(Received for publication, January 10, 1995)
From the
Biochemical and immunocytochemical analyses were performed to
evaluate the composition of the amyloid protein (A
)
deposited in the brains of patients with Alzheimer's disease
(AD). To quantitate all A
s present, cerebral cortex was
homogenized in 70% formic acid, and the supernatant was analyzed by
sandwich enzyme-linked immunoabsorbent assays specific for various
forms of A
. In 9 of 27 AD brains examined, there was minimal
congophilic angiopathy and virtually all A
(96%) ended at
A
42(43). The other 18 AD brains contained increasing amounts of
A
ending at A
40. From this set, 6 brains with substantial
congophilic angiopathy were separately analyzed. In these brains, the
amount of A
ending at A
42(43) was much the same as in brains
with minimal congophilic angiopathy, but a large amount of A
ending at A
40 (76% of total A
) was also present.
Immunocytochemical analysis with monoclonal antibodies selective for
A
s ending at A
42(43) or A
40 confirmed that, in brains
with minimal congophilic angiopathy, virtually all A
is A
ending at A
42(43) and showed that this A
is deposited in
senile plaques of all types. In the remaining AD brains, A
42(43)
was deposited in a similar fashion in plaques, but, in addition, widely
varying amounts of A
ending at A
40 were deposited, primarily
in blood vessel walls, where some A
ending at A
42(43) was
also present. These observations indicate that A
s ending at
A
42(43), which are a minor component of the A
in human
cerebrospinal fluid and plasma, are critically important in AD where
they deposit selectively in plaques of all kinds.
In the brains of patients with Alzheimer's disease (AD), ()amyloid composed of 4-kDa amyloid
protein (A
)
is deposited in senile plaques and in blood vessel walls.
Immunocytochemical studies using antisera to A
have established
that A
is deposited not only in neuritic plaques, which are
spherical clusters of altered neurites that in some cases surround well
defined amyloid cores, but also in large numbers of diffuse plaques,
which are poorly circumscribed, immunoreactive lesions showing minimal
neuritic change(1, 2) . Recently, we and others have
shown that normal processing of the large amyloid
protein
precursor (
-APP) secretes 4-kDa A
that is readily detected in
human cerebrospinal fluid and in medium conditioned by cultured
cells(3, 4, 5, 6) . This soluble
4-kDa A
is primarily A
1-40, although minor amounts of
A
1-42 and other species are also
secreted(7, 8, 9) . A
1-42 has been
shown to form insoluble amyloid fibrils more rapidly than
A
1-40(10, 11, 12, 13) .
Moreover, we have recently used transfected cultured cells to show that
-APP mutations (
I,
F) linked to familial AD increase the
percentage of long A
1-42(43) secreted(9) . Several
groups have examined the insoluble A
that remains after the AD
brain is extracted with high concentrations of
SDS(14, 15, 16, 17, 18) .
Recent reports using this approach, particularly those of Roher and his
colleagues, indicate that the SDS-insoluble amyloid in senile plaque
cores is primarily A
1-42(15, 17) , that
diffuse plaques are primarily A
17-42(18) , and that
vascular amyloid is a mixture of A
1-40 and
A
1-42(16) . Thus, the minor, secreted
A
1-42 may be critically important in the pathogenesis of AD.
Analysis of the SDS-insoluble A in AD brain will substantially
underestimate species ending at A
40 if these species are
selectively solubilized in high concentrations of SDS. Thus, to obtain
a better quantitation of the total A
in AD brain, we homogenized
AD cerebral cortex directly in 70% formic acid or sequentially
extracted first into Tris-saline buffer and then into formic acid and
analyzed the resultant fractions with sandwich ELISAs that specifically
detect A
s ending at A
40 (A
) or at
A
42(43) (A
). To determine where the A
s
that were quantitated are localized in AD brain, we immunostained
sections with BA-27 and BC-05, the monoclonal antibodies in our
sandwich ELISAs that specifically detect A
and
A
, respectively.
Figure 1:
Analysis of formic acid-extractable
A in AD and control brain by Superose 12 chromatography. The
results shown here for 1 AD and 1 control brain are typical of 3 AD and
3 control brains analyzed similarly. A, standard curve for
BC-05/4G8 assay. B, standard curve of A
for BA-27/4G8
assay.
, A
1-42;
, A
1-40. C,
BC-05/4G8 assay of A
in control brain. D, BA-27/4G8 assay
of A
in control brain. E, BC-05/4G8 assay of control
brain + synthetic A
. F, BA-27/4G8 assay of control
brain + synthetic A
. G, BC-05/4G8 assay of AD brain. H, BA-27/4G8 assay of AD brain. Synthetic A
s (500 pmol of
A
1-40 and 500 pmol of A
1-42) were added to 70%
formic acid at the same time as the control tissue, which was at
-70 °C. Values for the standard curves represent the mean of
three determinations ± S.E.
Having established the utility of this method,
we used it to analyze the A in 3 plaque-free control brains and in
3 AD brains with minimal cerebrovascular amyloid. All of the protein
detected by our sandwich ELISAs eluted from the Superose 12 column at
low molecular weight (Fig. 1, G and H) as
previously reported for A
analyzed by other
methods(8, 10) . Thus the sandwich ELISAs did not
detect full-length
-APP or large
-APP fragments containing
internal A
domains, as expected from the known specificity of
BC-05 and BA-27 for species that end at A
40 or A
42(43). Very
little A
was detected in plaque-free control brain (Fig. 1, C and D). In the 3 AD brains, the BA-27/4G8 ELISA
showed a total of 93 ± 45 pmol of A
/g whereas the BC-05/4G8
ELISA showed 3200 ± 1350 pmol of A
/g (Table 1). Thus
97% of the A
in these AD brains ended at A
42(43). The
A
-containing fractions were also analyzed using
BAN-50(anti-A
1-16)/BC-05 and BAN-50/BA-27 sandwich ELISAs
that we employed previously(9) . The BAN-50/BA-27 ELISA showed
a total of 73 ± 28 pmol of A
/g in AD brain, whereas the
BAN-50/BC-05 ELISA detected 725 ± 347 pmol/g (Table 1).
The sandwich ELISAs employing BAN-50 for capture do not provide
information on the total A in AD brain because BAN-50
(anti-A
1-16) cannot capture A
s beginning at or beyond
A
17. It was for this reason that we used horseradish
peroxidase-linked 4G8, a monoclonal to A
17-24, for detection
in BA-27/4G8 and BC-05/4G8 sandwich ELISAs. The increased amount of
A
detected with 4G8 suggests that there may be considerable
amino-terminally truncated A
in AD brain, consistent with the
results of others who have examined the SDS-insoluble A
in AD
brain(15, 17, 18) . This result should be
interpreted cautiously, however, because other factors such as the
presence of L-isoaspartates at A
1 and A
7 (15) may account for the diminished signal observed when BAN-50
was used for capture.
Figure 2:
A detected by BA-27/4G8 and BC-05/4G8
ELISAs in 27 AD brains. Cases are ordered in terms of increasing
BA-27/4G8 signal.
Direct analysis of the
A in 9 brains with minimal congophilic angiopathy (Table 1)
gave results essentially identical to those obtained when A
was
first fractionated by Superose 12 chromatography. Virtually all A
in the formic acid extracts from these 9 brains (96%) ended at
A
42(43), and substantially more A
was detected with BC-05/4G8
(4663 ± 421 pmol/g) than with BAN-50/BC-05 (714 ± 68)
assay. In the formic acid extracts of 6 brains with substantial
congophilic angiopathy (Table 1), A
,
analyzed by BC-05/4G8 (5089 ± 409 pmol/g) and BAN-50/BC-05 (826
± 119 pmol/g) ELISA, was found to be present in amounts
essentially identical to those measured in brains with minimal
congophilic angiopathy. In these brains, A
, measured
with BA-27/4G8 (13607 ± 3397 pmol/g) and BAN-50/BA-27 (5001
± 2164 pmol/g) ELISAs, was dramatically increased. On average,
67% of the A
in brains with substantial congophilic angiopathy
ended at A
40, and substantially more A
was detected with
BA-27/4G8 than with BAN-50/BA-27 assay indicating that much of the
A
ending at A
40, like the A
ending at A
42(43), is
amino-terminally truncated or modified.
Using the BAN-50/BA-27 ELISA
to analyze A1-40 extracted into Tris-saline, Suzuki et
al. previously showed a good correlation between the extent of
congophilic angiopathy in AD brain and the amount of A
1-40
in the Tris-saline extract(20) . Consistent with this, the
amount of A
extracted into Tris-saline was
dramatically increased in brains with substantial as compared to
minimal congophilic angiopathy when measured by either BAN-50/BA-27
(161 ± 60 versus 3 ± 1 pmol/g) or BA-27/4G8 (472
± 103 versus 5 ± 1 pmol/g) assay.
Figure 3:
Immunostaining of AD brain. Similar
regions of adjacent sections from the inferior temporal region of an AD
brain fixed in formalin and photographed at magnification 25
are shown after staining with: A, 4G8 (1:1000), which
recognizes A
17-24; B, BA-27 (1:100), which is
specific for A
s ending at A
40; C, BC-05 (1:20,000),
which is specific for A
s ending at A
42(43)(9) . Closedarrows show the cores of classic neuritic
plaques, and openarrows show vessels that are
stained by the antibodies employed. Note the many uncored (diffuse and
uncored neuritic) plaques that are intensely labeled by 4G8 and BC-05
but not BA-27.
The immunocytochemical results
shown here and in a recent report by Iwatsubo et al.(21) demonstrate that BC-05 intensely stains all types of
senile plaques, that vessels are stained by both BC-05 and BA-27, and
that BA-27 stains plaques poorly, in many cases only faintly staining
occasional cored plaques (Fig. 3). The apparent paucity of
A in senile plaques is remarkable because
A
1-40 is the major A
peptide in human cerebrospinal
fluid and it readily forms amyloid fibrils alone or in combination with
A
1-42 in vitro. Thus it seemed there might be
selective destruction of the BA-27 epitope during fixation that was
causing misleading immunocytochemical data on the relative amounts of
A
and A
in plaques and
vessels. To examine this possibility, we focused our initial
biochemical examination of A
on cases with minimal congophilic
angiopathy where immunocytochemistry predicted a near absence of
A
, and, to be sure that A
was not
lost as tissue was processed for biochemistry, we used a procedure that
evaluated the total A
in AD brain. Our results show unequivocally
that in brains with minimal congophilic angiopathy virtually all A
ends at A
42(43). Thus in many AD brains virtually all A
that
is deposited in senile plaques is A
.
Previous
reports (14, 15, 16, 17, 18) have
been confusing as to whether the A deposited in AD brains is
primarily A
or A
. Our
analysis of the total A
in 27 AD brains showed that in 9 cases,
all with minimal congophilic angiopathy, virtually all A
was
A
, that the remaining brains had increasing
amounts of A
, and that in 6 cases with substantial
congophilic angiopathy A
was the major form
deposited. Furthermore, the amount of A
in AD brains is at least
500-4400 times that in plaque-free control brains, and less than 2% of
the total A
in AD brain is extracted into Tris-saline. This
finding is consistent with previous reports (15, 17, 18) that most of the A
in AD
brains is amino-terminally truncated or modified.
Remarkably, the
amount of A in AD brains with minimal
congophilic angiopathy and little A
was similar to
that in brains with substantial congophilic angiopathy and large
amounts of A
. Thus, it appears that deposition of
A
in senile plaques is fundamental to AD
pathogenesis with additional deposition of A
,
primarily in blood vessel walls, occurring in some cases. This view is
supported by an immunocytochemical study of brains from trisomy 21
patients of various ages that was recently reported by Iwatsubo et
al.(22) . In these brains, A
stained by BC-05 was deposited first in diffuse plaques, and
there was essentially no staining by BA-27. Immature neuritic and
mature cored plaques then developed along with congophilic angiopathy.
As this occurred, BC-05 invariably showed intense staining of all
plaques as well as some staining of blood vessel walls whereas BA-27
intensely stained blood vessels and showed increasing staining of
A
in plaques, particularly mature cored plaques,
although it never stained plaques to the extent observed with BC-05.
Collectively, the findings reported here, the immunocytochemical data
of Iwatsubo et al.(21, 22) , the recent
analyses showing that most of the A
in isolated plaque cores ends
at A
42(15, 17) , the demonstration that
A
1-42(43) forms fibrils more rapidly than
A
1-40(10, 11, 12, 13) ,
and the finding that the familial AD-linked
-APP717 mutant
(
I,
F) increases secretion of A
1-42 but not total
4-kDa A
(9) indicate that A
, which
appears to be a minor component of the A
that is normally
secreted, is critically important in AD where it accumulates
selectively in senile plaques of all kinds.