(Received for publication, June 21, 1995; and in revised form, November 27, 1995)
From the
Ultracentrifugation and graded molecular sieving, as well as a
sensitive sandwich enzyme-linked immunosorbent assay were used to
isolate and quantitate the amounts of water-soluble oligomers of
amyloid (A
) peptides N-40 and N-42 in cerebral cortex of normal
and Alzheimer disease (AD) brains. AD brains contained 6-fold more
water-soluble A
(wsA
) than control brains. The majority of
water-soluble peptides in most AD cases was A
N-42, representing
12 times the amount found in control brains. The wsA
was present
in the form of monomers and oligomers ranging from less than 10 kDa to
greater than 100 kDa. The amount of wsA
N-42 in AD brains is about
50 times greater than the level of soluble A
N-42 found in the CSF
of AD patients. This disparity may be due to the rapid association of
wsA
N-42 into fibrillar deposits and/or to the integrity of the
anatomical barriers which separate the two extracellular spaces. In
this paper, we consider soluble any form of A
which has not yet
polymerized into its insoluble, filamentous form. This includes both
the newly synthesized forms of A
and those peptides which may be
loosely attached to insoluble filaments but which can, nevertheless,
still be considered soluble. It has been previously shown that, once it
has aggregated into its filamentous form, the A
peptides are
resistant to disaggregation and degradation by a number of denaturing
agents and aqueous buffers containing proteolytic enzymes. Therefore,
it is likely that the water-soluble A
peptides we quantified are
precursors to its insoluble, filamentous form. Consequently, reducing
the levels of soluble A
in AD brains could have profound effects
on AD pathophysiology.
The pathology of Alzheimer disease (AD) ()is
characterized by the deposition of the
amyloid (A
) peptides
in the extracellular space of the brain parenchyma and in the walls of
the cerebral blood vessels. These peptides are derived by proteolytic
degradation of a larger molecule, the A
precursor protein, whose
gene is localized on chromosome 21 (reviewed in (1) ). From a
chemical point of view, A
is represented by polypeptide chains 40
to 42 amino acid residues long with a M
of
4,500(2, 3, 4) . Another class of amyloid
associated with diffuse deposits contains the amino acid sequence of
residues 17-42 (3 kDa), corresponding to the C-terminal sequence
of the 1-42 A
form(5) . All of these A
peptides
are very insoluble and resistant to proteolytic degradation. These
physicochemical properties primarily result from the C-terminal amino
acid sequence of 12 to 14 hydrophobic residues which play an important
role in the initial aggregation and insolubility of A
. The
amphipathic N-terminal region of the A
molecule, consisting of 28
amino acids, appears to be necessary for the polymerization of this
peptide into cytotoxic 10-nm filaments. In addition, the N-terminal
portion of the A
molecule is probably responsible for the binding
to ancillary molecules such as apolipoproteins(6) ,
glycosaminoglycans (7) ,
1-antichymotrypsin(8) ,
the complement protein C1q(9) , and metal ions(10) .
According to their morphology, the A peptide deposits can be
classified as either fibrillar or amorphous. The fibrillar forms are
usually observed at the center of and around the neuritic plaques where
they are surrounded by dystrophic neurites and reactive glial cells.
They are also observed in the parenchymal and leptomeningeal blood
vessels, where they cause destruction of the vascular walls and myocyte
degeneration(11, 12) . The amorphous forms of A
are mainly localized to diffuse deposits scattered throughout the
brain's gray matter and apparently do not cause any observable
pathological alterations. Interestingly, numerous cell lines in culture
produce small quantities of soluble A
1-40, which suggests a
physiological role for the shorter A
peptide(13, 14, 15) . The A
1-42,
on the other hand, seems to be the major component of the parenchymal
and, to a lesser extent, the vascular deposits of
AD(2, 3, 4, 16) . The abundance of
this peptide suggests that it plays a relevant role in the
pathophysiology of AD.
Recently, small amounts of apparently soluble
low molecular mass A peptides (3.0, 3.7, and 4.0 kDa) were
isolated by immunoprecipitation from AD brain homogenates, which were
not detected in normal brains (17, 18) . Utilizing the
more sensitive sandwich ELISA soluble A
peptides have been
identified in normal and AD brains(19) . In the present paper,
we have further investigated the A
water-soluble fractions based
on a more rigorous centrifugal separation of AD and control brain
homogenates free of detergents or chaotropic agents. In addition, we
quantified the water-soluble oligomeric A
by sandwich ELISA in
fractions obtained by ultracentrifugation and graded membrane
filtration.
Figure 1:
Flow chart showing the
purification procedures utilized to separate the water-soluble and
insoluble fractions of A. The sandwich ELISA was performed in all boxed fractions. P and S refer to pellets
and supernatants, respectively. Brain homogenates were spun at 135,000 g for 2 h instead of the accepted criteria of 100,000
g for 1 h. This higher centrifugation yielded a better
separation between insoluble A
filaments and the more soluble
monomeric and oligomeric forms of this peptide. The water-soluble
material was further spun at 220,000
g and the
resulting supernatant subjected to ultrafiltration and
ultracentrifugation. The water-insoluble pellet, obtained from the
initial brain homogenate, was lysed in 10% SDS and centrifuged at
135,000
g. The resulting pellet and supernatant (P135
and S135, respectively) were further analyzed. The former contained
large quantities of insoluble fibrillar amyloid A
1-40,
1-42(5) . The latter fraction (see large shaded
box) also carried the 3-kDa A
17-42 peptide which was
purified by HPLC as described previously(5) . The A
17-42 is the most insoluble of all A
species. This peptide
only disaggregates into smaller micelle-like particles in SDS or into
monomers in the presence of strong denaturing agents such as 80% formic
acid(5
The pellet
retrieved after the 135,000 g centrifugation of the
initial homogenate, containing the insoluble A
, was dissolved in
10% SDS, Tris-HCl, pH 8. After standing 6 h at room temperature, the
lysate was centrifuged at 135,000
g for 2 h (Beckman
SW28 rotor) at 20 °C. The ensuing pellet (P135) was washed twice
with 20 mM Tris-HCl, pH 8, dissolved in 5 ml of 80% glass
distilled formic acid, and centrifuged at 275,000
g for 30 min at 4 °C. This step permitted the separation of
insoluble lipofuscin from the solubilized A
. The supernatant
(S275) was concentrated by vacuum centrifugation, dialyzed (1,000 Da
cutoff) against Tris buffer, and analyzed for A
by ELISA. The
supernatant derived from the SDS-135,000
g centrifugation (S135) was spun at 275,000
g (Beckman 41 Ti rotor) for 2 h at 4 °C. The resulting pellet
was suspended in 100 mM Tris-HCl, pH 8, containing 2 mM CaCl
and digested with 10 µg/ml DNase I
(Worthington). Following centrifugation at 275,000
g for 2 h, the pellet (P275) was dissolved in 80% formic acid and
submitted to size exclusion HPLC in a TSK-3000 SW column (0.7
60 cm, Altex). The chromatography was developed in 80% formic acid as
described previously(5) . In addition, the S275 formic acid
supernatant derived from P135 was also investigated by HPLC as
described above.
The quantitation of A 1-42
in samples was done with the capture antibody 266 and the rabbit
polyclonal antibody 277.2 developed to A
residues
39-43(23) . An alkaline phosphatase labeled-affinity
purified F(ab`)
fragment of donkey anti-rabbit IgG
(H+L), absorbed against mouse IgG, was added to the wells and
incubated for 1 h. After aspirating and washing the plates, a
chemiluminescent substrate, AMPPD, and an enhancer, emerald green, were
added and the chemiluminescence was detected using a chemiluminometer.
Standard curves for the assay were generated from samples of known
concentrations (0.125 to 2.0 ng/ml) of A
1-42. This assay
fails to detect A
1-40 or shorter A
species and has
less than 5% cross-reactivity with A
1-41 and A
1-43. The values obtained from the above assays were subtracted
from each other to determine the amount of A
containing amino
acids 1-28 through 1-40(N-40).
Previous studies from our laboratory and others have focused
on the chemical analysis of the fibrillar A present in neuritic
plaques and in the walls of the blood vessels. Due to the extreme
insolubility of these peptides, their purification required the
utilization of a variety of detergents and/or strong chaotropic and
denaturing agents. While these methods have been effective in isolating
the A
from the amyloid deposits, they have prevented the
characterization of the soluble A
which is the precursor of the
insoluble, filamentous form. This soluble pool of A
is
operationally defined as A
extracted from brain tissue under mild
condition (i.e. homogenated in 20 mM Tris-HCl, pH
8.5) and is hereafter referred to as water-soluble A
(wsA
).
The isolation and analysis of these water-soluble low molecular weight
forms, in the absence of the aforementioned agents, may help to
determine the degree to which they participate in the pathophysiology
of AD.
In the present study, we separated the pools of water soluble
and insoluble A from AD and control brains (Fig. 1). The
detection and quantitation of A
in our study, using sensitive
sandwich ELISA, revealed that wsA
N-40 and N-42 are present in the
aqueous extractable fraction of AD and control brains (Fig. 2A and Table 2). The level of insoluble
A
in AD brains is, on the average, 100 times that of control
brains, whereas the amount of wsA
in AD brains is about 6 times
that detected in control brains (Fig. 2, A and B, and Table 2). An interesting observation is that
there is about 50 times the quantity of wsA
N-42 found in AD brain
cortex as that detected by Vigo-Pelfrey et al.(
)in
the CSF of AD patients (A
N-42 = 0.4 ng/ml) using the same
ELISA technique. A possible explanation for the discrepancy between
these two pools may be that in AD brain parenchyma, this peptide is
rapidly incorporated into the insoluble 10-nm filaments characteristic
of this disease, drastically decreasing its opportunity of being
translocated into the CSF. An alternative explanation for the huge
difference found could be due to the integrity of the physical barriers
which exist between the extracellular space of the brain parenchyma and
the subarachnoidal space. These barriers are represented by the glia
limitans at the surface of the cerebral cortex and by the outer wall of
the perivascular spaces which is derived from the
leptomeninges(24) . In support of this possibility are the
presence of wisps of amyloid frequently observed in the molecular layer
of the cerebral cortex, just beneath the glia limitans and also in
adjoining perivascular spaces.
Figure 2:
Histograms depicting the amounts of
water-soluble and insoluble A in AD and control brains as
quantitated by sandwich ELISA. A, the distribution of wsA
recovered in the S220 and P220 fractions. The amount of amyloid in the
control cases (A-D) ranged from 5.3 to 10.2 ng/g and in the AD
cases (1-8) ranged from 21.0 to 89.1 ng/g. A
in the
P220 control group is not shown in the histograms since it amounts to
only 0.1 ng/g on average. B, total amount of insoluble A
(P135) in the control and AD cases. The absence of value in AD case 4
is due to its total investment in the chromatographic study. The
quantities of amyloid in the control cases (A-D) ranged from
5.1 to 20.8 ng/g and in the AD cases (1-8) ranged from
377.3 to 3000.0 ng/g. C, ultrafiltration values of A
.
Centricon membranes partitioned S220 into an apparent continuous
distribution of wsA
which could range from monomeric and dimeric
(<10 kDa) to polymeric (>100 kDa). The amounts of wsA
N-40
and N-42 in each of the filtrates and final retentate is shown in Table 3.
Analysis of the relative amounts of
wsA N-40 and N-42 revealed that the predominant form of wsA
in control brains is N-40. On the average, the N-40:N-42 ratio of
wsA
in control brains is 75:25, whereas in AD brains this ratio is
48:52 (Table 3). The amount of wsA
N-40 found in 5 of the 8
AD brains studied (cases 2, 5, 6, 7, and 8) fell within the same range
as that detected in the control group. Pathology reports of the three
remaining AD brains (cases 1, 3, and 4), which contained higher
quantities of wsA
N-40, indicated a significantly higher level of
cerebrovascular amyloidosis than the other five cases. This elevated
level of A
N-40 in cerebrovascular amyloidosis is in agreement
with data recently reported by Younkin's group(25) .
Significantly, since the average amount of wsA
N-42 in the AD
brain (19.0 ng/g of cortex) is higher than in the control group (1.6
ng/g of cortex), and the amount of N-40 was comparable in the
aforementioned 5 cases, it is reasonable to conclude that it is the
water-soluble N-42 peptide which is the main contributor to the
fibrillar deposits of A
in AD.
Molecular sieving techniques on
the S220 fraction demonstrated what appears to be a continuous
distribution of monomeric and oligomeric wsA ranging in size from
less than 10 kDa to more than 100 kDa (Fig. 2C). On the
average, the proportion of wsA
N-42 in the AD brains decreased
from about 70% in the <10 kDa (monomeric and dimeric) fraction to
about 40% in the 100-30 kDa and >100 kDa (octameric and larger)
fraction (Table 3). This may provide evidence that the
incorporation of wsA
N-42 into insoluble filaments occurs only
after it has oligomerized into octameric and larger molecules. However,
it is unclear if the apparent decrease in the proportion of N-42 in the
higher molecular weight fractions is actually due to a reduction in the
level of the peptide or to the inability of the ELISA to measure
accurately the level of N-42 due to steric hindrance caused by antibody
binding to the aggregated peptides. The results of our
ultracentrifugation studies also lend support to the presence of low M
wsA
in S220 fraction, since 50% of these
peptides were sedimented at 435,000
g. Under this
experimental condition, 73% of myoglobin (17 kDa) is pelleted. Hence,
it could be assumed that a large quantity of wsA
in S220 is
smaller than 17 kDa.
In a previous article, Tamaoka et al.(19) recovered soluble A from successive Tris buffer
extractions of brain homogenates in which they utilized a 1:2 ratio of
cortical tissue to buffer. They suggested that the apparently
``newly'' solubilized A
was derived from a
``reversible'' depolymerization of this peptide from
insoluble amyloid deposits. In our experience, the high density of the
homogenate and the lower centrifugal forces (100,000
g, 15 min) do not permit extraction and separation of the
soluble A
fraction. It is also possible that the magnitude of the
mechanical forces necessary to disperse such a dense homogenate may
account for the shearing of insoluble A
into their soluble
fraction.
In this study we considered the aqueous-extracted A
to be soluble if it failed to form a pellet after centrifugation at
220,000
g for 2 h. A pathway must exist by which newly
synthesized water-soluble A
polymerizes into its fibrillar,
insoluble form. It is yet to be determined at what stage in this
polymerization process insolubility is conferred upon the A
peptide, but there is ample evidence to conclude that once it achieves
its fibrillar form it is extremely insoluble in a variety of denaturing
agents and is non-degradable by proteolytic enzymes. Both of these
phenomena have been independently documented by both Selkoe's
group (26) and by this laboratory(27) , and very
recently communicated by Harigaya et al.(18) as the
aqueous insoluble A
fraction or SDS-formic acid soluble A
component. It is likely that during the polymerization process there
exists an intermediate, oligomeric A
which is still soluble but
may be loosely associated to the insoluble filaments prior to its
complete incorporation. Both soluble forms of this peptide could
potentially be precursors to the insoluble, filamentous A
prominent in AD.
We attempted in this study to determine the extent
to which the wsA quantitated by ELISA could actually be A
peptides which had dissociated from insoluble filaments during
purification procedures. For this reason, we conducted correlation
analyses between the observed amyloid burden and wsA
, amyloid
burden and insoluble A
, and between water-soluble and insoluble
A
, to estimate the extent to which this may have occurred. If the
wsA
quantitated is that which has dissociated from plaques, a
strong correlation between ELISA-measured wsA
and A
deposition (as measured by either A
burden or total insoluble
A
) would be expected. Although there is a weak correlation between
wsA
and A
burden (Fig. 3A), the absence of
correlation between insoluble A
and A
burden (Fig. 3B) would suggest that the former correlation may
lack relevance. The most likely explanation for this is that amyloid is
unevenly distributed from region to region in the cerebral cortex. It
is also possible that a weakly positive correlation does, in fact,
exist between wsA
and amyloid burden. However, since amyloid
formation is a dynamic process, the size of this water-soluble pool
will likely fluctuate and the existence of a weakly positive
correlation may or may not have pathophysiological significance. Fig. 3C shows that no correlation exists between
wsA
and insoluble A
. These data suggest that the majority of
the ELISA-measured wsA
may be newly synthesized A
and a
smaller percentage consists of loosely attached peptides which have not
yet been incorporated into the insoluble A
filaments.
Interestingly, when cases 3 and 8, possibly perceived as outliers, are
removed from the analysis, the correlation between insoluble A
and
A
burden becomes significant (
= 0.923; p < 0.05). However, removal of these two cases results in no
significant correlation between wsA
and A
burden (
= 0.528, p = 0.361). This lends support that
most of the A
present in this soluble pool is that which has been
newly synthesized. It is not yet known if all of the A
in this
water-soluble pool, whether newly synthesized or dissociated from
insoluble filaments, is ultimately incorporated into insoluble
filaments or if some percentage of this pool is subject to degradation
by proteolytic enzymes.
Figure 3:
Correlation analyses of quantitated A
in AD brains. A, correlation between A
burden and
ELISA-measured wsA
. Correlation coefficient
= 0.647; p = 0.116. B, correlation between A
burden and ELISA-measured insoluble A
. Correlation coefficient
= 0.364; p = 0.423. C, correlation between ELISA-measured wsA
and ELISA-measured
insoluble A
. Correlation coefficient
= -0.204; p = 0.661.
The identification of a water-soluble pool
of A N-42 allows us to contemplate its possible role in AD
pathology, independent of its potential contribution to the formation
of neuritic plaques and vascular deposits. There is a discordance
between the amount of fibrillar amyloid deposits and the extent of
neuronal pathology in AD brains. The location of the neuritic plaques
appears separate and distinct from the sites of greatest neuronal loss
and synaptic pathology. These observations have caused many to question
the relevance of the fibrillar A
deposits to the dementing process
of AD. In our opinion, a possible element which could reconcile this
seeming discordance is the existence of a potentially toxic,
water-soluble pool of oligomeric A
1-42. Our results show
that, on the average, the oligomeric wsA
N-42 pool is uniquely
elevated (12-fold) in AD compared to that of normal brains. The M
of these oligomers would favor their diffusion
throughout the brain parenchyma, however, this soluble pool would
likely go undetected using conventional immunochemical techniques.
Therefore, given our observation that a pool of water-soluble A
is
present in the cerebral cortex of control and AD brains, the potential
toxic effects of oligomeric A
upon nerve cells calls for further
investigation.