(Received for publication, February 2, 1995; and in revised form, January 26, 1996)
From the
The mutation at codons 670/671 of -amyloid precursor
protein (
PP) dramatically elevates amyloid
-protein (A
)
production. Since increased A
may be responsible for the disease
phenotype identified from a Swedish kindred with familial
Alzheimer's disease, evaluation of the cellular mechanism(s)
responsible for the enhanced A
release may suggest potential
therapies for Alzheimer's disease. In this study, we analyzed
Chinese hamster ovary cells stably transfected with either wild type
PP (
PP-wt) or ``Swedish'' mutant
PP
(
PP-sw) for potential differences in
PP processing. We
confirmed that increased amounts of A
and a
-secretase-cleaved COOH-terminally truncated soluble
PP
(
PP
) were secreted from
PP-sw cells. As shown
previously for
PP-wt cells, A
was released more slowly than
the secretion of
PP
from surface-labeled
PP-sw
cells, indicating that endocytosis of cell surface
PP is one
source of A
production. In contrast, by
[
S]methionine metabolic labeling, the rates of
A
and
PP
release were virtually identical for
both cell lines. In addition, the identification of intracellular
PP
and A
shortly after pulse labeling suggests
that A
is produced in the secretory pathway. Interestingly, more
A
was present in medium from
PP-sw cells than
PP-wt
cells after either cell surface iodination or
[
S]methionine labeling, indicating that
PP-sw cells have enhanced A
release in both the endocytic and
secretory pathways. Furthermore, a variety of drug treatments known to
affect protein processing similarly reduced A
release from both
PP-wt and
PP-sw cells. Taken together, the data suggest that
the processing pathway for
PP is similar for both
PP-wt and
PP-sw cells and that increased A
production by
PP-sw
cells arises from enhanced cleavage of mutant
PP by
-secretase, the as-yet unidentified enzyme(s) that cleaves at the
NH
terminus of A
.
In Alzheimer's disease a characteristic pathological
finding in the brains of affected individuals is the deposition of
amyloid -protein (A
) (
)in senile
plaques(1) . A
is the 39-43-amino acid proteolytic
cleavage product of the type I integral membrane protein
-amyloid
precursor protein (
PP). The
PP gene is encoded on chromosome
21, and alternative exon splicing produces three major isoforms of 695,
751, or 770 amino acids(2) . During constitutive secretion some
full-length
PP molecules are proteolytically cleaved between
lysine and leucine residues at positions 16 and 17 of A
(Fig. 1) by an enzyme termed
-secretase(3, 4) . Cleavage of
PP at this
position creates a soluble
100-120-kDa
NH
-terminal fragment (
PP
) (5) and
a COOH-terminal membrane-retained fragment of
10 kDa(6) .
Generation of these fragments by
-secretase precludes formation of
an intact A
sequence from full-length
PP.
Figure 1:
PP structure, enzymatic cleavage
sites, COOH-terminal fragments, and antibody epitopes. Schematic
diagram of
PP
. The vertical cross-hatched box represents the plasma membrane. The white box labeled A
represents the A
peptide (also shown enlarged with
the amino acid sequence listed). The horizontally striped box labeled KPI represents the Kunitz protease inhibitor
domain alongside the adjacent exon indicated by the small open
box; the NH
-terminal black box represents the
signal sequence.
,
, and
mark the sites of the
enzymatic cleavages by
-,
-, and
-secretases,
respectively. Also indicated are the
10-kDa fragment (including
the p3 region, transmembrane region, and COOH terminus) and the
12-kDa fragment (including the A
region, transmembrane
region, and COOH terminus). -NPTY- indicates the putative
clathrin internalization signal. Horizontal black bars indicate the approximate epitopes of antibodies B5, C7, 6E10,
MMAb, R1280 and R1282, and R1736.
A, however,
is known to be released during normal cellular metabolism both in
vivo(7, 8) and in a number of cell culture
systems(9, 10) . Cleavage of
PP at the NH
terminus of the A
sequence by an enzyme designated
-secretase creates a shortened form of
PP
and the
12-kDa COOH-terminal fragment(11, 12) . An
additional enzymatic cleavage at the COOH terminus of the A
sequence by the as yet unidentified enzyme designated
-secretase
generates the 4-kDa A
peptide. The
-secretase enzyme is also
hypothesized to generate p3, the 3-kDa NH
-terminal piece of
the membrane-retained
10-kDa COOH-terminal fragment of
PP
produced by
-secretase cleavage (7, 8, 9, 13) . In addition to the
secretory cleavage,
PP can also be processed in an
endosomal/lysosomal
pathway(14, 15, 16, 17) . Although
A
-containing COOH-terminal fragments are generated in lysosomes,
evidence suggests that these are not an important source of
A
(18) . Recently, it was shown that cell surface
PP
molecules can be processed in the endocytic pathway and may be the
direct precursors of A
, presumably by recycling internalized
molecules from the cell surface(19) .
Evidence that A
and
PP contribute to the pathogenesis of Alzheimer's disease
comes from the findings of missense mutations within and adjacent to
the A
region of the
PP gene in families with autosomal
dominant forms of Alzheimer's disease(20) . The
concurrence of the mutations with the disease phenotype suggests that
altered
PP function or processing may be pathogenic. A double
mutation at amino acids 670 and 671 (
PP
numbering)
changing Lys
to Asn
and Met
to Leu
(K670N/M671L) was identified in a Swedish
pedigree with familial Alzheimer's disease(21) . In
vitro analyses of transfected cells expressing the Swedish form of
PP (12, 22) and primary cell cultures of
fibroblasts obtained from affected individuals (23) reveal a
dramatic increase in A
production. However, the mechanism by which
A
generation is increased has not been elucidated. Furthermore, a
detailed analysis of cellular processing of
PP with this mutation
has not been reported. Because recent studies have implicated the
endocytic pathway in A
production(19) , we speculated that
A
production may be similarly enhanced in this pathway in cells
expressing the K670N/M671L
PP
mutation.
In this
report, biosynthetic analyses confirmed the increase in A
production and the abundant secretion of a shorter
PP
species by Chinese hamster ovary (CHO) cells stably transfected
with the
PP
K670N/M671L mutation. Furthermore,
A
generation was increased in both the secretory and endocytic
pathways. We postulate that this increase in A
production is the
result of enhanced proteolytic cleavage of the mutant
PP by the
-secretase enzyme.
Figure 2:
PP turnover,
PP
species, and precursor product relationship of
12-kDa
fragments and A
in CHO cells stably transfected with
PP
. Panel A, turnover of full-length
PP immunoprecipitated with antibody C7 from
PP-wt and
PP-sw cells pulse-labeled for 10 min with
[
S]methionine and chased for 0, 1, 2, or 4 h. Panel B, immunoprecipitation of
PP
from
conditioned medium with antibodies B5, R1736, and 6E10 from
PP-wt
and
PP-sw cells labeled for 4 h with
[
S]methionine. R1736 and 6E10 are specific for
-secretase-cleaved
PP
. Panel C,
COOH-terminal fragments and A
release from
PP-sw cells
following a 10-min pulse with [
S]methionine and
10- or 20-min chase. The COOH-terminal fragments were
immunoprecipitated with antibody C7 from cell lysates after the 10- or
20-min chase. The
12-kDa fragments (at arrowhead),
clearly apparent by 10 min, increased by 20 min. Media from parallel
cultures immunoprecipitated with antibody R1282 show an A
(at arrowhead) signal by 20 min. No A
signal is observed at
10 min even when the signal is intentionally amplified as in the lanes on the right. For comparison, the unamplified
A
image is presented to the left of the darkened
image. Molecular weights determined from prestained standards are
indicated. wt =
PP-wt cells; sw =
PP-sw cells.
Secretion of
a shortened PP
species has been reported from a
PP chimeric molecule expressing the ``Swedish'' mutation (31) . To confirm this finding with authentic
PP
molecules,
PP
was immunoprecipitated from conditioned
media using B5 antibody, which recognizes both
- and
-secretase species of
PP
, and two antibodies that
recognize only
-secretase-cleaved
PP
(R1736 and
6E10). As observed for untransfected CHO cells (not shown),
PP
from transfected CHO cells migrates as a doublet of
bands on low percentage polyacrylamide gels. As a result, the higher
molecular weight
PP
cleaved by
-secretase and the
slightly lower molecular weight
PP
cut by
-secretase can best be compared by observing the lower of the two
bands of each doublet (Fig. 2B). The
PP
from
PP-sw cells migrated at an M
consistently lower than that of
PP-wt cells, indicating the
secretion of a shorter
PP
species. Although both cell
lines secreted comparable levels of total
PP
by B5
antibody immunoprecipitation (Fig. 2B),
PP-sw
cells had dramatically reduced levels of
-secretase-cleaved
PP
(6 ± 1.3-fold less) than
PP-wt cells
using antibodies R1736 and 6E10 (Fig. 2B). Consistent
with this finding, and as reported by
others(12, 31, 32, 33) ,
PP-sw
cells also had correspondingly higher levels of
12-kDa
COOH-terminal
PP fragments (see below).
The
onset of secretion of total PP
was first detectable at
10 min as determined with B5 immunoprecipitation (Fig. 3B). However, at this first time interval only
minute amounts of
PP
were secreted from both
PP-wt and
PP-sw cells because the signal could be seen in the
10-min lane only after prolonged autoradiographic exposures (Fig. 3B).
PP
became pronounced at 20
min for both cell lines with peak secretion at approximately 30 min (Fig. 3A and Fig. 4). The profile of
PP
secretion as a function of time was essentially
identical for the two cell lines (Fig. 4). In the experiment
shown, although
PP
secretion by
PP-sw cells was
lower because of diminished expression of full-length
PP, the
profile of secretion is essentially identical to that of
PP-wt
cells. This profile of
PP
secretion did not depend on
the level of
PP expression because other wild type and Swedish
cell lines exhibited the same patterns of release (not shown).
Furthermore, comparison of
PP-wt and
PP-sw cells that
expressed equivalent levels of
PP confirmed that
PP
secretion by both cell lines was essentially the same (within
10% of each other as determined by Phosphorimage analysis of media
from triplicate cultures from each cell line, Student's t test, p = 0.49).
Figure 3:
Incremental release of
PP
, A
, and p3 from CHO cells transfected with
wild type or K670N/M671L mutant
PP
.
Immunoprecipitations of
PP
, A
, and p3 from
conditioned chase media from single cultures of
PP-wt and
PP-sw cells following a 10-min
[
S]methionine pulse label were collected at
10-min intervals. The level of
PP holoprotein expression was
somewhat lower in
PP-sw cells in this experiment. On low
percentage polyacrylamide gels,
PP
from CHO cells
migrates as a doublet. Panel A, total
PP
was
immunoprecipitated with antibody B5. Note the lower molecular weight
species of
PP
, indicated by the arrowhead,
from
PP-sw cell media. Panel B, the presence of
PP
at 10 min is confirmed by this long exposure of the
gel shown in panel A. The shortened
PP
form,
at the arrowhead, is apparent at the earliest time point. Panel C, A
and p3 immunoprecipitated by R1282 from the
same media as panels A and B. Positions of A
and
p3 are indicated. Molecular weights determined from prestained
standards are indicated on the right. wt =
PP-wt cells; sw =
PP-sw
cells.
Figure 4:
Profiles of PP
, A
,
and p3 release from CHO cells transfected with wild type or K670N/M671L
mutant
PP
. Data from Phosphorimage analysis of gels
in Fig. 3represent the percent secretion for each time point
relative to the cumulative (100%) secretion during the entire 60-min
chase. The top panel shows the
PP
release
from
PP-wt (designated by the solid line and circles in all graphs) and
PP-sw cells (designated by the dotted
line and triangles in all graphs) from antibody B5
immunoprecipitation. A
(middle panel) and p3 (bottom
panel) release from
PP-wt and
PP-sw cells,
immunoprecipitated with R1282 antibody, are also shown. wt =
PP-wt cells; sw =
PP-sw
cells.
Regarding A release, the
timing of A
secretion during the 1st h from
PP-wt and
PP-sw cells was also identical (Fig. 3C and Fig. 4). The A
signal was first apparent at the 20-min
collection time by autoradiography (Fig. 3C) and
reached a peak at 30-40 min. Although no discernible A
signal was ever seen on either autoradiograms or Phosphorimages at the
10-min chase time, after long exposures a few Phosphorimage counts
higher than background were detected in the 10-min lane (Fig. 4). At each chase time,
PP-sw cells consistently
released more A
than
PP-wt cells. The timing of p3 secretion
mirrored that of A
in both
PP-wt and
PP-sw cells
throughout the chase period (Fig. 3C), although
PP-wt cells consistently released more p3 relative to A
than
did
PP-sw cells. Authentication of A
(beginning at
Asp
) and p3 (beginning at Lys
) was obtained by
radiosequencing (not shown), as reported
previously(15, 19) . Thus, a difference in the ratios
of
-secretase- and
-secretase-generated molecules was also
reflected by the levels of p3 and A
released by these cell lines.
Finally, the formation of the
-secretase-generated
12-kDa
PP COOH-terminal fragments preceded the release of A
from
pulse-labeled
PP-sw cells (Fig. 2C). After a
10-min labeling with [
S]methionine, the
12-kDa fragment was apparent by the 10-min chase time in
PP-sw cells and increased at 20 min (Fig. 2C).
Consistent with the above results, A
was not apparent in the
corresponding media until 20 min of the chase period (Fig. 2C). This earlier generation of the
12-kDa
fragment prior to A
release, consistently seen in three
experiments, indicates a precursor-product relationship between the two
molecules.
Figure 5:
Generation of intracellular PP
and A
from CHO cells transfected with wild type or
K670N/M671L mutant
PP
. Panel A, B5 antibody
immunoprecipitation of
PP
from chase media and saponin
buffers of
PP-wt and
PP-sw cells pulse-labeled for 10 min
with [
S]methionine and chased for 20 min. Since
PP
from CHO cells migrates as a doublet, the higher
molecular weight
PP
cleaved by
-secretase (
at arrow) and the slightly lower molecular weight
PP
cut by
-secretase (
at arrow)
can best be appreciated by observing the lower of the two
bands. The shorter
PP
species is observed both
intracellularly (intra) and secreted into the medium (sec) of
PP-sw cells. The faint bands that run
below 97 kDa in the saponin lanes are degradation products.
Molecular weights determined from prestained standards are indicated on
the right. Panel B, antibody B5 immunoprecipitations
of intracellular and secreted
PP
from
PP-wt and
PP-sw cells following a 10-min pulse with
[
S]methionine and 10-60-min chase.
Intracellular
PP
was immunoprecipitated from saponin
buffers; secreted
PP
was obtained from chase media. Panel C, immunoprecipitation of
PP-wt and
PP-sw
control (cont) cell lysates after 4 h
[
S]methionine labeling with antibody R1280 or
R1280 that had been preabsorbed (abs) with the A
1-40 peptide. The positions of the
12-kDa COOH-terminal
fragments and A
are indicated at arrows on the left. wt =
PP-wt cells; sw =
PP-sw cells.
These results suggested that A can be formed within the
secretory pathway. Indeed, intracellular A
appeared to be present
in both
PP-wt and
PP-sw cell lysates labeled for 4 h (Fig. 5C). Preabsorption of R1280 antibody with the
A
1-40 peptide totally eliminated immunoprecipitation of
A
from the cell lysates by R1280 antibody (Fig. 5C), and no 4-kDa band was observed from the same
lysate using antibody C7. Treatment with trypsin prior to
immunoprecipitation did not diminish the A
signal (not shown),
indicating that A
was present inside the cells. In addition, cells
pulse labeled with [
S]methionine followed by a
20-min or 30-min chase had both A
and p3 isolated from cell
lysates (not shown). Thus, the immunoprecipitated A
had not been
derived from secreted molecules present on the extracellular plasma
membrane at the time of cell lysis. Furthermore, the appearance of
these intracellular A
and p3 molecules after short pulse-chase
intervals provides indirect evidence of their production in the
secretory and not the endosomal/lysosomal pathway. Nevertheless, A
and p3 bands were visualized only after 8-10 weeks of
autoradiographic exposure, suggesting that only very low levels of
A
were ever present intracellularly. The minute amounts of
intracellular A
precluded definitive identification by amino acid
radiosequencing.
Figure 6:
Release of A and
PP
from cell surface-iodinated
PP molecules. Panel A,
immunoprecipitation of A
with antibody R1280 from chase media of
PP-wt and
PP-sw cells following iodination of cell surface
PP. The timing of release of A
was the same from both cell
lines. Molecular weights determined from prestained standards are
indicated. Panel B, rapid release of
PP
was
observed from both
PP-wt and
PP-sw cells after surface
iodination and immunoprecipitation by antibody B5. Panel C,
immunoprecipitation of cell lysates with antibody C7 after iodination
revealed more full-length
PP on the surface of
PP-wt cells
than
PP-sw cells.
PP-sw cells, however, had more iodinated
12-kDa COOH-terminal fragments and fewer
10-kDa fragments
than
PP-wt cells. wt =
PP-wt cells; sw =
PP-sw cells.
Two additional observations are noteworthy from
these experiments. First, PP
derived from cell surface
PP by
PP-sw cells had an M
compatible
with
-secretase-cleaved
PP
(Fig. 6B). A lower M
-secretase-cleaved
PP
species was not
readily apparent after surface labeling. However, resolution of the
labeled bands is significantly less distinct from an iodine signal
because of radiographic intensification, and minor differences may be
undetectable. Second, we consistently observed more full-length
PP
on the surface of
PP-wt cells than
PP-sw cells (Fig. 6C) expressing the same amount of
PP. To
confirm and quantitate this difference, the levels of cell surface and
total
PP were measured by an antibody binding assay using
radioiodinated antibody 5A3 Fab fragments, which bind to an
extracellular
PP epitope(19) . Treatment with 0.1% saponin
permitted labeling of both cell surface and intracellular
PP.
Multiple repetitions of this experiment showed that
PP-sw cells
had approximately 50% less cell surface
PP than
PP-wt cells
(49.8% ± 0.7, p < 0.0001). Interestingly,
PP-sw
cells showed more of the COOH-terminal
12-kDa fragment and less of
the
10-kDa fragment than
PP-wt cells (Fig. 6C) present on the cell surface.
Figure 7:
Effects of various treatments on A
production and COOH-terminal fragments from CHO cells transfected with
wild type or K670N/M671L mutant
PP
. Panel
A, immunoprecipitation of
PP-wt and
PP-sw conditioned
media with antibody R1282, and cell lysates with antibody C7 from a 2-h
[
S]methionine label followed by a 2-h chase
containing either no drug (Cont), brefeldin A (Bref),
chloroquine (Cq), or bafilomycin A1 (Balfilo). Less
A
was released in the presence of drugs compared with the control
condition for both cell lines. Panel B, antibody C7
immunoprecipitation of
PP-sw cell lysates after a 10-min
[
S]methionine pulse followed by a 20-min chase
in the absence (0) or presence (.25) of bafilomycin
A1. Note the presence of the
12-kDa COOH-terminal fragment
generated by
-secretase cleavage at 20 min in the control lane (0) and its near absence after bafilomycin A1 treatment. wt =
PP-wt cells; sw =
PP-sw.
A double mutation in the PP gene from a Swedish kindred
with familial Alzheimer's disease is invariably linked with
Alzheimer's disease(21) . All cells reported to date
which express the
PP mutation produce dramatically more A
peptide than do cells expressing wild type
PP(12, 22, 23, 31, 32, 33) .
Since excess A
production may be causally related to the
Alzheimer's phenotype in individuals affected with the
``Swedish'' mutation(21) , it is important to
evaluate the mechanism by which A
is produced from
PP with
this alteration. In this study we performed a detailed analysis of the
biosynthetic processing of
PP in
PP-wt and
PP-sw CHO
cells.
Our results showed that, as anticipated, PP-sw cells
released substantially more A
than
PP-wt cells.
Interestingly, the timing of onset and the duration of A
secretion
during the 1st h following a short pulse labeling were coincident with
p3 release for both cell lines. Only the amounts of A
and p3
varied between
PP-wt and
PP-sw cells. Furthermore, treatments
known to decrease A
in
PP-wt cells (8, 13, 38) also affected
PP-sw cells.
We interpret our data to suggest that the pathway of A
production
is similar for
PP-wt and
PP-sw cells. In contrast, however,
the timing of A
release differed substantially depending on
whether cells were [
S]methionine-labeled or
surface-iodinated. In both cell lines A
was released with a
shorter time course from [
S]methionine-labeled
cells than from cells that were surface-iodinated. This difference in
the timing of A
secretion leads us to propose that A
is
generated in both the secretory and endocytic pathways from both
PP-wt and
PP-sw cells.
A number of observations suggest
that A is generated in the secretory
pathway(10, 39) . First, the timing of secretion of
PP
, A
, and p3 was essentially identical at early
chase times in both cell lines. Specifically, within the first 30 min
in a short pulse-chase experiment, the profiles of
PP
,
A
, and p3 secretion were remarkably similar. This chase paradigm
was chosen specifically to reveal the incremental release of these
early secretory products. Second, permeabilization of
[
S]methionine pulse-labeled cells followed by
immunoprecipitation with a
PP midregion antibody (B5) showed that
intracellular soluble
PP
was present in both
PP-wt (34, 35, 36) and
PP-sw cells
as reported previously(32) . The major intracellular species of
soluble
PP
from
PP-sw cells had a lower M
than
PP
from
PP-wt cells,
consistent with production by
-secretase cleavage. Significantly,
intracellular
PP
was present before abundant
PP
was secreted into the culture medium, thus
demonstrating a precursor-product relationship. Third, the
12-kDa
COOH-terminal fragment of
PP and A
showed a precursor-product
relationship, with the
12-kDa molecules apparent 10 min prior to
the appearance of A
. Moreover, consistent with a recent
report(12) , this COOH-terminal
12-kDa fragment was
specifically increased in
PP-sw cells compared with
PP-wt
cells. Fourth, our data suggest that intracellular A
is present in
both
PP-wt and
PP-sw cells. Based on results from trypsin
digestion using a short pulse-chase paradigm, A
in cell lysates
did not appear to represent extracellular A
attached to the cell
surface or to be derived from the lysosomal pathway. However, the
exceedingly small amount of intracellular A
suggests that A
turnover and secretion are rapid. This is consistent with the earlier
postulation that A
is released from cells soon after it is formed
and suggests that
-secretase cleavage occurs at or near the cell
surface(19) . Previously, intracellular A
has only been
detected in neurons(40) . Thus our preliminary findings suggest
that the pathways of A
production in neurons and non-neuronal
cells may be more similar than was previously thought.
Regarding the
endocytic processing of PP, cells labeled by selective cell
surface iodination confirmed that
PP-sw cells produced more A
from cell surface precursors than did
PP-wt cells. However, the
timing of A
release after surface labeling was essentially
identical for both
PP-wt and
PP-sw cells. As shown previously
for cells expressing wild type
PP(19) , A
generated
from surface-labeled molecules was released more slowly than
PP
by
PP-sw cells. These profiles of A
and
PP
release from surface-labeled molecules are
dramatically different from the [
S]methionine
labeling experiments in which A
and
PP
were
released simultaneously. Interestingly,
PP
and A
release from [
S]methionine pulse-chase
experiments showed that
PP
secretion peaked at
30
min followed by a sharp decrease, whereas A
release continued at
the same level until later chase times. We interpret the sustained
A
release into the medium at a time when
PP
secretion decreased (40-50 min) to represent the addition
of newly generated A
, derived from the endocytic pool, after the
contribution of the secretory pool of A
has peaked. Therefore, our
data indicate that A
can be derived from both the secretory and
endocytic pathways and that more A
is formed within each pathway
by
PP-sw cells.
Our studies have defined a number of
similarities in PP processing between
PP-wt and
PP-sw
cells. First, the timing of secretion of
PP
, A
,
and p3 is essentially the same for both cell lines within the 1st hour
following a 10-min pulse label. Second, various drug treatments
decrease A
in both
PP-wt and
PP-sw CHO cells. Third,
intracellular
PP
species and A
appear to be
present in both cell lines. Fourth, both secretory and endocytic
pathways appear to contribute to A
generation and release. Fifth,
both
PP-wt and
PP-sw cells secrete primarily
-secretase-cleaved
PP
from surface-labeled
PP. Thus, within the limits and sensitivity of our experimental
system, the timing and the pathway of A
secretion appear to be
identical in
PP-wt and
PP-sw cells. Only the amounts of
A
and
-secretase-cleaved precursors differed in
PP-wt
and
PP-sw cells. Our data and interpretation are therefore
consistent with the results of previous investigators who have
suggested that the ``Swedish'' mutation at the NH
terminus of A
enhances
-secretase
cleavage(12, 22, 23) . This altered
-secretase cleavage produces abundant
-secretase-cleaved
PP
in the secretory pathway in
PP-sw cells,
leading to excess A
production. However, it remains unclear at
present which pathway, secretory or endocytic, plays the greater role
in A
production.
PP-sw cells did show some differences in
PP processing from
PP-wt cells. In addition to the increase
in
-secretase-cleaved products described above, there was a 50%
reduction in the amount of cell surface
PP in
PP-sw cells.
Concomitantly, there was an increase in the
12-kDa
membrane-retained
PP fragments present on the cell surface of
PP-sw cells. Whether this increase in
12-kDa fragments is
sufficient to account for the decrease in full-length
PP molecules
at the cell surface of
PP-sw cells is unclear. Because secreted
PP
levels are similar between
PP-wt and
PP-sw cells, the reduction in full-length
PP at the surface
of
PP-sw cells suggests that the amount of
PP targeted to the
cell surface may represent a minor fraction of the total
PP
processed in the secretory pathway. Otherwise, one would expect to see
a substantial increase in
PP
released into the medium
from
PP-sw cells, which was not detected. Furthermore, this
interpretation is also consistent with reports of other cell types that
express little or no
PP on the cell
surface(34, 35, 36) .
In summary, our data
suggest that there is a similar mechanism for A generation in both
PP-wt and
PP-sw cells. The increased A
production from
PP-sw cells appears to result from enhanced
-secretase
cleavage of the mutant
PP in both the secretory and endocytic
pathways. A recent report has demonstrated altered
PP processing
in mutant
PP molecules with natural or designed mutations in codon
692(41) , whereas another report demonstrated an increased
percentage of longer A
peptides from
PP with codon 717
mutations (42) . Taken together, it appears that FAD
PP
mutations lead to pleiotropic effects on
PP and A
metabolism.
The Alzheimer phenotype associated with these dominant mutations may
therefore result from different cellular perturbations that
specifically modify
PP processing.
Note Added in Proof-Similar findings of
intracellular A recently have been reported by Martin et al. (Martin, B. L., Schrader-Fischer, G., Busciglio, J., Duke, M.,
Paganetti, P., and Yankner, B. A.(1995) J. Biol. Chem.270, 26727-26730) in cells transfected with Swedish mutant
[Abstract/Full Text]
PP.