(Received for publication, December 4, 1996, and in revised form, January 14, 1997)
From the Departments of Neurology and ** Pathology,
Harvard Medical School and Center for Neurologic Diseases, Brigham and
Women's Hospital, Boston, Massachusetts 02115, ¶ Athena
Neurosciences, Inc., South San Francisco, California 94080, and the
University of Heidelberg, Central Institute of Mental Health,
J5-D-68159 Mannheim, Germany
Mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes
cause the most common and aggressive form of early onset familial
Alzheimer's disease. To elucidate their pathogenic mechanism, wild-type (wt) or mutant (M146L, C410Y) PS1 and wt or
mutant (M239V) PS2 genes were stably transfected into
Chinese hamster ovary cells that overexpress the -amyloid precursor
protein (APP). The identity of the 43-45-kDa PS1 holoproteins was
confirmed by N-terminal radiosequencing. PS1 was rapidly processed
(t1/2 = 40 min) in the endoplasmic reticulum
into stable fragments. Wild-type and mutant PS2 holoproteins exhibited
similar half lives (1.5 h); however, their endoproteolytic fragments
showed both mutation-specific and cell type-specific differences.
Mutant PS1 or PS2 consistently induced a 1.4-2.5-fold increase
(p < 0.001) in the relative production of the highly
amyloidogenic 42-residue form of amyloid
-protein (A
42) as determined by quantitative immunoprecipitation
and by enzyme-linked immunosorbent assay. In mutant PS1 and PS2 cell lines with high increases in A
42/A
total
ratios, spontaneous formation of low molecular weight oligomers of
A
42 was observed in media, suggesting enhanced A
aggregation from the elevation of A
42. We conclude that
mutant PS1 and PS2 proteins enhance the proteolysis of
-amyloid
precursor protein by the
-secretase cleaving at A
residue 42, thereby promoting amyloidogenesis.
All patients with Alzheimer's disease
(AD)1 develop extracellular amyloid
deposits composed of the 40- and 42-residue amyloid -proteins (A
)
in brain areas subserving memory and cognition. Many of the deposits
are intimately associated with degenerating axons and dendrites,
activated microglial cells, and reactive astrocytes. Mutations or
polymorphisms in four genes that strongly predispose individuals to the
premature development of AD have been identified to date. First,
missense mutations in the APP gene (1-5), which encodes the
precursor of A
, increase the production of A
peptides,
particularly A
42, in vitro and in
vivo (6-10). Second, inheritance of the
4 polymorphism of the
apolipoprotein E gene increases the number and density of A
deposits
in the brain (11-15). The third and fourth genes to be linked to AD,
presenilin 1 (PS1) and presenilin 2 (PS2), cause the most common form of early onset
familial AD (16-18). These genes encode highly homologous proteins
predicted to span the membrane 7-8 times (19). Missense mutations in
PS1 and PS2, more than 30 of which have already been identified (20),
result in markedly accelerated clinical and neuropathological features
of AD.
A clue to the mechanism of the presenilins has come from the recent
report of selective elevations in A42 levels in plasma and skin fibroblast media of subjects harboring PS1 or
PS2 mutations (10). Because primary fibroblasts expressing
different PS1 or PS2 mutations show very low A
secretion that cannot be easily studied mechanistically, we examined
stably transfected Chinese hamster ovary (CHO) cell lines in which the
sole variable is the introduction of normal or mutant presenilin genes,
and any other host-derived factors are eliminated (21). Moreover, the
formation of A
oligomers can be detected in the conditioned media of
CHO cells (22). We stably introduced different PS1 and
PS2 mutant genes into CHO cells, and characterized the
expressed proteins by radiosequencing, pulse-chase experiments and
pharmacological treatment. In contrast to PS1-expressing cells, cells
expressing mutant PS2 showed presenilin endoproteolytic patterns that
differed from wt PS2 cells in both mutation-specific and cell
type-specific ways. Using two methods of quantitation, we found that a
direct effect of the familial AD-linked presenilin mutations is to
increase selectively and significantly the cellular production of the
highly amyloidogenic A
42 peptide. In mutant PS1 and PS2
cell lines with high increases of A
42 secretion,
spontaneous A
oligomer formation was observed, demonstrating that
heightened production of A
42 by cells results in
enhanced A
aggregation.
A cDNA encoding human PS1 was obtained by polymerase chain reaction from a human placental library (Clontech). Mutant PS1 (M146L, C410Y) and PS2 (M239V, N141I) cDNAs were also generated by polymerase chain reaction from wt cDNAs. Wild-type and mutant PS1 or PS2 cDNAs were subcloned into CMV-based mammalian expression vectors PCI-neo (PS1) (Promega) or pZeo (PS2) (InVitrogen) and transfected into CHO cells stably transfected with wt APP751 (7W cells; Ref. 23) using Lipofectin (Life Technologies, Inc.). Cells were maintained in 200 µg/ml G418 (Life Technologies, Inc.), 25 µg/ml puromycin (PS1) (Calbiochem), or 250 µg/ml Zeocin (PS2) (InVitrogen).
AntibodiesPS1 polyclonal antibodies J27 and 4627 were
raised against synthetic peptides corresponding to residues 27-42
(J27) or 457-467 (4627) of PS1 (47), respectively. Polyclonal antibody
2972 was raised to residues 1-75 of PS2. Polyclonal antibody C7 is
directed against the last 20 residues of APP (24). Polyclonal antibody R1282 is to synthetic A1-40; it precipitates A
and
p3 peptides from cell media (25). Monoclonal antibody 21F12 to a
synthetic peptide of residues 33-42 of A
specifically precipitates A
and p3 peptides ending at residue 42 from conditioned media (26).
In pulse-chase
experiments, cells were first incubated in methionine-free, fetal
bovine serum-free media for 45 min before pulse-labeling with 100 µCi/ml [35S]methionine for 5 to 20 min. Cells were then
changed to regular Dulbecco's modified Eagle's medium and chased for
0.5-5 h. For some experiments, brefeldin A (10 µg/ml) was present
during the chase period. Cells were lysed in a buffer containing 50 mM Tris, pH 7.6, 150 mM NaCl, 2 mM
EDTA, 1% Nonidet P-40, 12 mM CHAPS (Pierce), and a
protease inhibitor mixture (5 µg/ml leupeptin, 5 µg/ml aprotinin, 2 µg/ml pepstatin A, and 0.25 mM phenylmethylsulfonyl
fluoride; Sigma). Immunoprecipitation was performed as
described (27). All quantitation of A immunoprecipitates were
performed with a PhosphorImager 400A, using ImageQuant software
(Molecular Dynamics).
A sandwich ELISAs were performed as described
(28), except that the capture antibodies were 266 (to A
residues
13-28) for A
total and 21F12 (to A
residues 33-42)
for species ending at A
42. The reporter antibody was 3D6
(to A
residues 1-5) in both assays.
Wild-type and mutant (M146L, C410Y) PS1 genes
were stably transfected into CHO cells that stably overexpress APP
(cell line 7W) (see "Materials and Methods"). Wt PS1/APP
transfectants (PS70) were pulse-labeled for 5 min and chased for 0-5 h
with or without BFA. In the absence of BFA, we found that the half-life
of APP remained unchanged (~30 min), compared with that in the 7W
parental cell line (29). The expected N- and
O
-glycosylation of APP was observed, and the characteristic
10- and 12-kDa C-terminal fragments of APP appeared when cells were
chased for more than 0.5 h (Fig. 1). In the
presence of BFA, APP did not undergo normal maturation by glycosylation
(Fig. 1), in agreement with published data (30). BFA also blocked the
proteolytic processing of full-length APP into C-terminal fragments
(Fig. 1). In the same cell lysates, the PS1 holoprotein was identified
as a 43-45-kDa doublet, and this holoprotein had a half-life of ~40
min. BFA treatment did not change the electrophoretic mobility of the
PS1 proteins or alter the rate of PS1 turnover (Fig. 1), suggesting
that full-length PS1 is primarily processed within the endoplasmic
reticulum (ER). Consistent with this conclusion, we observed no
[3H]glucosamine or [35S]sulfate
incorporation into PS1 proteins, in contrast to the modification of APP
by these groups in the same cells (data not shown). The identity of the
43- and 45-kDa PS1 holoproteins was confirmed by radiolabeled
sequencing with [35S]methionine. We found methionines at
positions 1 and 16 in both bands, clearly demonstrating that both
proteins begin with the N-terminal methionine predicted from the
PS1 cDNA sequence (16). These data, together with the
consistent precipitation of both bands by an extreme C-terminal PS1
antibody (4627) (Fig. 1), suggest that the two polypeptides are
SDS-stable conformers of full-length PS1.
Stable 7W CHO cells expressing human PS2 proteins were also established
(see "Materials and Methods"), and the metabolism of PS2 was
examined by pulse-labeling for 20 min and chasing for 0.5-5 h,
followed by immunoprecipitation with the N-terminal PS2-specific antibody, 2972 (Fig. 2). PS2 transfectants overexpressed
full-length PS2 proteins as a broad doublet at 46-55 kDa, as well as
the characteristic higher molecular weight PS2 aggregates (31). Both wt
and mutant (M239V) full-length PS2 holoproteins had a half-life of
~1.5 h. We also searched for endoproteolytic products of PS2, which
have not been previously reported. We observed the appearance of a 35-kDa N-terminal fragment in both wt and mutant cells after chasing for 0.5 h. Once formed, the 35-kDa fragment was very stable, and there was no degradation after 5 h in both cell lines. In
addition, an 18-kDa band was precipitated by the N-terminal antibody in the wt PS2 transfectants but not in the M239V mutant PS2 cells (Fig.
2). This 18-kDa band was absent when the antibody was preabsorbed with
its peptide immunogen. The 18-kDa fragment was immediately detected
after 20-min pulse labeling of wt PS2 cells and then rapidly
disappeared during the chase period. To confirm its identity, we
performed radiosequencing of the 18-kDa fragment with
[35S]methionine and found methionines at positions 1 and
5, entirely consistent with the N-terminal amino acid sequence
predicted from the PS2 cDNA. Similar pulse-chase
experiments were conducted to assess any alteration of APP maturation
in the PS2 stable cell lines, and we detected no change in the
half-life and posttranslational modification of APP molecules in wt and
mutant PS2 transfectants. We also failed to observe any incorporation
of [3H]glucosamine or [35S]sulfate into PS2
proteins, compared with the expected modification of APP by these
groups in the same cells (data not shown).
Differential Endoproteolysis of Mutant PS2 Proteins in CHO and 293 Cells
The above results (Fig. 2) raised the possibility that the
processing of mutant PS2 proteins differs from that of wt PS2. This
phenomenon was not observed for the PS1 mutants examined here. As
reported previously (32), PS1 holoproteins underwent endoproteolysis to
form stable 27-28-kDa N-terminal and 17-18-kDa C-terminal fragments,
and the fragment pattern did not differ between wt and mutant PS1
proteins expressed in the CHO cells (data not shown). In the case of
the PS2 stable cell lines, we detected both the 35-kDa and 18-kDa
N-terminal fragments in wt PS2 transfectants, but not in untransfected
CHO cells, as determined under steady-state conditions by combined
immunoprecipitation-Western blotting of unlabeled cell lysates with
antibody 2972 (Fig. 3). The 35-kDa fragment was the
major endoproteolytic product, whereas the 18-kDa fragment occurred at
low abundance. The latter result was consistent with that of the
pulse-chase experiments, which indicated a quick turnover of the 18-kDa
fragment versus considerable stability of the 35-kDa
fragment (Fig. 2). In CHO cells expressing the M239V (Italian) or N141I
(Volga-German) mutations, only the 35-kDa fragment was detected (Fig.
3), suggesting a different proteolytic processing mechanism for wt and
mutant PS2 proteins. To confirm that this difference was not confined
to CHO cells, we examined the endoproteolysis of PS2 in human kidney
293 cells stably expressing wt or each of the mutant isoforms (Fig. 3). Here, we observed the 35-kDa fragment in both untransfected and PS2-transfected cells, suggesting a greater level of endogenous PS2
expression in 293 than CHO cells. No 18-kDa fragment was detected in
any of the 293 cell lines. Moreover, the Italian PS2 mutation selectively induced a significant reduction of the 35-kDa fragment in
both cell types, but this was associated with the formation of an
additional 30-kDa N-terminal fragment only in the 293 cells (Fig. 3).
Taken together, these results demonstrate a striking variability in the
proteolytic processing of PS2 holoproteins as a function both of
different mutations (i.e. the Volga-German versus
Italian mutation) and of different cell types (e.g. CHO versus 293 cells).
Comparison of the Levels of Total A
To
determine whether mutant PS proteins alter APP processing, we examined
numerous CHO cell lines stably overexpressing wt and mutant PS genes. A
total of 13 clones expressing wt PS1 (clones PS70, PS106,
and PS111), mutant PS1 (ML45, ML60, ML86, CY6, CY10, and
CY11), wt PS2 (PS2-1 and PS2-2), or mutant PS2
(MV31 and MV42) were used for quantitative analysis of APP processing
and A production. The various clones of wt or mutant PS1 or PS2 cell
lines expressed substantially different levels of PS protein (Fig.
4, A and B). In the PS70 and PS106
wt lines and the ML45, ML60, CY6, and CY10 mutant lines, the PS doublet
at 43-45 kDa and the characteristic PS oligomers at ~100-140 kDa
(31) were readily seen (Fig. 4A). Cell lines PS111, ML86,
and CY11 expressed the same set of proteins but at lower levels (data
not shown). The difference between the clones with the lowest and the
highest PS1 expression levels was about 10-fold. Two wt PS2 clones and
two M239V mutant PS2 clones expressed high levels of PS2 holoproteins
at ~46-55 kDa and higher molecular weight oligomers at ~110-140
kDa that were similar to those of PS1 (Fig. 4B). The higher
molecular weight bands that were consistently detected in all
immunoprecipitations have been shown to represent aggregates of PS
proteins in transfected cells (31).
We quantitated APP expression levels in all of these PS1 and PS2 stable
transfectants. Compared with the parental 7W cells that were
untransfected with PS genes, the biosynthesis and the steady
state levels of full-length APP did not change significantly in any of
these 13 clones (data not shown). The levels of Atotal and A
42 were then compared by immunoprecipitation of
conditioned media with antibodies that are highly specific for each
derivative. Medium from the same culture dish was aliquoted and
precipitated with antibodies R1282 (for A
total) or 21F12
(for A
peptides ending at residue 42). Phosphorimaging of the
A
total signal showed no significant alteration in cells
expressing mutant PS1 or PS2 (data not shown). However, when the same
media were precipitated by 21F12, we observed an increase in the
amounts of the A
42 and p342 gel bands in the
mutant cells (Fig. 4C). The ratio of A
42 to
total A
was calculated, and the fold increase in this ratio was
established by normalizing the mean ratios obtained in all wt PS1
(n = 40) or wt PS2 (n = 10)
determinations to 1.0 (Fig. 5A and Table
I). A 1.4-2.5-fold increase in mean
A
42/A
total ratios was obtained in the
mutant PS1 and PS2 clones (p < 0.001, except in MV31,
p < 0.005; two-tailed Student's t test).
Cell lines ML60, MV31, and MV42 secreted more A
42 (Figs.
4C and 5A and Table I) than the other lines. The
fold increase in A
42/A
total ratio in the
latter three lines was in the same range as that of a cell line
expressing the APP V717F mutation (Fig. 4C), which is known
to induce substantial overproduction of A
42 (8).
|
To confirm these results, the concentrations of A42 and
A
total in the conditioned media of PS1 and PS2
transfectants were measured by sensitive and specific sandwich ELISAs.
A scattergraph of all ELISA results is shown in Fig. 5B.
When the mean ratios obtained in all wt PS1 (n = 24) or
wt PS2 (n = 11) determinations were normalized to 1.0, the relative A
42/A
total ratios were increased 1.3-2.2-fold in the mutant PS1 and PS2 lines. The
differences between the wt and mutant lines were highly statistically
significant (p < 0.001, except in CY10,
p < 0.02), and correlated well with the quantitation
by immunoprecipitation in the same cell lines.
In the course of the immunoprecipitation studies, we
observed the occurrence of low molecular weight oligomers of A in
the conditioned media of the APP V717F cell line, as reported
previously (22). The V717F line produced more A
42 than
the APP wild-type 7W cell line, as expected (8), and it showed
42-specific oligomeric A
bands by immunoprecipitation with 21F12 and
SDS-PAGE (Fig. 4C, lane V717F). Interestingly,
the three PS1 or PS2 mutant clones with the highest level of
A
42 production, ML60, MV31, and MV42 (Fig. 5,
A and B), showed low molecular weight oligomeric
species indistinguishable from those of APPV717F (Fig.
4C, lanes ML60, MV31, and
MV42). Because the presence of two additional hydrophobic residues at the C terminus of A
42 is known to increase
its fibrillogenic potential in vitro and lead to its seeding
the aggregation of the A
40 peptide (33), the appearance
of A
42 containing oligomers in the medium further
supports the substantially heightened production of A
42
by cell lines expressing mutant PS1 or PS2.
The PS1- and PS2-linked cases of AD are distinguishable from
common sporadic AD cases principally by their earlier clinical onset
and more severe neuropathology (34-36). Therefore, elucidating the
genotype-to-phenotype relationship of the presenilin mutations should
shed light on factors that are important in the pathogenesis of AD in
general. Here, we show that expression of a mutant presenilin gene in
cultured cells results in a selective and statistically significant
increase in the secretion of the highly amyloidogenic A42 peptide. For most clones, the increase in the
A
42/A
total ratio obtained by
immunoprecipitation was closely similar to that obtained by ELISA
(Table I). Thus, the results were internally consistent. Our results
are in agreement with the selective elevation of A
42
levels in plasma and skin fibroblast media obtained from living PS1 and
PS2 patients (10). One of the PS1 mutations examined in the
latter studies is at the same codon (146) as one we tested. Moreover,
the degree of increase we observed, 1.3-2.5-fold, is the same as that
obtained in vivo (10) and in the same range as that
resulting from the APP 717 mutation (8).
We observed no obvious relationship between the level of PS1 expression
and the degree of increase in A42/A
total
ratio. For example, the clone with the highest increase (M146L-60) had lower PS1 protein levels than clone C410Y-6, which showed a relatively small but still significant (p < 0.001) increase. This
result strongly suggests that mutant presenilin proteins confer a
dominant negative gain of function, which can be seen even at low
levels of expression and in the presence of the endogenous wt
presenilins.
The fact that expressing solely a mutant presenilin cDNA in a
cultured peripheral cell leads directly to a selective increase in
A42 similar to that observed in vivo
(i.e. in plasma and primary fibroblasts) indicates that
factors unique to the brain or to the AD state are not required for
this A
overproduction. Excessive A
42 production thus
appears to be an initial consequence of mutant presenilin expression, a
conclusion that is supported by the findings that carriers of
PS1 mutations can show elevated A
42 levels in plasma presymptomatically, while the majority of symptomatic subjects with sporadic AD show no increase in A
42 levels despite
advanced clinical disease (10). Our results are consistent with the
recent demonstration of a significant 2-fold increase in the density of
A
42 plaques in the brains of subjects bearing a
PS1 mutation, compared with their density in severe sporadic
AD cases (34). Furthermore, A
42 has been shown to be the
initial constituent of plaques in AD and Down's syndrome, preceding
the development of the other cytopathological features of AD by many
years or decades (37-39).
Of particular interest in this study was the spontaneous appearance of
A42 oligomers in the conditioned media of PS1 or PS2 mutant cells with high (>2-fold) elevation in A
42
secretion. Peptides ending at A
42 appear to be a major
constituent of the diffuse plaques seen initially in AD and Down's
syndrome (34, 36, 40-44), and they have been proposed to serve as a
nidus for the aggregation of the more abundant A
40
peptides (33). In contrast to wt APP expressing cells, cells expressing
V717F mutated APP consistently showed A
42 oligomer
formation in the media, and these cells are known to undergo a
selective increase in A
42 secretion. Similar rises in
A
42 secretion caused by mutant PS1 or PS2 also led to
A
42 oligomer formation. The appearance of A
42 oligomers correlated with the relative increase in
A
42 levels; in cells expressing the same PS1 mutation
but showing A
42 elevations of less than 2-fold, no
A
42 oligomer was detected under the same in
vitro condition. Based on these findings, we postulate that PS1
and PS2 mutations, expressed throughout the lifetime of the host,
gradually lead to sufficient elevation of extracellular A
42 levels in the brain to induce A
42
oligomerization, with subsequent diffuse plaque formation and later
accrual of A
40 peptides. The sequential appearance of
first A
42 and then A
40 immunoreactive
plaques in Down's patients of increasing age (38, 39) are consistent
with this hypothesis.
The mechanism of the selective increase in A42
production caused by mutant presenilins remains to be elucidated. It is
unlikely that PS proteins generally affect APP synthesis or metabolism, as the posttranslational modification and turnover of total APP proteins were not changed by introduction of wt or mutant PS
genes. Rather, we speculate that APP proteins may interact directly
with PS1 or PS2 proteins in a presenilin-rich compartment of the cell (e.g. the ER or early Golgi) and that APP interacts with
mutant presenilin in a manner that leads to its increased exposure to the
-secretase cleaving specifically after residue 42 of the A
region. Evidence for the existence of 40- and 42-specific
-secretases in mammalian cells has recently been presented (26).
Wild-type PS might, for example, form a complex with APP (or the 12-kDa C-terminal fragment arising from
-secretase cleavage), and this complex might prevent access of APP to a 42-specific
-secretase in
the ER; mutations in PS could prevent the proper formation of such
complexes.
Since PS1 is known to undergo constitutive endoproteolysis, and very
little intact holoprotein is detectable in cells or tissues expressing
this gene (32),2 we searched for evidence of a change in
endoproteolytic patterns in cells bearing mutant presenilins. We did
not observe a significant change in fragment formation in the two
PS1 missense mutations we examined, despite a consistent and
significant increase of A42/A
total in all
clones expressing these mutations. However, the mutant form of PS1 that
has a deletion of exon 9 undergoes no endoproteolysis (32), and yet
this mutation also causes a selective increase in A
42
secretion in transfected cells (45).3
Therefore, interference with the normal endoproteolysis of PS1 appears
not be an obligatory step in producing the mutant phenotype. In the
case of our analyses of PS2 mutations, we searched for PS2
endoproteolytic products and identified a major 35-kDa N-terminal fragment. Its size is larger than the 28-kDa derivative, which is the
major N-terminal fragment of PS1, indicating that the size of PS2
endoproteolysis is substantially C-terminal to that of PS1, which
occurs at and around residue 298 (46). The proteolytic fragment
patterns varied when two distinct mutations were expressed in the same
cell type (293) and also varied when the same mutation (M239V) was
expressed in two different cell types (CHO and 293). Despite these
complex differences in PS2 endoproteolysis, the PS2 M239V mutant cell
line still showed increased A
42 secretion in both CHO
and 293 cells (this study).3 Our findings, therefore, point
to the need for caution in interpreting changes in presenilin
processing as a result of PS mutations. Any mutation should be analyzed
in at least two cell types, and multiple mutations should be examined
before any conclusion about the pathogenic role of altered
endoproteolysis is reached.
In summary, our results strongly support the hypothesis that familial
AD-linked mutations in PS1, PS2, and APP all cause AD by increasing the
cellular production of A42, thereby accelerating the
polymerization of this and other A
peptides and promoting cerebral
accumulation of A
as an essential early event in AD pathogenesis.
These findings provide further impetus for current efforts to identify
compounds which inhibit the production or the aggregation of A
as
therapeutic agents for AD.
We thank Dr. P. St George-Hyslop for providing pZeo-PS2 plasmids.