From the
Filamentous aggregates of the 40-42-residue amyloid
Amyloid
Although A
We have
observed that immunoprecipitation of the conditioned media of certain
To confirm the
structural identity of A
We initially asked whether secreted A
Confirmation of the
identity of the 3 most abundant bands as A
The data presented so far
indicate that SDS-stable low oligomers of secreted A
Experiments conducted in numerous laboratories on the
neurotoxic effects of synthetic A
It is important to
characterize and model accurately the process of aggregation of A
We have also
conducted several control experiments to exclude the possibility that
A
The extent to
which secreted A
Relatively more hydrophobic A
In addition to
peptide length, a large number of variables that may influence A
Finally, the ability to detect and quantitate
dimers, trimers, and tetramers of native A
We thank Drs. Christian Haass and Wei Qiao Qiu for
helpful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-protein (A
) accumulate progressively in the limbic and
cerebral cortex in Alzheimer's disease, where they are intimately
associated with neuronal and glial cytopathology. Attempts to model
this cytotoxicity in vitro using synthetic peptides have shown
that monomeric A
is relatively inert, whereas aggregated A
reproducibly exerts a variety of neurotoxic effects. The processes that
mediate the conversion of monomeric A
into a toxic aggregated
state are thus of great interest. Previous studies of this conversion
have employed high concentrations
(10
-10
M) of
synthetic A
peptides under nonbiological conditions. We report
here the detection of small amounts (<10
M) of SDS-stable A
oligomers in the culture media
of Chinese hamster ovary cells expressing endogenous or transfected
amyloid
-protein precursor genes. The identity of these oligomers
(primarily dimers and trimers) was established by immunoprecipitation
with a panel of A
antibodies, by electrophoretic comigration with
synthetic A
oligomers, and by amino acid sequencing. The
oligomeric A
species comprised
10-20% of the total
immunoprecipitable A
in these cultures. A truncated A
species
beginning at Arg 5 was enriched in the oligomers, suggesting that
amino-terminal heterogeneity can influence A
oligomerization in
this system. Addition of Congo red (10 µ
M) during
metabolic labeling of the cells led to increased monomeric and
decreased oligomeric A
. The ability to detect and quantitate
oligomers of secreted A
peptides in cell culture should facilitate
dynamic studies of the critical process of initial A
aggregation
under physiological conditions.
-protein (A
)
(
)
is a
hydrophobic proteolytic fragment of a ubiquitously expressed integral
membrane polypeptide termed the amyloid
-protein precursor
(
PP). Progressive cerebral deposition of A
is an invariant
feature of Alzheimer's disease (AD) that appears to precede the
development of the characteristic neuronal and glial cytopathology of
AD
(1) . One established molecular cause of familial (autosomal
dominant) AD (FAD) is the occurrence of missense mutations within or
immediately flanking the A
coding region of the
PP gene on
chromosome 21
(2) . Cerebral deposits of A
can exist as
dense, filamentous aggregates of the peptide surrounded by dystrophic
neurites and glial cells (classical or neuritic amyloid plaques) and as
loose, amorphous deposits associated with little or no local cellular
alteration (diffuse or preamyloid plaques). Recently, similar A
deposits, including some associated with neuritic dystrophy and
synaptic loss, have been described in transgenic mice overexpressing a
FAD-linked mutant
PP, strongly supporting the primacy of
PP
processing to A
in AD
(3) .
was
originally identified as the protein comprising the insoluble filaments
in AD amyloid deposits
(4) , it also occurs as a normal, soluble
product of cellular metabolism that is constitutively secreted by
PP-expressing cells and is found in cerebrospinal fluid and plasma
(5, 6, 7, 8) . As a result, the
mechanism by which soluble, monomeric A
gradually accumulates as
insoluble aggregates associated with surrounding cytotoxicity has
become a central question in AD pathobiology. Attempts to model the
toxicity of A
by adding synthetic peptides to neural cultures have
provided strong evidence that freshly solubilized, monomeric A
is
relatively inert, whereas aggregated, oligomeric, and polymeric A
that forms after incubating the peptide in vitro ( e.g. at 37° for 3-7 days) is cytotoxic ( e.g. see
Refs. 9-12). Many biophysical studies of the aggregation and/or
cytotoxicity of synthetic A
have been reported, but these have
several limitations. These studies employ high doses of synthetic
peptides, usually in the 10
-10
M range, whereas concentrations of A
in
physiological fluids are 10
-10
M(6, 13) , and normal concentrations in
brain are believed to be even lower
(14) . Aggregation is
generally examined under nonbiological conditions, e.g. using
synthetic peptides solubilized in organic solvents and then studied in
water or simple aqueous buffers free of other proteins. Peptides of a
single, specified length ( e.g. 28, 40, or 42 residues) are
generally used in contrast to the rich array of endogenous A
peptides having heterogeneous N and C termini that are produced by
cultured human cells
(5, 13, 15, 16) and found in human cerebrospinal fluid
(6, 17) and brain
(18, 19, 20) .
PP-transfected cells with A
-specific antibodies reveals, in
addition to monomeric 4-kDa A
and a related 3-kDa fragment (p3),
peptides migrating at
6, 8, and 12 kDa
(21) . We report
here the characterization of these peptides and show that they
represent stable oligomers that form from A
peptides secreted by
cells at high picomolar to low nanomolar levels. The ability to detect
these oligomers and to quantify their abundance among different
PP-expressing cell types will facilitate dynamic analyses of the
factors influencing the critical process of initiation of A
aggregation under physiological conditions.
Chinese hamster
ovary (CHO) cells were grown in Dulbecco's modified Eagle's
medium containing 10% fetal clone II (Hyclone). CHO cell lines
expressing wild-type PP-expressing Cell Lines
PP
were generated with the
expression vector pCMV751
(22) using the Lipofectin (Life
Technologies, Inc.) transfection method and selected by G418
resistance. Additional stable cell lines expressing certain
PP
missense mutations (for review see Ref. 2) were generated by
oligonucleotide-directed mutagenesis of pCMV751 and transfection into
CHO cells. These included the Val
Phe or Val
Ile
mutations at codon 717 (
PP
numbering) and the Lys
Asn and Met
Leu double mutation at codons 670 and 671
that are linked to early onset familial AD, and the Glu
Gln
mutation at codon 693 that causes hereditary cerebral hemorrhage with
amyloidosis, Dutch type. Multiple stable single clones were established
from each of these constructs.
Antibodies
The epitopes for many of the PP
antibodies used in this study are diagrammed (see Fig. 2). Rabbit
antisera to synthetic A
peptides include R1280 raised against
A
1-40
(5, 23) , antiserum Y to
A
1-38
(24) , and R1963 to A
21-37
(5) . Antiserum R1282 to synthetic A
1-40 was produced
as described for antiserum Y
(24) characterized
immunocytochemically on AD brain sections and by immunoprecipitation of
culture media and found to be highly similar to R1280. Monoclonal
antibodies 266 to A
13-28
(6) and 4G8 to
A
17-24
(25) were also used. Polyclonal antisera to
synthetic peptides of
PP outside the A
region included those
raised to
PP602-615
(26) ,
PP652-671,
PP724-739
(27) ,
PP751-770 (
C7)
(28) , and a bacterial recombinant protein of human
PP519-667 (
5)
(29) (all numbered according to
the
PP
isoform). Antiserum
PP652-671 was
generated similarly to
C7
(28) and characterized on
immunoblots of
PP-transfected cells and by immunoprecipitation of
cell lysates. Antibodies 266 and
5 were generously provided by
Athena Neurosciences, Inc.
Figure 2:
The 6-14-kDa proteins are
specifically immunoprecipitated by several A antibodies but not by
antibodies to
PP epitopes flanking A
. The positions of the
epitopes for the seven antibodies are indicated on the
PP
schematic. Antiserum dilution was 1:300 except for R1282 (1:150) and
R1963 (1:100).
PP antibodies flanking A
failed to precipitate
A
, p3, and the higher M
bands, while four
different A
antisera precipitated all of these
bands.
Immunoprecipitation, Gel Fluorography, and
Radiosequencing
Confluent 100-mm dishes of CHO cells were
incubated for 30-45 min in Met-free medium and then labeled
overnight with 300 µCi of [S]Met (75
µCi/ml; DuPont NEN) in 10% dialyzed fetal bovine serum (FBS)
(Hyclone). The conditioned media (CM) were spun at 2100
g for 30 min, precleared with protein A-Sepharose, and
immunoprecipitated overnight with the relevant antiserum (1:300 or as
noted), as described previously
(26) , except that all washes
were centrifuged at 5000
g. Immunoprecipitated samples
were then boiled for 3 min in SDS/
-mercaptoethanol (
ME)
sample buffer (Novex, San Diego, CA), electrophoresed on 10-20 or
16% Tris-tricine gels (Novex) and subjected to autofluorography. In
some control experiments, instead of eluting the immunoprecipitates
from protein A-Sepharose by boiling in SDS sample buffer, 0.2
M glycine, pH 2.5, was used, after which the samples were
neutralized with Tris base, mixed with SDS sample buffer lacking
ME, and directly electrophoresed without heating. Synthetic A
peptides used as positive controls for comigration studies were
A
1-42 (Bachem) and A
1-43 (generously provided by
S. Little, Lilly Research Labs). Antibodies were adsorbed at a
concentration of 10-15 µg of synthetic peptide/µl of
undiluted antiserum in 1% bovine serum albumin/phosphate-buffered
saline for 2-4 h at 4 °C and cleared at 16,000
g for 10 min. Gel bands were quantitated using a Molecular Dynamics
PhosphorImager 400A and ImageQuant software.
and related oligomers, cells were labeled
with [
H]Phe, and radiosequencing was performed as
described previously
(5, 30) .
Congo Red Experiments
Cells were labeled as above,
except in serum-free Dulbecco's modified Eagle's medium.
Congo red (Aldrich, Milwaukee, WI) was prepared as a stock solution of
10 m
M in MeSO and added to tissue culture medium
at a 1:1000 dilution during metabolic labeling.
peptides underwent
polymerization in cell culture, analogous to the aggregation,
precipitation, and seeding experiments reported for synthetic A
peptides in vitro ( e.g. see Refs. 10-12 and
31-35). Conditioned media from
[
S]methionine-labeled CHO cells stably
transfected with
PP
cDNA (CHO
) were
immunoprecipitated with R1280, a high-titer polyclonal antibody to
synthetic human A
, and electrophoresed in
Tris-tricine gels. Autofluorographs revealed bands of
6, 8, and 12
kDa, in addition to the characteristic 4- (A
) and 3-kDa (p3)
peptides (Fig. 1 A, lane 3). Absorption
of R1280 with synthetic A
abolished the
precipitation of A
and p3 as well as the three larger proteins
(Fig. 1 A, lane 4). As in numerous
previous reports
(31, 33) , an A
synthetic peptide
spontaneously formed SDS-stable oligomers upon storage; comparison of
an R1280 immunoblot of this peptide preparation
(Fig. 1 A, lane 2) with the fluorograph
of the R1280 immunoprecipitate of CHO
medium run in the
adjacent lane (Fig. 1 A, lane 3)
revealed that the 4-, 8-, and 12-kDa CHO-derived peptides comigrated
with bands of these sizes in the synthetic peptide sample, strongly
suggesting that the bands of molecular mass greater than 4 kDa
represent A
oligomers. Examination of several CHO lines stably
expressing mutant (
PPV717I,
PPV717F, and
PPE693Q) or
wild-type
PP
always revealed the 6, 8, and 12 kDa
R1280-reactive bands in all lines (Fig. 1 B).
Quantitation of these A
-specific bands by PhosphorImaging
demonstrated that differences among the cell lines examined to date
were not consistently dependent on the mutation expressed (data not
shown). The average percent of each A
-specific band as a function
of the total signal for all four A
bands was calculated for all of
the above cell lines. The mean percents ± S.D. ( n = 32) were: 4 kDa, 65 ± 11%; 6 kDa, 19 ± 7%;
8 kDa, 11 ± 4%; and 12 kDa, 4 ± 4%. Nontransfected CHO
cells, which endogenously express low levels of
PP, showed
substantially smaller amounts of these bands, as expected
(Fig. 1 C). In the experiments that follow, high
PP
expressing stable cell lines were utilized to enhance detection of the
oligomers. On longer fluorographic exposures of our highest
PP
expressing cell lines, faint A
-specific bands of
10, 14, and
sometimes 16 kDa were detected (Fig. 1 D).
Figure 1:
CM from labeled CHO cells
immunoprecipitated with A
antibodies contains proteins of
6-16 kDa in addition to A
and p3. A,
R1280-immunoprecipitated CM from [
S]Met-labeled
CHO
cells ( wt-1 line; see B below) was
electrophoresed on a 16% Tris-tricine gel ( lanes 3 and 4); purified synthetic A
peptides were loaded
directly in adjacent lanes ( 1 and 2). The gel was
transferred to polyvinylidine difluoride membrane and either
immunoblotted with R1280 ( lanes 1 and 2) or
subjected to autoradiography ( lanes 3 and
4). Lane 1, synthetic A
(100 ng); lane 2, synthetic
A
(100 ng). The extent of oligomerization of
the two peptides is not directly comparable, because the preparations
had different histories of in vitro handling and storage.
Lane 3, CHO
CM precipitated by R1280;
lane 4, CHO
CM precipitated by R1280
preabsorbed with synthetic A
. B,
comparison of wild-type and mutant
PP
stably
transfected CHO lines reveals the 6, 8, and 12 kDa bands in all lines.
Cell lysates (above) and CM (below) were immunoprecipitated with
anti-
PP
( C7) or R1280,
respectively, and electrophoresed on a 10-20% Tris-tricine gel.
Arrowhead indicates position of the full-length
PP in the
lysates. Quantitation of the A
-specific bands by PhosphorImaging
( n = 4) demonstrated that differences among the cell
lines in these experiments were not consistently dependent on the
mutation (data not shown). wt, wild-type; E693Q, Glu
to Gln at
PP
(
PP
numbering);
V717I ( V717F), Val to Ile (or Phe) at
PP
. C, R1280 immunoprecipitation of CM from
nontransfected CHO cells reveals smaller amounts of A
-related
bands. Lane 1, nontransfected cells; lane 2, CHO
wt-1 cells. D, longer
exposures of R1280-precipitated CM from the highest expressing cell
line ( V717F- 2; see B) reveal additional
bands of 10, 14, and 16 kDa.
To
determine whether the bands migrating between 6 and 14 kDa represented
A oligomers or larger fragments of
PP containing the A
region, we precipitated CHO
-conditioned media with
antibodies directed at
PP epitopes flanking the A
sequence
(residues 672-713 of
PP
).
Anti-
PP
(
5),
anti-
PP
,
anti-
PP
,
anti-
PP
, and
anti-
PP
(
C7) all
failed to precipitate A
, p3, and the higher M
peptides ( Fig. 2and data not shown). In contrast, six
additional A
antisera specific for either synthetic
A
, A
, or
A
peptides (Fig. 2) or to
native A
purified from AD amyloid filament fractions
(36) (not shown), precipitated each of the 3-5 higher
M
bands, A
, and in some cases, p3. Adsorption
of each of the synthetic A
antisera with its respective peptide
immunogen abolished the reaction (not shown).
oligomers was obtained
by sequencing them after metabolically labeling the cells with
[
H]phenylalanine
(5, 30) .
Radiosequence analysis of the 4-kDa band demonstrated peaks of
[
H]Phe in cycles 4, 19, and 20, consistent with
an A
peptide beginning at Asp
(Fig. 3 A), as has been found by radiosequencing in
other cell types
(5, 30) . Analysis of the 6 kDa band
revealed a profile similar to that of the 4 kDa band, as well as a peak
centered at cycle 15, consistent with an A
peptide beginning at
Arg
(as described previously in other cells
(30, 37) ) (Fig. 3 B). Minor
[
H]Phe peaks centered at cycles 10 and
24-25 consistent with A
species beginning at
Ile
and/or Glu
were also seen
(5, 30) . This result suggests that the 6 kDa band
represents an A
monomer or dimer with N-terminal heterogeneity
that migrates anomalously in this gel system. Radiosequence analysis of
the 8 kDa band revealed major peaks of [
H]Phe at
cycles 4, 19, and 20, consistent with A
beginning at
Asp
, and at cycles 15 and 16, consistent with an A
peptide beginning at Arg
. Minor peaks at cycles 9 and 10
are consistent with an Ile
and/or a Glu
species (Fig. 3 C). This result suggests that the 8
kDa band represents a dimeric species composed in large part of the
A
Asp 1 peptide but including dimers that are N-terminally
heterogeneous. In this regard, the 8 kDa gel band can sometimes be seen
to comprise a tightly spaced doublet ( e.g. Fig. 2
),
presumably representing structurally different A
dimers.
Radiosequencing of the 12 kDa band revealed
[
H]Phe at cycles 4, 19, and 20, again consistent
with A
beginning at Asp
, as well as at cycles 15 and
16, consistent with A
beginning at Arg
. In addition, a
major peak at cycle 3 is consistent with an A
species starting at
Ala
(Fig. 3 D). These results suggest that the 12
kDa band comprises A
trimers having N-terminal heterogeneity.
Figure 3:
Radiosequencing of 6-, 8-, and 12-kDa
A-reactive proteins confirms their identity as oligomers.
[
H]Phenylalanine radioactivity obtained at each
cycle of Edman degradation is graphed for the 4 (A),
6 ( B), 8 ( C), and 12 kDa ( D) bands
precipitated by R1280 from CHO
-conditioned
medium.
Because aggregation of synthetic A peptides may be modified by
several serum proteins, including apolipoprotein E, apolipoprotein J,
and transthyretin
(38, 39, 40, 41) , we
investigated the effect of serum on A
oligomer formation in our
system. CHO cells labeled overnight in the absence of fetal bovine
serum showed markedly reduced levels of the 4 and 6 kDa A
bands
and an increase in the 8-12 kDa bands when compared with cells
labeled conventionally in 10% serum (Fig. 4 A). Importantly,
the addition of serum to labeled cell-free conditioned medium just
prior to the immunoprecipitation reaction resulted in no change in the
monomer/oligomer pattern (Fig. 4 B), indicating that
serum does not alter the immunoprecipitation. These results suggest
that proteins in serum can stabilize the amount of A
monomer and
decrease the formation of A
oligomers in our cultures.
Figure 4:
Conditions affecting the relative amounts
of A monomer and oligomers. A, the omission of FBS during
overnight labeling resulted in marked decreases in the A
and 6 kDa
bands, and a concurrent increase in the amounts of the 8 and 12 kDa
oligomeric bands. Preabsorption of R1280 with synthetic
A
( lanes marked -)
shows the specificity of all bands. B, the addition
of FBS to conditioned medium just prior to the immunoprecipitation
reaction had no effect. Lanes 1 and 2,
overnight labeling in the presence ( lane 1) or
absence ( lane 2) of 10% FBS. Lane 3, 10% FBS added to serum-free conditioned medium prior
to immunoprecipitation. C, the addition of 10 µ
M
Congo red ( lane 2) during overnight labeling in
serum-free medium resulted in substantial increases in the A
and 6
kDa bands, with a slight decrease in the amount of the 8 and 12 kDa
bands when compared with the addition of the Me
SO vehicle
alone ( lane 1). Lane 3, overnight
labeling in serum-free medium alone; lane 4,
overnight labeling in serum-free medium plus Me
SO at 1:1000
caused no change in A
and the oligomers; lane 5,
overnight labeling in 10 µ
M Congo red; lane 6, conditioned serum-free medium plus 10 µ
M
Congo red added just prior to immunoprecipitation also caused no change
in the A
/oligomer pattern. D, elution of R1280
immunoprecipitates from protein A-Sepharose with 0.2
M
glycine, pH 2.5 ( lane 2) gave the same A
monomer
and oligomer pattern as conventional elution by boiling in SDS/
ME
sample buffer ( lane 1). V717F-2 cells used in
A-D.
To
assess whether the extent of A oligomerization in culture can be
altered pharmacologically, we chose to investigate the effect of the
dye Congo red. Congo red has been shown to decrease the accumulation of
the protease-resistant form of the prion protein and scrapie
infectivity in vitro, resulting in the inhibition of amyloid
fibril formation in this model
(42) . Addition of 10
µ
M Congo red during serum-free metabolic labeling of our
cells overnight produced a dramatic increase of the 4 and 6 kDa A
bands and a concurrent slight decrease of the 8 and 12 kDa oligomeric
bands (Fig. 4 C, lanes 1 and
2). At lower doses (2.5 and 5 µ
M), a
dose-dependent increase of the 4 and 6 kDa bands occurred (data not
shown). Addition of the Congo red vehicle, Me
SO (1:1000),
to the labeling medium had no effect on the A
pattern
(Fig. 4 C, lanes 3 and 4).
Addition of 10 µ
M Congo red to the labeled conditioned
medium just prior to immunoprecipitation also had no effect on the
A
pattern (Fig. 4 C, lane 6).
Experiments are underway to determine whether the consistent
A
-raising/oligomer-lowering effect of Congo red is due to
inhibition of the conversion of monomeric A
to oligomers,
stabilization of the A
monomer from degradation, altered A
production, or some combination of these.
can be
detected in CHO-conditioned medium by immunoprecipitation. To exclude
the possibility that these oligomers formed only during the
immunoprecipitation reaction, several control experiments were
performed. Synthetic A
was added to either
fresh Dulbecco's modified Eagle's medium with 10% FBS or to
this medium conditioned overnight by CHO
cells at
concentrations of the peptide that matched or exceeded those of
secreted A
(
5 n
M). Incubation at 37 °C for 4 h
followed by R1280 precipitation under conditions identical to those
that demonstrated A
oligomers in cell culture revealed only the
A
monomer and no oligomeric bands on immunoblots (data not shown),
indicating that our immunoprecipitation procedure does not induce
oligomerization of synthetic A
. To exclude the possibility that
boiling the samples in SDS sample buffer prior to electrophoresis
induced the aggregation, immunoprecipitated samples were eluted from
protein A-Sepharose with glycine, pH 2.5, neutralized and loaded
without heating in a sample buffer devoid of
ME; gel fluorography
revealed the usual pattern of monomeric and oligomeric A
bands
(Fig. 4 D). Furthermore, addition of either FBS or Congo red to
serum-free labeled conditioned medium just prior to the
immunoprecipitation reaction had no effect, whereas their presence
during cell labeling consistently resulted in dramatically altered
patterns of A
monomer and oligomers (Fig. 4, B and
C).
peptides in hippocampal cortical
cultures ( e.g. see Refs. 43-45) and in brain-injected
animals ( e.g. see Refs. 46-48) have led to widely
divergent results. In some studies, neuronal toxicity and loss were
reproducibly seen when synthetic A
was
compared with control peptides administered in the same vehicle. Other
studies using putatively highly similar or identical peptides showed
little or no induction of specific toxicity (for review, see Ref. 49).
Subsequent analyses of different lots of peptides synthesized by the
same or different facilities have revealed that A
peptides of
identical sequence can adopt varying degrees of random coil,
-helical, and
-pleated sheet secondary structure as well as
varying states of aggregation ( e.g. see Refs. 10-12).
Freshly prepared A
peptides can exist in a largely random coil
monomeric state that is unassociated with significant cellular effects,
but they can be converted into a principally
-pleated sheet
conformation and a partially aggregated state that induces
neurotoxicity by incubating (``aging'') the peptide in buffer
for several days
(10, 11, 12) . There is
evidence that a conformational transition to increasing
-sheet
structure and the subsequent polymerization of the peptide to fibrils
underlies its conversion to a toxic moiety
(11, 12, 32, 50) . It is possible that
an analogous slow aggregation/polymerization process may explain in
part the differences between the apparently pathologically inert,
largely nonfibrillar A
found in diffuse plaques
(51) , and
the fibrillar deposits found in plaques that are intimately associated
with neuritic and glial dystrophy
(52) .
peptides that occurs under physiological conditions in cerebral tissue.
To this end, we have searched for evidence of spontaneous aggregation
of the native A
peptides that are produced by cells. Several lines
of evidence support the conclusion that the 8-16-kDa
R1280-reactive proteins we have detected in conditioned medium are
oligomers of A
: ( a) their specific, adsorbable
immunochemical reaction with all 6 A
antibodies examined but not
with various antibodies to flanking
PP regions; ( b) their
electrophoretic co-migration with oligomers of synthetic A
peptide; ( c) the substantially higher amounts of these
proteins in
PP-transfected than untransfected cells; and
( d) the direct confirmation of the A
composition of the
6-, 8-, and 12-kDa proteins by radiosequencing.
aggregation arises only during the immunoprecipitation reaction.
First, synthetic A
, which is the same length
as the principal A
peptide secreted by cultured cells
(15) , does not undergo oligomer formation during
immunoprecipitation when added to CHO-conditioned medium at endogenous
or higher concentrations. Second, elimination of boiling and
ME in
the immunoprecipitation protocol does not change the appearance of the
oligomers. Third, and most importantly, we describe two manipulations
(omission of serum and addition of Congo red) that reproducibly alter
the relative amounts of A
oligomers versus monomers in
the CHO medium when present during metabolic labeling ( i.e. during putative formation of the oligomers) but have no effects
when performed just prior to the immunoprecipitation ( i.e. after the oligomers have formed). This finding clearly indicates
that the oligomers exist in the medium prior to the immunoprecipitation
reaction rather than only after it. However, because there is no method
to assay unconcentrated conditioned medium directly for these very low
abundance (picomolar level) oligomers, we cannot exclude the
possibility that some additional A
oligomerization could occur
during concentration. We are currently examining the effects of Congo
red and serum proteins on the A
oligomer to monomer ratio; these
mechanisms are likely to be heterogeneous and complex.
peptides of various lengths participate in A
oligomerization in vivo and in cell culture remains to be
determined. It has been proposed that A
peptides longer than 40
residues, particularly the A
peptide believed
to be a major constituent of the compacted plaque cores
(19, 20, 53, 54) , could serve as a
nidus for the aggregation of shorter species, including the much more
abundantly secreted A
peptide
(34, 55) . Small amounts of A
peptides ending at
residue 42 have been detected in the media of
PP-transfected cells
(13, 15) and in human cerebrospinal fluid
(17) .
Moreover, FAD-linked
PP missense mutations located near the
carboxyl end of the A
region have been shown to cause enhanced
secretion of the longer (42 residue) A
species
(13) .
Synthetic A
has been shown to aggregate
preferentially into fibrillar amyloid
(34) . The role of
A
in the formation of the 6-16-kDa
A
oligomers in cell culture needs to be elucidated using
carboxyl-terminal specific antibodies for immunoprecipitation and
enzyme-linked immunosorbent analysis. We have not yet observed a
consistant effect of these mutations on oligomer levels in our
transfected cells. This could be due to the use of antibodies that do
not preferentially detect longer A
peptides or to the short period
of time over which we assayed in vitro A
aggregation.
In vivo, although A
1-42 is made throughout life, it
takes at least several decades before one can detect any aggregated
A
deposits in human brain.
peptides, such as species beginning at Arg
or
Glu
, have also been detected in the medium of
PP-expressing cells and hypothesized to be important in enhanced
A
aggregation
(30, 56) . In this regard, we could
detect virtually no Arg
A
in the 4 kDa monomeric band
from our cultures, and yet we found small amounts in the SDS-stable
multimers we obtained from the same cells (Fig. 3), supporting
the hypothesis of enhanced participation of the heterogeneous A
monomeric species in oligomerization. That A
N-terminal
heterogeneity influences the aggregation of A
into amyloid in
vivo has recently been shown in AD brain tissue by the use of
N-terminal specific A
antibodies
(57) .
oligomerization could be assessed under physiological conditions and
concentrations in the cell culture system we describe. These variables
include the effects of
amyloid-associated proteins
(``pathological chaperones'') ( e.g. heparan sulfate
proteoglycan
(58) and apolipoproteins E and J
(38, 39) ), metal ions
(35, 59) , and
oxidizing and anti-oxidizing agents
(60, 61, 62) . The addition of these molecules
to the cultures, the use of doubly transfected CHO cell lines
expressing
PP and a chaperone protein of interest, and the study
of cell lines genetically deficient in a particular chaperone could
lead to new insights about how such molecules influence the earliest
stages of A
aggregation. It is also likely that certain cell types
and certain culture conditions may be found that allow much more
extensive aggregation of endogenous A
than we have observed in
this initial study. We have recently detected small amounts of A
oligomers after long autofluorographic exposures of the
R1280-precipitated media of
PP-transfected human kidney 293
cells.
(
)
The levels of the putative oligomers
were far lower than those in CHO transfectants secreting similar
amounts of A
monomer. This result suggests that in vitro oligomerization of A
can occur in different cell types and
that cell-specific factors are likely to promote or retard the extent
of A
aggregation.
at submicromolar
concentrations similar to those likely to exist within early A
deposits could allow such cultures to be used to screen and
characterize small molecules that inhibit the formation, or proteases
that enhance the clearance, of neurotoxic A
aggregates. We have
observed a consistent change in A
monomer to oligomer ratios after
treatment with Congo red, providing an example of such an amyloid
binding compound. Further characterization of the cellular and
extracellular effects of this and similar compounds on the production,
initial aggregation, and clearance of A
can now be conducted under
physiological conditions.
, amyloid
-protein;
PP,
-protein precursor; AD, Alzheimer's
disease; FAD, familial (autosomal dominant) Alzheimer's disease;
CHO, Chinese hamster ovary; FBS, fetal bovine serum; CM, conditioned
medium;
ME,
-mercaptoethanol.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.