Inhibition of amyloid precursor protein processing
by ?-secretase through site-directed antibodies
Michal Arbel*, Iftach Yacoby*, and Beka Solomon†
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel
Communicated by Ephraim Katchalski-Katzir, Weizmann Institute of Science, Rehovot, Israel, March 23, 2005 (received for review September 26, 2004)
Amyloid-? peptide (A?P) that accumulates in the Alzheimer’s
diseased brain is derived from proteolytic processing of the amy-
loid precursor protein (APP) by means of ?- and ?-secretases. The
?-secretase APP cleaving enzyme (BACE), which generates the N
terminus of A?P, has become a target of intense research aimed at
blocking the enzyme activity, thus reducing A?P and, subse-
quently, plaque formation. The search for specific inhibitors of
?-secretase activity as a possible treatment for Alzheimer’s disease
intensified with the discovery that BACE may be involved in
processing other non-APP substrates. The presence of the APP–
BACE complex in early endosomes highlights the cell surface as a
potential therapeutic target, suggesting that interference in APP–
BACE interaction at the cell surface may affect amyloid-? produc-
tion. We present here a unique approach to inhibit A?P production
by means of antibodies against the ?-secretase cleavage site of
APP. These antibodies were found to bind human APP overex-
pressed by CHO cells, and the formed immunocomplex was visu-
alized in the early endosomes. Indeed, blocking of the ?-secretase
both intracellular and extracellular A?P formation in these cells.
Alzheimer’s disease ? ?-secretase site ? monoclonal antibodies ?
amyloid ?-peptide production ? endocytic pathway
intraneuronal accumulation of neurofibrillary tangles. The se-
nile plaques are composed of deposited amyloid-? peptides
(A?Ps), which are derived from the enzymatic processing of a
type I transmembrane protein called amyloid precursor protein
(APP) (1). The ?-secretase APP cleaving enzyme (BACE)
generates the N terminus of the A?P peptide and produces a
membrane-bound C-terminal fragment (CTF), C99. This mem-
brane-bound product serves as a substrate for ?-secretase com-
plex processing, which releases amyloid peptides of 40 or 42 aa.
Pharmacologic and cell biology studies demonstrated that the
three major enzymatic activities involved in APP processing, ?-
?- and ?-secretases, are distinct in their subcellular localization
and in their respective cleavage products (2, 3). It was shown that
?-secretase activity must reside both in the endosomes (4) and
in the secretory pathway (5). Antibody uptake and biotinylation
studies showed that most cell surface-located BACE is reinter-
nalized into the early endosomal compartments, from where it
can recycle back to the cell surface or can later be retrieved to
endosomal?lysosomal compartments and?or to the trans-Golgi
network (6, 7). The endocytic pathway, responsible for internal-
ization and initial processing of cell surface APP in endosomes,
is well established (4, 8–10). Indeed, the mutagenesis of the APP
internalization signal (11) and expression of the dominant-
negative dynamin mutant that prevents endocytosis in the trans-
fected cells (10) reduced both A?P 40 and A?P 42 secretion
levels. Recent morphological evidence from living cells tests the
and in early endosomes. Free resonance energy transfer analysis
showed that there is a strong and previously unemphasized
interaction at the cell surface where APP and BACE dramati-
cally colocalize and appear to be internalized together after 15
lzheimer’s disease (AD) is characterized by the accumula-
tion of senile plaques in the brain extracellular space and by
min into early endosomes (12). Colocalization of APP and
BACE in early endosomes highlights the cell surface as an
additional potential site for APP–BACE interaction. All these
studies suggest that A?P is derived through processing of APP
endocytosed from the cell surface in addition to the secretory
We report here the preparation of a specific mAb against the
?-secretase cleavage site of APP (blocking ?-site 1, mAb BBS1).
levels in CHO cells overexpressing human APP, whereas unrelated
antibodies directed to the N-terminal of APP had no effect.
Materials and Methods
All animals were treated according to the regulations of the
Animal Care and Use Committee of Tel Aviv University.
Antigen Preparation. The antigen used in this study for immuniza-
tion mimics the ?-secretase cleavage site of APP. The ?-secretase
cleavage site, which resides between amino acids 663 and 671, is
highly conserved through evolution, whereas the double Swedish
of KM (amino acids 670–671). To overcome the poor immunoge-
nicity of short peptides and the tolerance against self-antigens, we
of peptides that mimic the WT APP ?-secretase cleavage site
(MAP-[ISEVKMDA]8) as well as the half-Swedish mutation
Immunization Protocol. BALB?c mice, 8 weeks old and weighing
20–30 g, were challenged with MAP displaying eight copies of
either the half-Swedish mutation or the WT sequence of the
?-secretase cleavage site. Mice were immunized with 100 ?g of
14-day intervals. Blood samples were drawn before the first immu-
and analyzed for IgG levels against MAP-[ISEVKMDA]8express-
ing the WT sequences of the ?-secretase cleavage site of APP.
Evaluation of IgG Levels by ELISA. Microtiter plates (Nalge Nunc)
were coated with 0.1 ?g per well of MAP-[ISEVKMDA]8, which
mimics the WT ?-secretase cleavage site of APP [diluted in 0.1 M
Na2CO3(pH 9.6)] and incubated overnight at 4°C. The plates were
washed with PBS (0.05% Tween 20) and then blocked with 3%
BSA?PBS for 1 h at 37°C. Serial sera dilutions were added for an
additional hour at 37°C, and the sera end-point titer, defined as the
search) followed by O-phenylenediamine (Sigma). After the redox
Abbreviations: A?, amyloid-?; A?P, A? peptide; AD, Alzheimer’s disease; APP, amyloid
precursor protein; BACE, ?-secretase APP cleaving enzyme; BBS1, blocking ?-site 1; EEA1,
early endosome antigen 1; MAP, multiple antigenic peptide; MTT, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide; CTF, C-terminal fragment.
*M.A. and I.Y. contributed equally to this work.
†To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
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reaction was stopped by using 4 M HCl, substrate degradation was
monitored by absorption at 492 nm.
Generation of a mAb Against the ?-Secretase Cleavage Site on APP.
Immunized mice with the highest titer against MAP conjugated to
the WT sequence of the ?-secretase cleavage site (MAP-
[ISEVKMDA]8) were killed, and lymphocytes isolated from their
spleens were fused with NSO myeloma cells as described for
hybridoma technique (13). The generated clones were screened for
MAP-[ISEVKMDA]8binding by ELISA as described above. Pos-
itive clones were isolated and further analyzed for binding of
full-length WT APP as expressed by CHOhAPP751 cells by using
immunofluorescence techniques as detailed below. The selected
both on the cell surface and inside the cell and was chosen for
Specificity of mAb BBS1 Binding. mAb BBS1 purification from
ascetic fluids was performed by protein G affinity chromatography
The antibody ability to bind MAP-[ISEVKMDA]8 at different
concentrations was analyzed by using ELISA as described above.
Antibody specificity was analyzed by a competitive ELISA.
Decreasing concentrations of MAP-[ISEVKMDA]8(0.125 mM to
29 pM) were preincubated for 1 h at room temperature with mAb
BBS1 (13.3 nM). The immunocomplex was then added to a
microtiter plate precoated with 0.1 ?g per well of MAP-
mAb BSS1 was incubated with nonrelevant MAP expressing eight
copies of a prion antigen.
Stability of the Immunocomplex at Different pH Values. The stability
of the mAb BBS1 and MAP-[ISEVKMDA]8immunocomplex was
evaluated at different pH values in the range of 3–7. Microtiter
plates were coated with 0.1 ?g per well of MAP-[ISEVKMDA]8as
described above. After blocking, mAb BBS1 (13.3 nM) diluted in
in triplicate for an additional hour at 37°C. Antibody ability to bind
pH conditions was tested by incubation of the antigen-coated wells
with buffers at different pH values.
Antibody Recognition of the ?-Secretase Cleavage Site. Cellline.CHO
cells stably transfected with WT human APP 751 isoform
(CHOhAPP751) were kindly provided by D. Selkoe (Harvard
Medical School, Boston). Cells were grown in DMEM (F-12)
containing 10% FCS and 2.5 mM L-glutamine. hAPP 751-express-
cells growing medium.
Western blot. Lysates extracted from CHOhAPP751 cells were used
for Western blot analysis. Cells were lysed with ice-cold Triton-doc
lysis buffer (0.5% Triton X-100?0.25% Na-deoxycholate?150 mM
NaCl?10 mM Tris?HCl, pH 7.5?10 mM EDTA) and then centri-
fuged at 21,000 ? g for 1 min. Supernatants were collected,
incubated for 20 min on ice, subjected to 10% SDS?PAGE, and
then blotted onto nitrocellulose membrane (Schleicher & Schuell).
Tween 20), was further incubated overnight with different concen-
trations of mAb BBS1 (6.6–26.6 nM) and mAb AMY33 (20 nM,
Zymed) that bind APP in the midregion of amyloid-? (A?).
Anti-mouse IgG horseradish peroxidase-conjugated secondary an-
tibody was added for 45 min after the membrane was thoroughly
washed. Blots were developed by using the enhanced chemilumi-
nescence system according to the manufacturer’s instructions
Cell immunofluorescence. CHOhAPP751 cells (2 ? 105) were seeded
on coverslips in 24-well plates. At ?80% confluence, cells were
washed twice with PBS and fixed with 4% paraformaldehyde (in
X-100 in PBS for 2 min. After washes with PBS, cells were blocked
with 10% normal goat serum in 3% BSA for 30 min and incubated
with mAb BBS1 (80 nM) for 1 h, followed by an additional hour of
incubation with Cy2-conjugated goat anti-mouse IgG (Jackson
ImmunoResearch). After being thoroughly washed with PBS, cells
were mounted by using Prolong Antifade (Molecular Probes).
Antibody Internalization into the Cell. The antibody internalization
At 80% confluence, mAb BBS1 (13.3 nM) was added to the cell
medium. Cells were fixed and permeabilized as mentioned above
after 30, 60, or 90 min of incubation with the antibody. After cell
blocking, rabbit anti-early endosome antigen 1 (EEA1) polyclonal
and rabbit anti-EEA1 were visualized by the addition of both
Cy2-conjugated goat anti-mouse IgG and Cy3-conjugated goat
anti-rabbit IgG, respectively, for 45 min.
Antibody Interference with A?P Production. CHOhAPP751 cells
(2.5–4 ? 106) were seeded in six-well plates. At 100% confluence,
cells were washed twice with PBS and administered with sera-free
media consisting of mAb BBS1 (13.3 nM), rabbit anti APP N-
terminal antibodies (residues APP 46–60) (13.3 nM, Sigma),
and?or 100 ?M chloroquine, which is known to inhibit cell endo-
cytosis. The basal level of A?P was monitored in cells treated with
each treatment. For extracellular A?P evaluation, media was
collected after 3, 9, and 24 h of incubation, and cells were further
incubated for an additional 4 days. Cells were then collected from
each well by using a cell scraper, centrifuged at 3,000 ? g for 2 min,
washed with PBS, and resuspended in 100 ?l of 70% formic acid,
followed by 10-s sonication. The solution was then centrifuged at
100,000 ? g for 20 min at 4°C to remove insoluble material, and
supernatant was collected and neutralized with 1.9 ml of 1 M Tris
(pH 9). All samples were analyzed for their protein concentration
by using Bradford reagent (Bio-Rad) and aligned for their protein
content before evaluation of A?P levels. One of six repeats from
each treatment group was used for 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) viability assay (see below).
were quantified by using a sandwich ELISA. The monoclonal
anti-A?P antibody AMY-33, used as the capture antibody, was
added to ELISA plates [0.23 ?g per well diluted in 0.1 M Na2CO3
(pH 9.6)] and incubated overnight at 4°C. The plates were washed
at 37°C. Media samples (40, 15, and 5 ?l collected after 3, 9, and
24 h, respectively) and equilibrated protein extracts from each
treatment were applied in triplicate and incubated overnight at 4°C
Biotinylated monoclonal anti-A?P antibody 196 (76 ng per well),
which recognizes the sequence 3–6 of A?P, and biotinylated mAb
8G7 (75 ng per well, Calbiochem) were used for detection of total
A?P and A?P42, respectively. Antibodies were diluted in 1%
BSA?PBS and incubated for 3 h at 37°C. Plates were thoroughly
washed with PBS (0.05% Tween 20) and administered with extra-
avidin-conjugated alkaline phosphatase (Sigma) for 1 h at 37°C.
The substrate p-nitrophenyl phosphate (Sigma) was used to mon-
42 (0.1–1 ng per well, Bachem) were used for construction of
C99 analysis by metabolic labeling.BecausethelevelsofC99produced
by CHOhAPP751 are below detection limits, evaluation of treat-
Arbel et al.
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ment ability to reduce C99 levels was performed in 100-mm-
combined from four identical plates. CHOhAPP751 cells were
grown to 100% confluence, washed with PBS, and administered
with sera-free media in the absence of methionine. After 30-min
starvation, cells were labeled with 125 ?Ci?ml (1 Ci ? 37 GBq)
[35S]methionine and administered with mAb BBS1 (13.3 nM) for
4 h. Labeled cells alone under the same conditions were used for
evaluation of the basal level of C99. Cells were then harvested and
lysed by using 0.05 M Tris?HCl, pH 8.0?0.15 M NaCl?0.005 M
EDTA containing 1% Nonidet P-40 and 200 ?g?ml PMSF. Ex-
tracted proteins were immunoprecipitated with anti-APP C-
terminal antibody (Calbiochem) and 50 ?l of protein G magnetic
beads (Dynal, Great Neck, NY). The immunocomplexes were
achieved by gel incubation with 2,5-diphenyloxazole (20% in acetic
acid, Sigma) for 1 h, after which the gel was dried and the
radioactive signal collected. C99 density was evaluated by using
IMAGEMASTER densitometry software (Amersham Pharmacia).
s?APP levels analysis by Western blot. Culture media (30 ?l) was
collected from cells treated with mAb BBS1, as well as from
untreated cells after 24 and 48 h, and electrophoresed as described
above for Western blot analysis. Visualization of s?APP was
achieved by using mAb 196, which binds amino acids 3–6 of A? (1
?g?ml) and thus binds s?APP and not s?APP.
MTT viability assay. CellviabilitywasmeasuredbycolorimetricMTT
each treatment group after 5 days of incubation was washed twice
with PBS and administered with fresh sera-free media containing
1 mg?ml MTT reagent (Sigma) for 2 h at 37°C. The dark-blue
solution [20% SDS (wt?vol) dissolved in 50% dimethylformamide
(pH 4.7)] and an overnight incubation at 37°C. The colorimetric
reaction was measured by absorption at 570 nm.
Generation of Immune Response Against the ?-Secretase Cleavage
Site of APP. Mice were immunized with MAP displaying eight
obtained was low after five immunizations, as can be expected for
a self-antigen. To stimulate the mice’s immune systems against the
of mice with MAP displaying eight copies of the partial Swedish
mutation that resides at the same site (M670L). The immunoreac-
tivity of sera isolated from the two groups of immunization was
analyzed against MAP displaying the WT epitope by using ELISA.
The one amino acid substitution, using MAP displaying the half-
Swedish mutation, enabled the generation of a high antibody titer
in a short period. High levels of IgG were evident after the first
immunization with MAP displaying half of the Swedish mutation,
reaching an end-point titer, defined as the maximal sera dilution in
immunization (Fig. 1A).
Generation of a mAb Against the ?-Secretase Cleavage Site of APP.
Mice with the highest antibody levels were killed, and their spleens
were used for preparation of monoclonal antibodies by the hybrid-
oma fusion technique. A series of mAbs was obtained by screening
the WT sequence of the ?-secretase cleavage site. mAb BBS1,
which showed the higher affinity in binding MAP expressing the
WT sequence of the ?-secretase cleavage site (MAP-[ISEVK-
MDA]8), was chosen for further analysis. The antibody ability to
immunized with MAP expressing eight copies of the partially Swedish mutated BACE cleavage site of APP (MAP-[ISEVKLDA]8). Sera fractions of immunized mice
were analyzed for their anti-MAP-[ISEVKMDA]8IgG levels displaying the WT sequence of the ?-secretase site of APP. Black bars, first immunization; gray bars,
second immunization; white bars, third immunization. Spleens were isolated from mice with the higher titers, and mAb was prepared by the hybridoma
technique. (B) mAb BBS1 binding properties. mAb BBS1’s ability to bind MAP-[ISEVKMDA]8was analyzed by ELISA. Percentage of the maximal antibody binding
is presented in different antibody concentrations. (C) Competition between soluble antigen (MAP-[ISEVKMDA]8) and antigen adsorbed to ELISA plates for mAb
BBS1 binding. Continuous line, binding of mAb BBS1 after incubation with increasing concentration of MAP-[ISEVKMDA]8; dashed line, antibody binding after
by ELISA after 1 h of incubation at different pH values as described in Materials and Methods. Diamonds, pH effect on mAb BBS1 antigen binding; squares, pH
effect on the coated antigen.
Generation and in vitro characterization of mAb BBS1 raised against the ?-secretase cleavage site of APP. (A) Immune response in BALB?c mice
www.pnas.org?cgi?doi?10.1073?pnas.0502427102Arbel et al.
bind MAP-[ISEVKMDA]8was analyzed by ELISA, showing an
IC50of 1.165 nM (Fig. 1B). mAb BBS1 was also analyzed for its
ability to bind A?Ps (A?P 1–16, A?P 40, and A?P 42) by using
ELISA. In all cases described, no immunoreactivity was detected
(data not shown).
Antibody specificity to MAP-[ISEVKMDA]8was confirmed in
a competitive ELISA performed after preincubation of increasing
antigen concentration with a constant amount of mAb BBS1 (Fig.
1C). Although antibody ability to bind the precoated plate was
reduced after incubation with the antigen in solution in a dose-
dependent manner, preincubation with nonrelevant MAP failed to
affect antibody binding.
Because optimal ?-secretase activity requires a mildly acidic pH
(?5), we tested the immunocomplex stability under a pH range of
3–7. At pH 5, mAb BBS1 retains 95% of its ability to bind the
antigen, and 20% of its binding abilities are still evident after 1 h of
incubation at pH 3 (Fig. 1D).
mAb BBS1 Recognizes Full-Length APP. Because mAb BBS1 was
generated against a small peptide from APP sequence, it was
essential to assure that the antibody binds full-length APP. The
ability of the antibody to bind the WT ?-site of full-length APP was
analyzed in the CHOhAPP751 cell line expressing high levels of
human WT APP by Western blot (Fig. 2Ai) and cell immunoflu-
orescence (Fig. 2Aii). In the Western blot analysis, mAb BBS1
specificity is demonstrated in a concentration-dependent manner.
mAb BBS1, generated against half of the Swedish mutated ?-site
sequence, recognizes the double Swedish mutated APP in Western
Mayo Clinic, Jacksonville, FL)] (data not shown). In the immuno-
fluorescence assay, cells were fixed and permeabilized, after which
mAb BBS1 was administered and visualized by using a Cy2-
conjugated secondary antibody. Antibody reactivity was evident at
the cell surface and in the perinuclear areas, such as the endoplas-
mic reticulum and Golgi apparatus, as well as in other cell com-
Because most ?-secretase activity is localized intracellularly, we
tested antibody ability to cointernalize into the early endosomes
the cell was evident after 60 min (Fig. 2B). Fig. 2B Upper, which is
mAb BBS1, as can be expected after APP internalization by means
evident in other intracellular areas.
mAb BBS1 Interferes with A?P Production. The effect of immuno-
complexation of mAb BBS1 with APP on A?P production was
measured by using 100% confluent CHOhAPP751 cells in the
presence of mAb BBS1 in sera-free media. Anti-N-terminal poly-
clonal antibodies were used as a negative control and chloroquine,
which is known to inhibit endocytosis and thus reduce A?P levels,
served as a positive control. Untreated cells were used to measure
the basal levels of A?P. Media were collected after 3, 9, and 24 h,
as described in Materials and Methods. These time points were
chosen, because the relatively high A?P concentration in growing
media is measurable above detection limits. The percentage of
secreted A?P in each treatment group compared with A?P se-
creted by the untreated group at the three time points measured is
shown in Fig. 3A. An ?13% reduction in secreted A?P levels was
evident after 3 h of incubation with mAb BBS1, reaching 22%
within 9 h. After 24 h, only 16% reduction was measured, probably
a result of antibody consumption. Anti-N-terminal polyclonal an-
tibodies had no significant effect on A?P production at any of the
measured time points. Chloroquine, as can be expected, displayed
the most profound effect on A?P levels; however, the reduction is
not only a result of endocytosis inhibition, but also of massive cell
After 5 days of incubation, cells were lysed with 70% formic acid
and sonicated. Fig. 3B shows the percentage of intracellular A?P
compared with that of the untreated group. A dramatic reduction
of ?50% is evident in cells treated with mAb BBS1 for 5 days.
reduction of 60% in the intracellular levels; however, by the time of
measurement (5 days), most of the chloroquine-treated cells had
A?P levels observed in the mAb BBS1 treatment group is not the
result of a lower number of cells producing A?P, as evident in the
chloroquine-treated cells, one repeat of the experiment (of six
change in viability is evident in the untreated and mAb BBS1-
The sandwich ELISA described here measures total A?P levels
cells. (A) Antibody ability to bind APP expressed by CHOhAPP751 is demon-
strated by Western blot (i) and immunofluorescence (ii). (i) CHOhAPP751 cells
were lysed, electrophoresed, and transferred to a nitrocellulose membrane.
mAb BBS1 was applied at different concentrations, and mAb AMY-33, which
binds the midregion of A?, was used as a positive control (left). (ii) Immuno-
labeling of cells with mAb BBS1. Antibody binding is detected by using a
Cy2-conjugated secondary antibody and visualized by using an LSM-510 Zeiss
CHOhAPP751 cells were administered with mAb BBS1 in the growing media
and incubated with the antibody for 60 min. After incubation with the
antibody, cells were fixed and permeabilized, and antibody presence inside
the cells was detected by using Cy2 secondary antibody (Left). Cells were
counterstained for early endosomes by using rabbit anti-EEA1 after Cy3
secondary antibody (Center), and the superposition is demonstrated in Right.
Upper and Lower represent two different fields at different magnitudes.
(Scale bar, 10 ?m.) Cell labeling was visualized by using an LSM-510 Zeiss
Antibody ability to bind full-length APP expressed by CHOhAPP751
Arbel et al.
May 24, 2005 ?
vol. 102 ?
no. 21 ?
for both secreted and intracellular A?P. Results obtained from
samples showed that most of the secreted A?P is A?P 40, whereas
reported by others (15, 16).
of the corresponding membrane-bound CTF, C99. For that pur-
pose, we analyzed the levels of C99 in mAb BBS1-treated cells
thionine. After 4 h of treatment with the antibody, the decrease in
C99 levels was estimated as 20% (Fig. 3C). Reduction in C99 levels
levels observed after 3 h of incubation with the antibody (Fig. 3A).
The most profound effect on A?P levels was evident by examining
However, evaluation of C99 by means of metabolic labeling could
not be performed for such a long period because, at later time
points (i.e., 12 h), cells were beginning to die in the two examined
groups, probably due to lack of methionine.
To evaluate whether blocking the ?-secretase site of APP, and
thus inhibiting A?P production, enhances ?-processing, s?APP
levels were analyzed. No significant change in s?APP levels was
observed between the untreated and mAb BBS1-treated cells after
both 24 and 48 h of incubation (Fig. 3E).
Altered processing of the APP is considered a major event in the
pathogenesis of AD, but what accelerates amyloidogenesis in
sporadic AD has not been identified. The A?P that accumulates
in Alzheimer’s diseased brain is derived from proteolytic pro-
cessing of the APP by means of ?- and ?-secretases through the
secretory and endocytic pathways. BACE, which generates the N
terminus of A?P, has become a main target of intense research
aimed at blocking the enzyme activity and, thus, production of
A?P toward inhibition of amyloid plaque formation (17–19).
However, recent evidence has shown that BACE may process
some non-APP substrates (20–22) and that other putative
?-secretases may be involved in APP cleavage, mainly in spo-
radic AD (23, 24). In this study, we describe a unique approach
to inhibit the mainly endocytic pathway of ?-secretase activity
based on blocking the ?-secretase cleavage of APP by site-
We generated a specific mAb against the ?-secretase cleavage
site of APP. Specificity of the antibody was established in a
competitive ELISA, as well as by Western blot analysis and immu-
nofluorescence, by using CHO cells expressing WT human APP.
The immunocomplex was found to remain 95% stable within pH 5,
the common pH in the ?-secretase environment (e.g., endosomes).
mAb BBS1, raised against the ?-site of APP, cointernalized with
and reduced the extra- and intracellular A?P levels in CHO cells
overexpressing human APP. The intracellular levels of A?P were
dramatically reduced compared with the basal level of A?P pro-
duced by these cells after 5 days of incubation with the antibody.
The secreted A?P levels were reduced by 22% after 9 h. CTF-?
was examined in the following way: CHOhAPP751 cells were administered with mAb BBS1, anti-APP N-terminal polyclonal antibodies, or chloroquine
diluted in sera-free media. Untreated cells were used to measure A?P basal level. (A) Secreted A?P levels were measured from the growing media at
different time points (white bars, 3 h; black bars, 9 h; gray bars, 24 h), and the ratio between secreted A?P in each treatment group and the untreated
group was calculated and presented in percentage. The experiment was repeated six times for each group. (B) Intracellular A?P measurements were
performed after 5 days of incubation. The ratio between the intracellular A?P levels in each treatment group and in the untreated group was calculated
and presented in percentage (black bar, untreated cells; white bar, mAb BBS1-treated cells; gray bars, anti-N-terminal antibody and chloroquine-treated
cells). The experiment was repeated five times for each group. (C) CTF? levels were measured after 4 h of metabolic labeling and immunoprecipitation
estimated a 20% reduction of CTF? levels in mAb BBS1-treated cells. (D) MTT cell viability assay was performed after 5 days of incubation (black bar,
untreated cells; white bar mAb BBS1-treated cells; gray bars, anti-N-terminal antibody and chloroquine-treated cells). (E) The levels of s?APP released to
mass of ?100 kDa in the supernatant of mAb BBS1-treated cells (?) as well as in that of untreated cells (?).
Inhibition of extra- and intracellular A?P accumulation. The ability of mAb BBS1 to interfere with the APP processing and thus reduce A?P levels
www.pnas.org?cgi?doi?10.1073?pnas.0502427102 Arbel et al.
levels,after4hoftreatmentwithmAbBBS1,werereducedby20%, Download full-text
in accordance with the 13% decrease observed in secreted A?P
after 3 h. It is worth mentioning that antibodies directed to the N
extra- and intracellular levels of A?P.
The ability of BBS1 antibody to interfere with A?P production
and thus with the disease progression in vivo is under investigation
by using AD transgenic mice models expressing either the WT or
Swedish mutated ?-site of APP.
We performed active immunization feasibility studies in a small
number of 8-month-old AD transgenic mice (Tg 2576, Taconic
Farms). Mice were immunized with the described antigen exhibit-
test compared with saline-treated mice (n ? 4). Moreover, brain
biochemistry and immunohistochemistry analysis demonstrated a
remarkable reduction in total A?P levels and plaque load, respec-
tively (Fig. 4, which is published as supporting information on the
PNAS web site). These data suggest that the generated antibodies
against the ?-secretase cleavage site do cross the blood–brain
the soluble A?P levels.
Experimental evidence that peripheral antibodies cross the
administered with anti-A? antibodies, and not in those adminis-
tered with a control IgG, antibodies labeled the remaining congo-
philic amyloid plaques in the brain. Seabrook et al. (26) also
reported immunohistochemical labeling of amyloid deposits for
paraffin-embedded tissues, confirm the observations of Bard et al.
(27), who used cryosections in demonstrating the ability of anti-A?
antibodies administered peripherally to enter the CNS, decorate
preexisting amyloid plaques, and mediate their clearance (27).
Despite the fact that AD pathogenesis is classically characterized
by the extracellular accumulation of A? peptides, the extent to
which those insoluble fibrils contribute to the neuronal death is still
unclear. Support for the intracellular origin of the extracellular A?
accumulates in the endosomal?lysosomal system before it appears
in the amyloid plaque formed in AD transgenic mice models (28).
Observation of early A?P 42 intraneuronal accumulation within
may explain AD progression within the brain. Intracellular A?P 42
cell body and along processes and terminals of affected neurons.
The resultant accumulation of A?P in the parenchyma may hasten
the pathological process, providing a potential mechanism for the
‘‘spread’’ of A?-related pathology (15).
All these studies emphasize the importance of limiting intra-
our knowledge, this study is the first attempt to inhibit ?-secre-
tase activity by blocking the BACE cleavage site of APP by
inhibition of BACE itself by various small synthetic molecules.
Antibody-mediated blockage of the ?-secretase site of APP by
either mAb BBS1 in a cellular model or by active immunization
A?P, suggesting that this approach may be applicable as a
prophylactic therapy for AD. Antibody internalization into the
A?P, may be beneficial in avoiding microglia and complement
activation, as was reported for anti-A? antibodies that bind the
senile plaques (reviewed in ref. 29).
The concept presented here for inhibiting ?-secretase activity on
APP primarily affects A?P generated by means of the endocytic
pathway, which is responsible for the internalization and processing
of cell surface APP. Because up-regulation of endocytosis may
represent a possible basis for accelerated ?-amyloidogenesis in
30), the immunological approach presented in this study may
become a therapeutic strategy in AD treatment that is worthy of
We thank Prof. D. Selkoe, Dr. T. Golde, and Dr. C. Eckman for
providing the different cell lines; Drs. Vered Lavie and Maria Becker for
analyzing amyloid burden in the transgenic mice experiment; F. Mar-
golin for manuscript editing; D. Galitzki for help with animal care; and
the members of our laboratory for helpful discussions.
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Arbel et al.
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