From the Department of Central Nervous System and Cardiovascular Research, Schering-Plough Research Institute, Kenilworth, New Jersey 07033
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ABSTRACT |
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The calpain inhibitor
N-acetyl-leucyl-leucyl-norleucinal (ALLN) has been reported
to have complex effects on the production of the The Familial AD has thus far been associated with autosomal dominant
mutations in the genes encoding APP, presenilin 1 (PS1), and presenilin
2 (2, 3). Multiple mutations in these three genes are associated with
increased A A In this study, the effects of ALLN on A Reagents--
Antibodies W02, G2-10, and G2-11 were obtained
from Dr. Konrad Beyreuther (University of Heidelberg, Heidelberg,
Germany). W02 recognizes an epitope at amino acids 5-8 of the A cDNA Constructs, Cell Culture, and Transfection of Cultured
Cells--
A human APP695 cDNA clone with the Swedish mutation
(APPsw) and a cDNA encoding the the C-terminal 99 amino acids of
APP plus an N-terminal methionine (hereafter referred to as C100) were obtained from Dr. Barry Greenberg. C100 was cloned into the expression vector pcDNA3.1 (Invitrogen, San Diego, CA). The SPC100 construct consists of the N-terminal 18 amino acids of APP appended to the N
terminus of C99 as described by others (17). To prepare the SPC100
construct, residues 19-596 of the APP695 cDNA were deleted by
using the Seamless Cloning Kit (Stratagene, La Jolla, CA). The
resulting SPC100 construct was cloned into the pcDNA3.1 vector. The
APP London mutation (18) was introduced into C100 and SPC100 constructs
using the QuickChangeTM site-directed mutagenesis kit
(Stratagene). The human cDNAs encoding wild type PS1 and mutant PS1
with the exon 9 deletion (PS1
Human embryonic kidney 293 cells were purchased from American Type
Culture Collection (Rockville, MD) and were grown in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin. For transient expression of C100 and SPC100, 293 cells were seeded into 6-well plates, and transfection was conducted 2 days later when the cells reached 60-70% confluence. Cells were transfected by means of LipofectAMINE Plus (Life Technologies, Inc.) according to the manufacturer's instructions. To prepare 293 cells stably expressing APPsw, 293 cells were transfected as described above. About 24 h
after transfection, cells were passed to media containing 0.4 mg/ml
G418, and G418-resistant clones were analyzed for A ALLN Treatment--
APPsw cells and cells transiently expressing
C100 or SPC100 were treated with various concentrations of ALLN for
16 h. The conditioned media were then collected, centrifuged at
10,000 × g for 5 min to remove cell debris, and stored
at Western Blot--
APPs ELISA Analysis of A Metabolic Labeling of C100 with
[35S]Methionine--
293 cells were seeded in 60-mm
dishes and transiently transfected with C100 as described above. About
40 h after transfection, cells were incubated in methionine- and
cysteine-free DMEM medium for 1 h and were then labeled for 1 h with 150 µCi/ml [35S]methionine. The cells were
subsequently washed and either kept frozen at Expression of C100 and SPC100--
In addition to full-length APP
with the Swedish mutation, two truncated APP constructs were used in
this study to examine the processing of APP by
293 cells transiently expressing SPC100 secreted 2-3-fold more A Modulation of A
Examination of cell lysates revealed that ALLN increased the cellular
level of C100 in a concentration-dependent manner in cells
expressing C100 and APPsw, whereas it did not affect C100 protein
levels in cells expressing SPC100 (Figs.
4 and
5A). The ALLN-induced increase
in C100 in APPsw cells was not due to increased In cells expressing APP, the protease inhibitor ALLN has recently
been shown to inhibit selectively the production of A The Treatment of cells expressing C100 or APPsw with low concentrations of
ALLN resulted in increased secretion of both A-amyloid peptide
(A
). In this study, the effects of ALLN on the processing of the
amyloid precursor protein (APP) to A
were examined in 293 cells
expressing APP or the C-terminal 100 amino acids of APP (C100). In
cells expressing APP or low levels of C100, ALLN increased A
40 and
A
42 secretion at low concentrations, decreased A
40 and A
42
secretion at high concentrations, and increased cellular levels of C100
in a concentration-dependent manner by inhibiting C100
degradation. Low concentrations of ALLN increased A
42 secretion more
dramatically than A
40 secretion. ALLN treatment of cells expressing
high levels of C100 did not alter cellular C100 levels and inhibited
A
40 and A
42 secretion with similar IC50 values. These
results suggest that C100 can be processed both by
-secretase and by
a degradation pathway that is inhibited by low concentrations of ALLN.
The data are consistent with inhibition of
-secretase by high
concentrations of ALLN but do not support previous assertions that ALLN
is a selective inhibitor of the
-secretase producing A
40. Rather, A
42 secretion may be more dependent on C100 substrate concentration than A
40 secretion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amyloid peptide
(A
)1 is the major protein
component of the senile plaques found in the brain of Alzheimer's
disease (AD) patients. A
is produced by proteolysis of a single
transmembrane domain protein known as the amyloid precursor protein
(APP) (reviewed in Refs. 1 and 2). The first step in A
production
involves the cleavage of APP by an uncharacterized protease termed
-secretase. Cleavage of APP by
-secretase produces a large
ectodomain protein known as APPs
, which is ultimately secreted, and
a C-terminal 14-kDa membrane-bound fragment known as C100 (also termed
C99 in some references). C100 is subsequently cleaved by
-secretase, another uncharacterized protease that cleaves within the transmembrane domain of C100 and produces the 39-43-amino acid A
peptides. A
40
is the dominant species of A
secreted from cultured cells and is
also more abundant in cerebrospinal fluid of normal and AD patients.
A
42, which comprises about 5-10% of total A
secreted from
cultured cells, is more amyloidogenic and is the major species of A
that is deposited at the early stage of senile plaques formation.
42 production (4-6). Collectively, these data suggest
that excessive A
42 production is critical for the development of AD.
Whereas the locations of mutations in the APP gene suggest that the
mutations lead to increased A
42 production by increasing cleavage of
APP by
- or
-secretase, the mechanism by which presenilin
mutations increase A
42 production remains unclear. Primary neuronal
cultures derived from PS1 knock-out mice exhibit marked reduction of
A
secretion (7), suggesting an essential role of PS1 in generating
A
. Understanding the cellular mechanisms that regulate A
production will be a key step to unraveling the pathogenesis of AD.
production can also be modulated by peptide aldehyde protease
inhibitors such as N-acetyl-leucyl-leucyl-norleucinal (ALLN, also known as calpain inhibitor I or LLnL) (5, 8-11). ALLN was first
identified as a cysteine protease inhibitor (12), but at high
concentrations it can also inhibit proteasome-associated activities
(13). It has been reported that ALLN inhibits A
40 production at
concentrations that have little effect on or even increase A
42
production (5, 9). These data are interpreted as evidence suggesting
that A
40 and A
42 are produced by distinct
-secretases. In
contrast, a recent study demonstrates that ALLN increased A
40 and
A
42 production at low concentrations and decreased A
40 and A
42
production at higher concentrations (10). Thus, the reported effects of
ALLN on A
production are conflicting, and the mechanism(s) by which
ALLN modulates A
production are not clear. Nevertheless, ALLN may
serve as an important tool to investigate the regulation of A
biosynthesis.
40 and A
42 secretion are
examined in detail and the mechanism by which ALLN modulates A
secretion is further defined. The data provide the novel insights that
substrate availability plays a major role in regulating A
40 and
A
42 production by
-secretase.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptide, and G2-10 and G2-11 specifically recognize the C terminus
of A
40 and A
42, respectively (14). Antibody 54 was obtained from
Dr. Barry Greenberg (Cephalon, Inc., West Chester, PA) and recognizes the secreted APP ectoprotein formed after
-secretase cleavage (APPs
) (15). Antibody 14, which recognizes an N-terminal domain of
PS1(16), was obtained from Dr. Samuel Gandy (Cornell University, New
York). N-Acetyl-leucyl-leucyl-norleucinal (ALLN) was
purchased from Boehringer Mannheim. All tissue culture reagents used in this work were from Life Technologies, Inc.
E9) (19) were obtained from Dr. Peter
St. George-Hyslop (University of Toronto, Toronto, Canada). PS1
E9
sometimes is referred to as exon 10 deletion (20), as an alternate
5'-untranslated region exon was missed in the initial characterization
of PS1 genomic structure (19).
secretion by
ELISA (see below). Clones secreting high levels of A
were expanded
and maintained in media supplemented with 0.2 mg/ml G418.
20 °C prior to ELISA and Western blot analysis. The cell
monolayers were washed with cold phosphate-buffered saline and stored
frozen at
20 °C prior to Western blot analysis.
was detected in conditioned media by
Western blot analysis with antibody 54. C100 and SPC100 were identified
in cell lysates with antibody W02. Visualization was performed with an ECL kit (Amersham Pharmacia Biotech) according to the manufacturer's procedure.
Peptides--
Sandwich ELISA assays were
developed to measure A
40 and A
42 using the combination of
antibodies G2-10/W02 and G2-11/W02, respectively. Both antibody G2-10
and G2-11 are more than 100-fold selective for A
40 and A
42,
respectively (14), and the sensitivity of these assays are about
50-100 pg/ml. Briefly, Nunc MaxiSorb immunoassay plates were coated
overnight at 4 °C with 0.4 µg/well G2-10 in 100 mM
NaHCO3 (pH 9.5) or with 1 µg/well G2-11 in 100 mM Tris-HCl (pH 7.4). Subsequently, the antibody solution
was removed, and the wells were incubated overnight at 4 °C with 5% bovine serum albumin in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl (TBS). The wells were then washed with TBS plus
0.05% Tween 20 (TTBS) and were stored at 4 °C for up to 6 months.
Conditioned media were diluted with 10% bovine serum albumin in TBS to
yield a final concentration of 2% bovine serum albumin, and 100 µl
of diluted media was added to each well along with 40 ng/well
biotinylated W02. Biotinylation of W02 was performed with the
EZ-LinkTM Sulfo-NHS-LC-Biotinylation kit (Pierce) according
to the manufacturer's instructions. The plate was incubated at 4 °C
with gentle shaking either overnight (for A
40 measurement) or for at
least 24 h (for A
42 measurement). The plate was then washed
five times with TTBS, and 100 µl of 0.5 µg/ml horseradish
peroxidase-conjugated NeutrAvidin (Pierce) was added to each well and
incubated at room temperature for 1 h. The color was developed
with the TMB-H2O2 system (Kirkegaard & Perry
Laboratories, Gaithersburg, MD) according to the manufacturer's instructions, and absorption at 450 nm was measured on a plate reader.
20 °C (pulse) or
incubated with fresh complete DMEM (chase) in the absence and presence
of 25 µM ALLN. The chase media and cell monolayers were
collected at different time points and kept frozen until further
analysis. C100 and A
peptide were immunoprecipitated with antibody
W02 from radiolabeled cells and chased media, respectively. The cells
were solubilized with 0.6 ml/dish of 1× RIPA buffer (50 mM
Tris-HCl (pH 7.5), 150 mM NaCl, 1% deoxycholate, 1%
Triton X-100, 0.1% SDS), and 0.5 ml of cell lysate was used for each immunoprecipitation assay with 3 µg of antibody W02. For
immunoprecipitation of A
from chased media, 0.2 ml of 5× RIPA was
added to 1 ml of media together with 3 µg of antibody W02. Forty
microliters of Protein G plus Protein A-agarose (Calbiochem) was added
to each immunoprecipitation reaction, and the mixtures were rocked
overnight at 4 °C before being centrifuged at 10,000 × g for 2 min. The pellets were then washed twice with 1×
RIPA buffer and once with 10 mM Tris-HCl (pH 7.5). The
immunocomplexes were denatured in SDS-polyacrylamide gel
electrophoresis sample buffer and boiled for 5 min before being
processed by electrophoresis and autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase (see Fig.
1A). The construct designated
as C100 consists of the methionine initiation codon plus the C-terminal
99 amino acids of APP. The N terminus of C100 corresponds to the
-secretase cleavage site, i.e. the N terminus of the A
peptide. The construct designated SPC100 consists of the methionine
initiation codon, the 16 amino acid signal peptide of APP, and the
first two amino acids of APP appended to the N terminus of C100. C100
and SPC100 are similar to the constructs previously reported by Dykes
et al. (17) except that the expression vector pcDNA3.1
was used instead of pCEP4. Western blot analysis of extracts from 293 cells transiently expressing SPC100 detected a 14-kDa band that
co-migrated with native C100 (Fig. 1B). These data confirm
that the signal peptide of SPC100 was removed. As reported previously
(21), the level of expression of C100 was much higher in cells
transiently expressing SPC100 than in cells transiently expressing C100
(Fig. 1B).
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Fig. 1.
Protein expression and A
secretion in cells expressing C100 and SPC100. A,
schematic diagram of the C100 and SPC100 expression constructs.
B, Western blot analysis of lysates prepared from cells
expressing C100 and SPC100. The Western blot was probed with antibody
W02 as described under "Experimental Procedures." C and
D, concentration of A
40 (C) and A
42
(D) in the conditioned media from cells expressing C100 and
SPC100. A
40 and A
42 were quantitated by ELISA assay as described
under "Experimental Procedures." The data shown are from one
transfection and are representative of more than five
transfections.
40
and A
42 than cells transiently expressing C100 (Fig. 1, C
and D), consistent with a previous report (21). The
increased A
secretion from cells expressing SPC100 relative to cells
expressing C100 is probably due to the higher level of C100 in these
cells (Fig. 1B). Despite the quantitative differences in
A
secretion from cells expressing C100 and SPC100, the relative
amounts of A
42 and A
40 secreted by cells expressing the two
constructs (i.e. the A
42:A
40 ratios) were similar
(Fig. 2A). The effects of APP
and PS1 FAD mutations on A
secretion from cells expressing C100 and
SPC100 were also tested. As reported previously (16), extracts from 293 cells expressing wild type PS1 displayed a 48-kDa product corresponding
to full-length PS1 as well as a 33-kDa N-terminal fragment of PS1 (Fig.
2B). Extracts from 293 cells expressing the PS1 mutant
PS1
E9 displayed a full-length PS1 protein with slightly greater
mobility than that of full-length wild type PS1 due to the deletion of
exon 9 in this mutant (Fig. 2B). The 33-kDa N-terminal
fragment of PS1, which is derived primarily from endogenous PS1, was
not increased significantly by overexpression of PS1wt or PS1
E9. As
previously reported for full-length APP (4), the secretion of A
42
from cells expressing either C100 or SPC100 was selectively increased
by co-expression of PS1
E9 or by introduction of the London mutation
into C100 or SPC100 (Fig. 2A). Co-expression of PS1
E9 did
not alter A
40 secretion from cells expressing either C100 or SPC100,
nor did co-expression of wild type PS1 affect A
secretion from cells
expressing either construct (data not shown). Co-expression of wild
type PS1 or PS1
E9 did not affect the levels of C100 protein in cells
expressing either C100, SPC100, C100-London, or SPC100-London (Fig.
2C and data not shown). Thus,
-secretase processing of
C100 derived from either the C100 or SPC100 constructs is qualitatively
similar as evidenced by the similar relative secretion of A
40 and
A
42 (i.e. the A
42:A
40 ratio) and the similar effect
of APP and PS1 FAD mutations on A
secretion.
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Fig. 2.
Modulation of A
production by APP and PS1 FAD mutations in cells expressing C100
and SPC100. Expression vectors containing cDNAs encoding
C100, 100-lon, SPC100, or SPC100-lon) were transiently transfected into
293 cells together with either an empty expression vector or an
expression vector containing cDNAs encoding PS1wt or PS1
E9.
About 24 h after transfection, the cells were fed with fresh
media, and conditioned media were collected the next day. The
concentrations of A
40 and A
42 in the conditioned media were
determined by ELISA assay as described under "Experimental
Procedures." The cell monolayers were washed with ice-cold PBS and
stored at
20 °C before analysis of protein expression by Western
blot as described under "Experimental Procedures." A,
A
42:A
40 ratio in the conditioned media. B, Western
blot analysis of PS1 expression with antibody 14. C, Western
blot analysis of C100 expression in the cell lysates with antibody W02.
The data shown here are from one experiment and are representative of
three independent transfections.
Production by ALLN--
ALLN and related
peptide aldehyde protease inhibitors have multiple effects on APP
processing. In addition to modulating A
production, it has been
observed that these inhibitors can also potentiate the
-secretase
pathway and enhance the secretion of APPs
(5, 8). The altered
-secretase processing may indirectly affect the
-secretase
pathway as the two proteases compete for a common substrate.
Consequently, the effect of ALLN on A
production in cells expressing
APP is complex, and it is very difficult to distinguish the specific
action of ALLN on the
-secretase cleavage step. Since C100 is the
product of
-secretase cleavage and is the native substrate for
-secretase (1), A
production by cells expressing C100 or SPC100
reflects only
-secretase processing. We therefore utilized cells
expressing C100 and SPC100 to study the effect of ALLN on the
-secretase cleavage reaction without the influence of this inhibitor
on
- and
-secretase processing. In cells expressing C100 or
APPsw, both A
40 and A
42 secretions were increased by treatment
with low concentrations of ALLN and decreased by treatment with high
concentrations of ALLN (Fig. 3,
A and C). A
42 secretion from cells expressing
C100 or APPsw was increased much more dramatically by low
concentrations of ALLN than was A
40 secretion (Fig. 3, A
and C). In contrast, ALLN had only
concentration-dependent inhibitory effects on A
40 and A
42 secretion from cells expressing SPC100 (Fig.
3B).
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Fig. 3.
Effects of ALLN treatment of
A production. About 24 h after
transfection, 293 cells transiently expressing C100 (A),
SPC100 (B), or APPsw (C) were treated overnight
with various concentrations of ALLN. Conditioned media were collected
and analyzed for A
40 (
) and A
42 (
) concentration by ELISA
as described under "Experimental Procedures." The data shown are
the average of duplicate transfections in a single experiment and are
representative of three independent experiments.
-secretase activity
since ALLN did not potentiate the secretion of APPs
from these cells
(Fig. 5B). The increase in C100 protein level induced by
ALLN in cells expressing C100 or APPsw was not a result of inhibition
of
-secretase since concentrations of ALLN that increased cellular
C100 protein levels also increased both A
40 and A
42 production.
In addition, pulse-chase experiments demonstrated that at
concentrations of ALLN that increased A
production (data not shown),
this compound decreased the rate of C100 turnover in cells expressing
C100 (Fig. 6), suggesting that ALLN
increases C100 protein levels by inhibiting C100 degradation.
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Fig. 4.
Effect of ALLN on cellular C100 levels in
cells expressing C100 and SPC100. 293 cells transiently expressing
C100 or SPC100 were incubated with various concentrations of ALLN as
described in Fig. 3. After conditioned media were collected, cell
monolayers were washed with cold PBS and lysed with SDS sample buffer.
C100 levels in the cell lysates were determined by Western blot
analysis with antibody W02 as described under "Experimental
Procedures." Lanes 1-6, cell lysates from cells
expressing C100 treated with the indicated concentrations of ALLN;
lanes 7-12, cell lysates from cells expressing SPC100
treated with the indicated concentrations of ALLN.
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Fig. 5.
Regulation of APP processing by ALLN in cells
expressing APPsw. 293 cells expressing APPsw were incubated
overnight with ALLN at the indicated concentrations. Conditioned media
were then collected, and cell monolayers were washed with cold PBS and
lysed in SDS sample buffer. A, Western blot analysis of APP
and C100 in lysates of cells expressing APPsw following ALLN treatment.
The Western blot was probed with antibody W02. B, Western
blot analysis of APPs in conditioned media collected from cells
expressing APPswe following ALLN treatment. APPs
was detected with
antibody 54.
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Fig. 6.
Pulse-chase labeling study of C100
metabolism. About 36 h after transient transfection, cells
expressing C100 were labeled with [35S]Met for 1 h.
The cell monolayers were then washed with PBS and chased for 1 and
3 h in the presence and absence of 25 µM ALLN.
Antibody W02 was used to precipitate C100 from cell lysates.
Immunoprecipitates were separated on 14% SDS-polyacrylamide gel
electrophoresis, and the gel was further processed for fluorography.
Lane 1, cells pulse-labeled for 1 h; lanes 2 and 3, cells chased for 1 and 3 h without ALLN;
lanes 4 and 5, cells chased for 1 and 3 h in
the presence of ALLN.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
40 at
concentrations that have little effect on the production of A
42 (5,
9). These data were interpreted as indicating that distinct
-secretases are responsible for A
40 and A
42 production. Although these data are intriguing, a recent study suggests that the
effects of ALLN on A
secretion from cells expressing APP are complex
(10). To elucidate the mechanisms by which ALLN modulates A
production and, by inference,
-secretase activity, this study
examined the effects of ALLN on A
production in more detail.
-secretase reaction was studied in 293 cells expressing either
APPsw or various amounts of C100, the C-terminal fragment of APP that
represents the immediate substrate of this enzyme. C100 was produced in
293 cells by expression of two constructs designated as C100 and SPC100
(Fig. 1). As reported previously, much higher levels of C100 were
produced in cells expressing SPC100 than in cells producing C100 (21),
presumably because the signal peptide present in SPC100 permits more
efficient processing or sorting of the protein. The characteristics of
A
production in cells expressing APPsw, C100 or SPC100 cells were
similar. The relative amounts of A
40 and A
42 secreted by cells
expressing C100 or SPC100 were similar as reflected by the similar
A
42:A
40 ratios, and like the APP expression cells (4, 18), the
secretion of A
42 was specifically increased by co-expression of
mutant PS1 or by introduction of the London mutation into the
constructs (Fig. 2). These observations argue that the same
-secretase(s) is(are) responsible for A
production in cells
expressing all three constructs.
40 and A
42 (Fig. 3,
A and D), which is consistent with results
reported by others (10). Interestingly, treatment of cells expressing either C100 or APPsw with low concentrations of ALLN also increased cellular C100 protein accumulation (Figs. 4 and 5A).
Pulse-chase experiments in cells expressing C100 demonstrated that a
low concentration of ALLN decreased the rate of C100 turnover (Fig. 6),
whereas A
secretion was increased during the same period (data not
shown). Taken together, these data suggest that low concentrations of ALLN increase A
production by inhibiting C100 turnover and, hence, increasing the amount of C100 substrate available for
-secretase cleavage (Fig. 7). This suggestion is
supported by the observation that ALLN did not affect cellular C100
levels in cells expressing SPC100 and correspondingly did not increase
A
secretion from these cells at any concentration. One implication
of these results is that in addition to cleavage by
-secretase, C100
is normally degraded by a distinct ALLN-sensitive pathway (Fig. 7).
Channeling of C100 into this alternative, ALLN-sensitive degradation
pathway would prevent A
production (Fig. 7). The fact that ALLN did
not increase cellular C100 levels in cells expressing SPC100 may be due
to the fact that the ALLN-sensitive degradation pathways is overwhelmed
by the much higher levels of cellular levels of C100 present in these
cells. Low concentrations of ALLN also increase A
secretion from
primary hippocampal cultures where only endogenous APP is
expressed,2 suggesting that
the ALLN-sensitive degradation of this APP intermediate represents a
normal metabolic process and is not merely an artifact of
overexpressing C100 in cultured cells. The presence of an
ALLN-sensitive catabolic pathway for C100 may provide a mechanism by
which cells regulate substrate availability for
-secretase and thus
regulate cellular A
production. In this regard, the regulation of
C100 metabolism may play an important role in AD pathogenesis.
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Fig. 7.
Model for metabolism of C100 by
-secretase and and ALLN-sensitive degradation
pathway.
An alternative mechanism has been proposed to explain the ability of
ALLN to increase A secretion (22). Several protease inhibitors,
including ALLN, are known to prevent the proteasome-mediated degradation of PS1 (22, 23). Based on this observation and the fact
that presenilin mutations are associated with increases in A
42
secretion, it has been suggested that the stabilization of presenilins
by protease inhibitors like ALLN may directly potentiate A
42
production in cells (22). Our data do not support this model for two
reasons. First, the effects of ALLN and PS1 mutations are additive,
i.e. low concentrations ALLN further increase the A
42:A
40 ratio in cells expressing mutant PS1
(22).3 Second, the fact that
A
42 production in cells expressing SPC100 can be increased by
co-expression of a mutant PS1 (Fig. 2A), but not by ALLN
(Fig. 3B), provides additional evidence that PS1 mutations and ALLN regulate A
42 production by independent mechanisms.
High concentrations of ALLN inhibited A40 and A
42 production by
cells expressing either APPsw or C100. High concentrations of ALLN may
directly inhibit
-secretase and, hence, decrease A
production,
but other effects of ALLN on cellular metabolism could also be
responsible for this effect. In cells expressing SPC100, which gives
rise to much higher cellular C100 levels than in cells expressing APPsw
or C100, ALLN inhibited A
40 and A
42 production at all
concentrations and the IC50 values of ALLN for inhibition
of A
40 and A
42 production were very similar. This observation
does not support the conclusion that ALLN selectively inhibits A
40
production (5, 9), a finding that was interpreted as implying the
existence of distinct
-secretases responsible for A
40 and A
42
production. Since ALLN modulates A
production by multiple
mechanisms, as documented in this study and Ref. 10, it is difficult to
use this compound to pharmacologically distinguish multiple
-secretases.
Whereas low concentrations of ALLN and other calpain/proteasome
inhibitors increase A40 and A
42 production, the increase in
A
42 production induced by these protease inhibitors is much more
pronounced (Ref. 10, Fig. 3, A and D). Although a
definitive explanation for this phenomenon cannot be discerned from the
existing data, a reasonable model can be proposed. As discussed above
and illustrated in the model diagrammed in Fig. 7, ALLN increases A
production by increasing the availability of the
-secretase substrate C100. Since ALLN increases A
42 secretion more dramatically than A
40 secretion, this model implies that the
-secretase
cleavage reaction producing A
42 is more dependent on substrate
concentration than the reaction producing A
40. In other words, the
-secretase that cleaves C100 to produce A
42 has a higher
Km for the substrate C100 than the
-secretase
that cleaves C100 to produce A
40. Thus, the ALLN-induced increase in
the cellular concentration of the
-secretase substrate C100 will
increase A
42 secretion more than A
40 secretion. The fact that
A
40 secretion was only slightly increased by ALLN would suggest that
the
-secretase that produces A
40 is nearly saturated with C100
under the conditions used in these experiments. This model would
explain why A
40 is the dominant A
species produced during normal
physiologic processing of APP. It should be pointed out that this model
and the experimental data that support it do not necessarily require
the existence of two distinct
-secretase enzymes that independently
produce A
40 and A
42. The major weakness of this model is the
observation that the higher cellular levels of C100 seen in cells
expressing SPC100 relative to cells expressing C100 is associated with
similar increases in A
40 and A
42 secretion rather than a more
selective increase in A
42 secretion as would be predicted by the
model. It is possible that the model is fundamentally correct but that increasing cellular C100 levels by expressing SPC100 rather than C100
is somehow biochemically or mechanistically different from increasing
cellular C100 levels with ALLN. In any case, the hypothesis that
substrate concentration is a more important determinant of A
42
production than A
40 production provides a novel framework for
further experiments aimed at understanding the mechanisms regulating
A
production.
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ACKNOWLEDGEMENTS |
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We thank Dr. Sam Gandy for providing antibody 14 and Drs. Peter St. George-Hyslop and Georges Levesque for presenilin cDNA constructs.
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FOOTNOTES |
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* 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.
To whom correspondence should be addressed: Schering-Plough
Research Institute, K15-3-3600, 2015 Galloping Hill Rd., Kenilworth, NJ
07033. Tel.: 908-740-6559; Fax: 908-740-2383; E-mail:
lili.zhang{at}spcorp.com.
2 P. Fraser and L. Zhang, unpublished data.
3 L. Zhang and L. Song, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
A,
-amyloid
peptide;
AD, Alzheimer's disease;
APP,
-amyloid
precursor
protein;
APPsw, APP carrying Swedish mutations;
ALLN, N-acetyl-leucyl-leucyl-norleucinal, also known as calpain
inhibitor I or LLnL;
C100, the C-terminal 100-amino acid fragment of
APP;
SPC100, C100 with an N-terminal signal peptide derived from the
N-terminal 18 amino acids of APP;
C100-lon and SPC100-lon, C100 and
SPC100 carrying london mutation;
DMEM, Dulbecco's modified Eagle's
medium;
FAD, familial Alzheimer's disease;
PBS, phosphate-buffered
saline;
PS1, presenilin 1;
ELISA, enzyme-linked immunosorbent
assay.
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REFERENCES |
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