage over
-cleavage in non-neuronal cells (13, 14), we detected less sAPP with
Ban50 from NT2
cells compared with NT2N neurons (Fig. 2C, top panel), consistent with the possibility
that NT2N neurons produce sAPP
' endogenously. This result was
confirmed with a second monoclonal antibody, NAB228, that recognizes
the first 11 amino acids of A
(Fig. 2C, 2nd
panel). C5A4/2, a polyclonal antibody that specifically recognizes
the C terminus of sAPP
, showed the presence of sAPP
in media
conditioned by NT2N neurons (Fig. 2C, 3rd panel).
In contrast, the amount of sAPP
in media conditioned by NT2
cells
was undetectable. Finally, a polyclonal antibody that specifically
recognizes the C terminus of sAPP
' demonstrated the presence of
endogenous sAPP
' produced by NT2N neurons (Fig. 2C,
bottom panel). Therefore, not only is
-secretase activity
relatively low in NT2N neurons, as determined pharmacologically, but
BACE cleavage at both Asp-1 and Glu-11 is readily detected as a product
of normal APP metabolism from NT2N neurons.
Cells and NT2N Neurons--
To better
understand the secretion and intracellular generation of BACE-derived
A
peptides in human neurons, we characterized BACE expression in
NT2
cells and NT2N neurons. Expression of BACE mRNA was
determined by Northern analysis of NT2
cells and NT2N neurons. BACE
mRNA expression was clearly present in NT2N neurons, as seen by the
presence of 7.0-, 4.4-, and 2.6-kb bands (Fig.
3A). NT2
cells, however,
showed markedly less BACE expression, most notably demonstrated by the
absence of a 7.0-kb band. The 4.4-kb band, possibly representing one of
the several identified BACE splice variants (39, 40), was somewhat
reduced in NT2
cells relative to NT2N neurons. Two different
radiolabeled BACE cDNA fragment probes yielded the same results
(Fig. 3A and data not shown).
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Fig. 3.
BACE expression in NT2 cells and NT2N
neurons. A, mRNA from NT2
cells and replate 3 NT2N neurons were electrophoresed on formaldehyde-agarose gels and
hybridized with a radiolabeled PstI BACE cDNA fragment
and visualized by PhosphorImager. Equal mRNA loading was determined
by glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
expression and by 28 S and 18 S ribosome levels. B, crude
membrane fractions from stably transduced NT2
cells and replate 2 NT2N neurons expressing BACE (NT2
/NT2B30
/NT2B17
, undifferentiated
cells; NT2N/NT2B30N/NT2B17N, neurons) were separated on a 7.5%
Tris-glycine gel and immunoblotted for BACE. C, total sAPP
from media conditioned for 24 h from NT2
cells or replate 3 NT2N
neurons was immunoprecipitated with Karen, electrophoresed on a 7.5%
Tris-glycine gel, and immunoblotted for sAPP
with C5A4/2, showing
sAPP
derived from either APP751/770 or APP695. D,
C-terminal APP fragments were immunoprecipitated with 2493 from NT2
cells and replate 2 NT2N neurons that were metabolically labeled for
2 h, separated on a 10/16.5% discontinuous gradient Tris-Tricine
gel, and exposed to a PhosphorImager screen. The bands corresponding to
C99, C89, and C83 are labeled.
cell lysates, whereas NT2N neuron lysates exhibited faint
immunoreactivity (Fig. 3B). This suggested that low BACE expression limits A
production in NT2
cells. To determine the effect of BACE expression on A
production from endogenous APP, NT2
cells were stably transduced using a pseudotyped self-inactivating lentiviral vector encoding BACE or GFP. This viral vector is non-toxic to NT2
cells and NT2N neurons and results in stable transgene expression in differentiated neurons for over 5 months in
vivo.3 Several subclones uniformly expressing GFP or
BACE were isolated (data not shown). As a control, GFP-expressing
NT2G7
cells were selected to ensure that the process of transduction
and subcloning did not alter APP processing. Two subclones, NT2B30
and NT2B17
, were chosen to represent low and high BACE-expressing
clones, respectively (Fig. 3B), and to control for transgene
insertion effects. Subclones retained their ability to differentiate
into neurons upon retinoic acid exposure, and transgene overexpression was maintained in differentiated neurons (Fig. 3B).
and NT2N conditioned media were
immunoprecipitated with polyclonal antisera to the APP ectodomain to
collect total sAPP. The immunoprecipitate was then subjected to
SDS-PAGE and immunoblotted with C5A4/2, a rabbit polyclonal antibody
specific for the C terminus of sAPP
(Fig. 3C). NT2
cells did not secrete appreciable levels of sAPP
, consistent with
the fact that BACE expression is nearly absent in NT2
cells. Overexpression of BACE in NT2
cells, however, resulted in the detection of sAPP
in a dose-dependent manner. Unlike
untransduced NT2
cells, sAPP
was detected from untransduced NT2N
neuron-conditioned media. The shift in electrophoretic mobility between
sAPP
recovered from BACE-expressing NT2
cells and NT2N neurons
reflects the shorter isoform of APP expressed in NT2N neurons.
Furthermore, sAPP
secretion by NT2B30N and NT2B17N neurons, which
express exogenous BACE as a consequence of retroviral transduction, was increased relative to control NT2N neurons. In addition to increased cleavage at Asp-1, we determined that
'-cleavage at Glu-11 was also
present in BACE-expressing cells by immunoprecipitating C-terminal APP
fragments from metabolically labeled NT2
cells, NT2N neurons, and
BACE subclones with 2493, a polyclonal antibody raised against the C
terminus of APP. BACE overexpression resulted in increased C99 and C89
relative to control cultures in both undifferentiated cells and
neurons, corresponding to increased proteolysis of endogenous APP at
residues Asp-1 and Glu-11, respectively. Significantly, C89 levels
increased in a dose-dependent manner in that higher BACE-expressing clones had higher C89 levels. GFP expression was found
to have no effect on sAPP
secretion or C-terminal fragment production in both undifferentiated cells and neurons (data not shown).
--
Given the differences in endogenous BACE expression between
NT2
cells and NT2N neurons, we sought to determine the effect of
overexpressing BACE on A
production in these cells and to demonstrate the presence of N-terminally truncated A
peptides derived from BACE cleavage of APP at Glu-11. Media conditioned for
24 h were subjected to sandwich ELISA analysis and normalized for
APP expression. Two sandwich ELISA systems were used in which either
Ban50 (anti-A
1-10) or BNT77 (anti-A
11-28) monoclonal antibodies
were used to capture A
. The Ban50 ELISA was used to detect
full-length A
, whereas the BNT77 ELISA was used to detect both
full-length and N-terminally truncated A
species. Both ELISA systems
demonstrated the production of A
in NT2
cells upon the overexpression of BACE (Fig.
4A) at levels similar to that
secreted by untransduced NT2N neurons. However, the effect of BACE
overexpression was more pronounced in NT2N neurons, increasing A
secretion 5-8-fold over untransduced NT2N neurons. Furthermore, the
differences in A
concentration as detected by Ban50 or BNT77
indicated that a large proportion of secreted A
peptides are
N-terminally truncated. To identify N-terminally truncated A
peptides secreted by NT2N neurons, conditioned media were
immunoprecipitated by 4G8 and subjected to MALDI-TOF mass spectrometry.
A control mixture of synthetic A
1-40 and A
11-40 yielded peaks
of expected mass (4329.27 and 3152.29 Da, respectively; Fig.
4B). Furthermore, both A
1-40 and A
11-40 were readily
detected from untransduced NT2N neurons (4328.56 and 3151.57 Da,
respectively; Fig. 4C) and BACE-overexpressing NT2N neurons
(4329.91 and 3150.94 Da, respectively; Fig. 4D), consistent
with the ELISA data. Other peaks corresponding to C-terminally truncated A
A
1-34, A
1-37, A
1-38, and A
1-39 with
masses of 3784.71, 4076.02, 4132.14, and 4230.10 Da, respectively) were also identified in the mass spectra of neuronal medium (Fig. 4, C and D). However, since these C-terminally
truncated A
peptides contain intact N termini, the increased
concentration detected by BNT77 is primarily due to the presence of
A
11-40/42 in culture media.
View larger version (22K):
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Fig. 4.
BACE expression increases full-length and
N-terminally truncated A secretion.
A, media conditioned for 24 h by NT2
cells, NT2N
neurons (5-week-old replate 2 neurons) and stably transduced subclones
(NT2G7
/NT2B30
/NT2B17
, undifferentiated cells;
NT2G7N/NT2B30N/NT2B17N, 5-week-old replate 2 neurons) were assayed for
A
production by sandwich ELISA. Full-length A
was measured by
Ban50 ELISA, whereas full-length and N-terminally truncated A
was
measured by BNT77 ELISA, shown as mean values corrected for APP
expression ± S.E. from six cultures over three independent
collections. A control mixture of A
1-40 and A
11-40
(B), NT2N conditioned media (C), and NT2B30N
conditioned media (D) were immunoprecipitated with 4G8 and
subjected to MALDI-TOF analysis to identify different secreted A
species. Several peaks corresponding to C-terminally truncated A
peptides, in addition to A
1-40 and A
11-40, are labeled.
--
Previous analysis
of several cell lines indicated that both high expression of APP and
high
-secretase activity were co-requisites for intracellular A
detection (18, 28). We therefore tested whether BACE overexpression
increases intracellular A
in NT2N neurons. Because the BNT77 ELISA
did not have the sensitivity required for accurate intracellular A
quantification, a more sensitive ELISA was utilized in which A
peptides from cell lysates were captured with either JRF/cA
40 or
JRF/cA
42, specific for A
40 and A
42, respectively. Captured
peptides were detected with either JRF/A
N, recognizing A
1-7 (to
measure full-length A
) or m266, recognizing A
13-28 (to measure
full-length and N-terminally truncated A
). BACE overexpression
resulted in the presence of intracellular A
from undifferentiated
NT2B30
and NT2B17
cells, in contrast with NT2
and NT2G7
cells
(Fig. 5). The amount of intracellular
A
from BACE-expressing non-neuronal cells was comparable with the
amount of intracellular A
from untransduced NT2N neurons. Similar to
secreted A
, BACE overexpression resulted in a marked increase in
intracellular A
in NT2N neurons. Furthermore, N-terminally truncated
A
peptides comprised a large proportion of intracellular A
. The
difference between A
concentration as determined by JRF/A
N and
m266 indicated that N-terminally truncated A
is present
intracellularly not only in BACE-overexpressing cells but in
untransduced NT2N neurons as a result of normal metabolism of
endogenous APP.
View larger version (17K):
[in a new window]
Fig. 5.
Intracellular accumulation of truncated
A peptides. A
from NT2
cells, NT2N
neurons (5-week-old replate 2 neurons), and stably transduced subclones
(NT2G7
/NT2B30
/NT2B17
, undifferentiated cells;
NT2G7N/NT2B30N/NT2B17N, 5-week-old replate 2 neurons) was extracted
with RIPA and assayed by sandwich ELISA for intracellular A
.
Full-length A
was measured by JRF/A
N ELISA, whereas full-length
and N-terminally truncated A
was measured by m266 ELISA, shown as
mean values ± S.E. from four to six cultures over three
independent collections. Undifferentiated cultures were corrected for
expression of APP, whereas neuronal cultures were corrected for
expression of neuronal specific enolase.
species, NT2N, NT2B30N, and NT2B17N neurons were
metabolically labeled, and both media and cell lysates were immunoprecipitated with 4G8. Due to the lower sensitivity of 4G8 immunoprecipitation, we overexpressed APP in NT2N neurons to increase the production of A
. As shown in Fig.
6A, overexpression of APP in NT2N neurons resulted in a large increase in secreted A
. However, by using both Ban50 and BNT77 ELISAs, we found that the relative amount
of truncated A
secreted by NT2N neurons upon APP overexpression was
reduced. That is, although 24.5% of A
produced from endogenous APP
is truncated, only 8.7% of A
is truncated upon APP overexpression. Despite this relative decrease in truncated A
, immunoprecipitates from NT2N media revealed three bands (Fig. 6B) corresponding
to A
1-40/42 (upper 4-kDa band), A
11-40/42 (middle 3.2-kDa
band), and p3 (A
17-40/42, lower 3-kDa band). As expected,
A
1-40/42 and A
11-40/42 were increased in a
dose-dependent manner upon BACE expression, consistent with
both the ELISA and mass spectral analysis. Immunoprecipitates from
neuronal lysates demonstrated that full-length A
increased as a
result of BACE expression. Furthermore, A
11-40/42 was also
recovered from BACE-expressing NT2N cell lysates in a
dose-dependent manner, as shown by the presence of a
3.2-kDa band that co-migrated with the A
11-40/42 recovered from
media. Importantly, p3 was not detected from NT2N neuron lysates,
indicating that p3 does not accumulate intraneuronally and that lysates
were not contaminated with media. Thus, although the relative amount of
truncated A
recovered by 4G8 immunoprecipitation was altered by APP
overexpression, these results nonetheless confirm that N-terminal
heterogeneity of A
is part of the intracellular processing pathway
of APP and not solely due to partial degradation of secreted A
peptides.
View larger version (28K):
[in a new window]
Fig. 6.
Secreted and intracellular
A 11-40/42 in NT2N neurons. A,
media conditioned overnight by NT2N neurons (left) or NT2N
neurons transduced with Semliki Forest virus to overexpress APP695
(right) were assayed by Ban50 or BNT77 ELISA to determine
the concentration of either full-length A
(Ban50) or full-length and
truncated A
(BNT77), shown as mean values ± S.E. from six
cultures over two collections. B, NT2N, NT2B30N, and NT2B17N
neurons were transduced with Semliki Forest virus to overexpress APP695
and metabolically labeled for 8 h. Media and RIPA lysates were
immunoprecipitated with 4G8, electrophoresed on a 10/16.5%
discontinuous gradient Tris-Tricine gel, and exposed to a
PhosphorImager screen. Full-length APP, C-terminal APP fragments,
full-length A
1-40/42, truncated A
11-40/42, and p3 are labeled.
The contrast of the bottom of the gel has been enhanced to demonstrate
the increase in A
production due to BACE overexpression. The
contrast was further enhanced (bottom) to demonstrate the
presence of N-terminally truncated A
.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cleavage has been implicated in the pathogenesis of AD, either due
to a pathogenic mutation (K595N/M596L, APPsw) at the
-cleavage site of APP in familial AD (41-43) or by increased BACE expression in sporadic AD (29). Alternatively, other familial AD-associated mutations
in either APP or presenilin increase the production of A
1-42
relative to A
1-40 (1). Despite the predominance of A
1-40 in
cerebral spinal fluid (CSF) (44), A
1-42 is more abundant in senile
plaques (3), consistent with its ability to aggregate more readily than
A
1-40 in vitro (2). A third class of familial AD
mutations, located in the middle of the A
domain, has been
postulated to increase the amyloidogenicity of A
peptides (9).
Interestingly, these mutations also appear to increase the production
of A
11-40/42 (8, 9). Similar to C-terminal extensions to A
,
N-terminal truncations have been shown to reduce solubility although
increasing sedimentation and
-pleated sheet structure of A
peptides relative to full-length A
(45-47). Furthermore,
cyclization of the N-terminal glutamate in A
11-40/42 protects the
peptide from degradation by most aminopeptidases (48). Therefore,
N-terminally truncated A
peptides may accelerate the seeding and
maturation of senile plaques and thus exacerbate the progression of
Alzheimer's disease, particularly in some genetic settings.
peptides are N-terminally
truncated has been unclear. A
peptides beginning at Glu-11 were
first identified from purification of A
peptides from human CSF (37)
and are found in insoluble fractions from AD brain (5, 12). The
discovery of BACE led to the realization that two alternative cleavage
sites are present in the N-terminal region of A
and that
'-cleavage is species-specific (10, 23). Therefore, although rodent
neuronal cells preferentially cleave endogenous APP at position 11 (22, 49), overexpression of human APP in rodent cells does not result in the
formation of human A
11-40/42 (22). Many modified A
peptides
accumulate in brains of tg2576 mice, a transgenic mouse model
overexpressing APPsw, including isomerized Asp-1
(L-iso-Asp), stereoisomerized Asp-1 (rectus Asp), and
pyroglutaminated Glu-3. However, pyroglutaminated Glu-11 is
conspicuously absent from tg2576 brains (50). Therefore, although many
of the mechanisms for N-terminal modification of human A
are
present, the generation of A
11-40/42 is currently missing from both
rodent cell culture and transgenic models. Given the limitations of
rodent models, we investigated the generation of A
11-40/42 in human
NT2N neurons. We found that the secretion of sAPP and A
correlates
with the expression of BACE that occurs upon neuronal differentiation. The increased secretion of sAPP was predominantly due to the secretion of sAPP
and sAPP
'. Additionally, NT2N neurons produce the
N-terminally truncated A
11-40/42 from normal metabolism of
endogenous APP. Furthermore, exogenous BACE expression increased the
secretion and intracellular generation of both A
1-40/42 and
A
11-40/42. Interestingly, increasing APP expression decreased the
relative amount of truncated A
produced by NT2N neurons. In
contrast, non-neuronal cells with higher levels of BACE overexpression
than reported here resulted in the preferential generation of
N-terminally truncated A
over full-length A
(11, 12). Taken
together, the ratio of APP to BACE expression may dictate the extent of
'-cleavage. Regardless, the intracellular generation of
A
11-40/42 from normal APP processing in NT2N neurons indicates that
this N-terminally truncated peptide is generated prior to deposition into insoluble aggregates in AD.
peptides were recovered from NT2N neuron medium with
truncated C termini. These C-terminally truncated A
species are also
found in human CSF (51) and AD brain homogenates (5, 12), indicating
that they may also contribute to amyloid formation. The close
correlation between A
peptides found in NT2N neuronal medium and
human CSF further validates the NT2N neuronal culture system as a
useful model to study the generation of N- and C-terminally truncated
A
peptides. Immunoprecipitation of intracellular A
from
BACE-expressing NT2N neurons yielded a faint band slightly smaller than
full-length A
(see Fig. 6). Although obscured somewhat by the
intense signal derived from full-length A
, this truncated A
peptide appeared to be present in NT2N media samples, indicating that
it corresponds to one of the C-terminally truncated A
peptides identified by mass spectrometry. APP and presenilin mutations that are
known to affect C-terminal
-secretase cleavage also result in the
increased accumulation of N-terminally truncated A
peptides (5, 6),
indicating that
- and
-secretase cleavage may influence each
other. Importantly, the magnitude of the increase in A
production
upon BACE expression indicates that the level of endogenous BACE
expression in NT2
cells and NT2N neurons is rate-limiting in terms of
A
generation. Although
-secretase activity was not addressed
directly in these experiments, the modest effect of BACE expression in
non-neuronal NT2
cells compared with the effect of BACE expression in
NT2N neurons indicates that
-secretase cleavage is enhanced in neurons.
from neuronal cells is high relative to
non-neuronal cells, the concentration of A
in human CSF is below the
threshold for A
aggregation in vitro (2). The stability
and insolubility of intraneuronal A
lead to the hypothesis that
intracellular A
may be the source of A
aggregates that seed
senile plaques. Indeed, insoluble A
accumulates in NT2N neurons with
age in culture (28), and SDS-stable oligomeric A
is found
intracellularly prior to secretion (52). Furthermore, prior to the
presence of amyloid pathology, A
can be detected biochemically from
tg2576 mice (50) and patients with early cognitive dysfunction (53).
Intracellular A
has been found in affected brain regions in AD
brains (54-56) and in animal models of AD amyloid pathology (57-60).
Finally, mRNA isolated from senile plaques is predominantly
neuronal (61). These reports suggest that the nidus for senile plaque
formation may be intraneuronal A
. Interestingly, although various
N-terminally truncated A
species, including A
11-40/42, are
readily detected from detergent-insoluble preparations from AD brain,
p3 is not detected (5). p3 is a major component of diffuse plaques in
AD (62, 63) and in diffuse plaques in the cerebellum of Down's
syndrome patients (64). However, cerebellar diffuse plaques of Down's
syndrome patients do not progress to form neuritic senile plaques even
though in vitro studies of the p3 peptide indicate that it
is highly hydrophobic, capable of forming fibrils, and has the
tinctoral properties of amyloid as determined by thioflavin T and Congo
Red staining (64). We could not detect intracellular p3 from NT2N
neurons, consistent with the generation of p3 at or near the plasma
membrane (65, 66). These observations are also consistent with the
hypothesis that the intracellular environment is necessary to convert
fibrillogenic A
peptides into a nidus for senile plaque formation.
peptides. The trans-Golgi network produces predominantly A
1-40 (67, 68), although the endoplasmic reticulum/intermediate compartment produces A
1-42 (28, 31, 68, 69). Endoplasmic reticulum/intermediate compartment-derived A
1-42 is not secreted but rather is retained intracellularly and contributes to the accumulation of a pool of insoluble A
that can be recovered with formic acid. Unfortunately, the sandwich ELISAs used in this study either do not have the sensitivity (BNT77) or are incompatible (JRF/A
N and m266) with formic acid lysates. However, the increased production of intracellular A
upon BACE expression is expected to
increase the accumulation of insoluble full-length and truncated A
peptides. Production of endoplasmic reticulum/intermediate compartment-derived A
is independent of presenilin, indicating that
multiple
-secretases may responsible for
-secretase cleavage in
different subcellular organelles (15). In contrast, BACE-deficient untransduced NT2
cells do not have appreciable levels of
intracellular A
, although BACE overexpression increases
intracellular A
. Therefore, BACE appears to be responsible for both
secreted and intracellular A
. The extent of
'-cleavage is also
dependent on the subcellular localization of BACE and APP in 293 cells
(12). The engineering of BACE-overexpressing NT2N neurons allows for
future investigations into the subcellular site of A
11-40/42
generation in neuronal cells. However, the downstream effect of
A
11-40/42 generation on plaque formation awaits the engineering of
transgenic mice co-expressing human BACE and human APP.
generation in NT2N neurons than in non-neuronal NT2
cells. Therefore,
even modest increases in BACE expression may precipitate amyloid
formation due to overproduction of A
. Conversely, mild inhibition of
BACE activity may have a large effect on A
generation, underscoring
the possibility of using BACE inhibitors as a therapy for AD. However,
given the endogenous production of A
11-40/42 by human NT2N neurons,
the effect of BACE inhibitors on both full-length and N-terminally
truncated A
peptides needs to be determined.
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ACKNOWLEDGEMENTS |
---|
We gratefully thank Takeda Pharmaceutical,
Janssen Pharmacia, and Lilly for providing monoclonal antibodies for
the A sandwich ELISA. We thank K. N. Liu and A. Crystal for
critical reading and suggestions in the preparation of this manuscript
and Dr. J. Huse and Dr. C. Wilson for valuable discussions. We thank
C. D. Page, J. Bruce, and C. Li for assistance with cultured cells and lentivirus production. We are grateful to Dr. L. J. Chang, Dr.
G. Kobinger, Dr. D. Watson, and Dr. J. H. Wolfe for providing transfer plasmids for lentivirus production.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Training Grants T32 AG00255 (to E. B. L.) and NIA AG11542 (to R. W. D. and V. M.-Y. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a Paul Beeson Faculty Scholar award.
¶ John H. Ware III professor of Alzheimer's research. To whom correspondence should be addressed: Center for Neurodegenerative Disease Research, Dept. of Pathology and Laboratory Medicine, Maloney 3, HUP, Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909; E-mail: vmylee@mail.med.upenn.edu.
Published, JBC Papers in Press, December 11, 2002, DOI 10.1074/jbc.M210105200
2 E. B. Lee and V. M.-Y. Lee, unpublished data.
3 Watson, D. J., Longhi, L., Lee, E. B., Fulp, C. T., Fujimoto, S., Royo, N. C., Passini, M. A., Trojanowski, J. Q., Lee, V. M.-Y., McIntosh, T. K., and Wolfe, J. H. (2003) J. Neuropath. Exp. Neurol., in press.
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ABBREVIATIONS |
---|
The abbreviations used are:
AD, Alzheimer's
disease;
A, amyloid-
peptide;
A
1-40, A
1-42, 40- and
42-amino acid forms of A
respectively;
A
11-40, A
11-42,
N-terminally truncated A
peptides starting at position Glu-11;
ADAM, a disintegrin and metalloprotease;
APP, amyloid-
precursor protein;
APP695, 695-amino acid isoform of APP;
APP751/770, 751- and 770-amino
acid isoforms of APP;
BACE,
-site APP-cleaving enzyme;
C83,
-secretase derived C-terminal fragment of APP;
C89,
-secretase
derived C-terminal fragment beginning at Glu-11;
C99,
-secretase
derived C-terminal fragment beginning at Asp-1;
ELISA, enzyme-linked
immunosorbent assay;
FBS, fetal bovine serum;
GFP, green fluorescent
protein;
NT2
, undifferentiated embryonal carcinoma NTera2/c1.D1;
NT2N, differentiated neuron derived from NTera2/c1.D1;
PMA, phorbol
12-myristate 13-acetate;
RIPA buffer, radioimmune precipitation assay
buffer;
sAPP, total secretase-derived N-terminal ectodomain of APP;
sAPP
,
-cleavage-derived N-terminal ectodomain of APP;
sAPP
,
-cleavage-derived N-terminal ectodomain of APP;
sAPP
',
'-cleavage-derived N-terminal ectodomain of APP;
TAPI, (N-R-(2-hydroxyaminocarbonyl)methyl)-4-methylpentanoyl-L-naphthylalanyl-L-alanine
2-aminoethyl amide;
Tricine, N-[2-hydroxyl-1,1-bis(hydroxymethyl)ethyl]glycine;
VSV-G, vesicular stomatitis virus surface glycoprotein;
DMEM, Dulbecco's
modified Eagle's medium;
MALDI-TOF, matrix-assisted laser desorption
ionization/time of flight;
CSF, cerebral spinal fluid.
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