From the Department of Psychiatry and Genetics and
Aging Unit and the § Department of Neurology and Genetics
and Aging Unit, Massachusetts General Hospital, Harvard Medical School,
Boston, Massachusetts 02114
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ABSTRACT |
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The cortical deposition of A is an event that
occurs in Alzheimer's disease, Down's syndrome, head injury, and
normal aging. Previously, in appraising the effects of different
neurochemical factors that impact upon the solubility of A
, we
observed that Zn2+ was the predominant bioessential
metal to induce the aggregation of soluble A
at pH 7.4 in
vitro and that this reaction is totally reversible with
chelation. We now report that unlike other biometals tested at maximal
biological concentrations, marked Cu2+-induced aggregation
of A
1-40 emerged as the solution pH was lowered from
7.4 to 6.8 and that the reaction was completely reversible with either
chelation or alkalinization. This interaction was comparable to the
pH-dependent effect of Cu2+ on insulin
aggregation but was not seen for aprotinin or albumin. A
1-40 bound three to four Cu2+ ions when
precipitated at pH 7.0. Rapid, pH-sensitive aggregation occurred at low
nanomolar concentrations of both A
1-40 and
A
1-42 with submicromolar concentrations of
Cu2+. Unlike A
1-40, A
1-42
was precipitated by submicromolar Cu2+ concentrations at pH
7.4. Rat A
1-40 and histidine-modified human
A
1-40 were not aggregated by Zn2+,
Cu2+, or Fe3+, indicating that histidine
residues are essential for metal-mediated A
assembly. These results
indicate that H+-induced conformational changes unmask a
metal-binding site on A
that mediates reversible assembly of the
peptide. Since a mildly acidic environment together with increased
Zn2+ and Cu2+ are common features of
inflammation, we propose that A
aggregation by these factors may be
a response to local injury. Cu2+, Zn2+, and
Fe3+ association with A
explains the recently reported
enrichment of these metal ions in amyloid plaques in Alzheimer's
disease.
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INTRODUCTION |
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A, a low molecular weight (39-43 amino acids) protein that is
a proteolytic product derived from the larger amyloid precursor protein
(1-3), is the major component of neocortical amyloid collections in
Alzheimer's disease (AD1;
see Refs. 4 and 5). As is the case with other amyloid proteins, A
originates as a normally soluble and constitutive protein found in
biological fluids and tissue (6-15). A
also aggregates to form
diffuse amorphous deposits in AD but also following head injury
(16-18) and in healthy aged individuals (19). Combined with other
inflammatory proteins such as proteoglycans (20), amyloid P-component
(21), and apolipoprotein E (22, 23), A
is found in the brains of
individuals affected by AD and Down's syndrome (DS) as dense
extracellular deposits of twisted
-pleated sheet fibrils in the
neuropil (senile plaques) and within cerebral blood vessels (amyloid
congophilic angiopathy; see Refs. 4 and 5). The deposition of A
,
however, is not confined to the brain parenchyma, having been detected
in amyloid diseases of the muscle (24-26), blood vessels (27, 28), and
in the kidneys, lungs, skin, subcutaneous tissue, and intestine of AD
patients (29, 30).
Cerebral A deposition occurs in other aged mammals that have the
human A
sequence (31) but is not a feature of aged rats (32, 33).
Although soluble A
1-40 is produced by rat neuronal
tissue (34), it contains three amino acid substitutions (Arg
Gly,
Tyr
Phe, and His
Arg at positions 5, 10, and 13, respectively
(32)) that appear to alter the physicochemical properties of the
peptide preventing it from precipitating in the neocortex.
A accumulates in the brain in AD and DS in forms that can be
resolubilized in water (35, 36) but also in forms that require harsher
conditions to extract and exhibit associated SDS-resistant polymerization on polyacrylamide gel electrophoresis (35, 37). A
1-40 is the predominant soluble species in biological fluids, whereas A
1-42, a minor species of A
that is
more insoluble in vitro, is the predominant species found in
plaques and deposits associated with AD and DS (35-41).
We have pursued studies of the physicochemical properties of synthetic
A peptides in order to appraise the potential of various neurochemical environments to induce A
deposition. To this end, we
had previously found that A
is strikingly precipitated by certain
metals in vitro, in particular Zn2+ (42-45).
This is important since zinc and other biometals are concentrated in
the brain neocortical parenchyma. A recent study, using micro
particle-induced x-ray emission analyses of the cortical and accessory
basal nuclei of the amygdala, demonstrated that levels of
Zn2+, Fe3+, and Cu2+ are
significantly elevated within AD neuropil compared with control neuropil and that these metal ions are significantly further
concentrated within the core and periphery of plaque deposits (46). We
have also found that the extraction of A
deposits from brain tissue into aqueous buffers is increased in the presence of chelators of
Zn2+ and Cu2+ (47), providing further evidence
that these metal ions participate in the deposition of A
within
amyloid plaques.
In common with the appearance of amorphous and amyloid deposits is the
observation that altered neuronal H+ homeostasis may
accompany Alzheimer's disease and head injury. AD is complicated by
cerebral acidosis (pH 6.6; see Ref. 48), which may be related to
impaired glucose metabolism (49) or to the inflammatory response seen
in AD-affected brain tissue (reviewed in Refs. 50 and 51). The release
of metal ions such as Cu+ and Fe2+ from
metalloproteins is induced by a mildly acidotic environment (52-58).
Therefore, we investigated the effect of several bioessential metal
ions on their ability to bind and alter the solubility of A1-40/42 under mildly acidic conditions (pH
6.6) to determine whether there are any unforeseen interactions that
may help explain the propensity for A
to precipitate under the
mildly acidotic conditions anticipated within the metabolically
diseased brain parenchyma. A striking and unexpected interaction
between pH and Cu2+ was observed for human A
.
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MATERIALS AND METHODS |
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Reagents and Preparation--
Human A1-40
peptide was synthesized, purified, and characterized by high pressure
liquid chromatography analysis (HPLC), amino acid analysis, and mass
spectroscopy by the W. M. Keck Foundation Biotechnology Resource
Laboratory (Yale University, New Haven, CT). Rat A
1-40
was obtained from Quality Control Biochemicals, Inc. (Hopkington, MA).
The HPLC elution profiles of both peptides in these working batches
were identified as a single peak. Amino acid analysis of the synthetic
peptides indicated no apparent chemical modifications in the amino acid
residues. Synthetic A
peptide solutions were dissolved in
trifluoroethanol (30% in Milli-Q water (Millipore Corp., Milford, MA))
or 20 mM Hepes (pH 8.5) at a concentration of 0.5-1.0
mg/ml, centrifuged for 20 min at 10,000 × g, and the
supernatants (stock A
) used for subsequent aggregation assays on the
day of the experiment. Prior to use all buffers and stock solutions of
metal ions were filtered though a 0.22-µm filter (Gelman Sciences,
Ann Arbor, MI) to remove any particulate matter. All metal ions were
the chloride salt, except lead nitrate.
Aggregation Assays--
To determine the centrifugation time
required to completely sediment aggregated proteins,
A1-40 (2.5 µM) in 150 mM NaCl
and 20 mM Hepes (pH 7.4) was incubated for 30 min at
37 °C with no metal, and under aggregating conditions of
Zn2+ (100 µM), Cu2+ (100 µM), or pH 5.5. Reaction mixtures were centrifuged at
10,000 × g for different times or ultracentrifuged at
100,000 × g for 1 h. Centrifugation for >10 min
at 10,000 × g was sufficient to completely sediment
Zn2+-, Cu2+-, and pH-induced aggregates of
A
1-40 when compared with ultracentrifugation
(100,000 × g for 1 h). Therefore, centrifugation for 10-20 min at 10,000 × g was used in subsequent
assays to sediment aggregated particles.
Stoichiometry and Binding Analyses of Cu for
A--
Cu2+ concentrations were determined using the
spectrophotometric method of Matsuba and Takahashi (59), adapted to
microtiter plate volumes. Sample Cu2+ concentrations were
determined from a Cu2+ standard curve (0-100
µM in 20 mM ammonium acetate, 150 mM NaCl (pH 7.4)).
Turbidometric Assays--
Turbidity measurements, as an assay
for aggregation, were performed in a flat-bottomed 96-well microtiter
plate (Corning Costar Corp.), and absorbances (405 nm) were measured
using a Spectramax Plus spectrophotometric microplate reader (Molecular
Devices, Sunnyvale, CA). Automatic 30-s plate agitation mode was
selected for the plate reader to evenly suspend the aggregates in the
wells before all readings. A1-40 stock was brought to
10 µM (300 µl) in 20 mM Hepes buffer, 150 mM NaCl (pH 6.6, 6.8 or 7.4) ± metal ions prior to
incubation (30 min, 37 °C) and absorbance measurement.
Modification of A1-40--
Blockage of histidine
residues was achieved by incubating A
1-40 (1.5 mg/ml
dissolved in distilled, deionized H2O, following centrifugation (10,000 × g for 20 min) to remove
undissolved peptide) with 5 mM diethyl pyrocarbonate (DEPC)
in 25 mM phosphate buffer (pH 7.4) for 30 min at 4 °C
(61). Formation of N-carbethoxyhistidine residues was
followed by the characteristic increase in absorbance at 240 nm (62).
The modified protein was dialyzed extensively to remove excess DEPC,
and its ability to aggregate at pH 7.4 and 6.6 with and without
Cu2+, Fe3+, and Zn2+ was tested as
described above. Subsequently, reversal of the histidine modification
was achieved by incubating the modified protein with 0.1 M
hydroxylamine hydrochloride in 25 mM phosphate buffer (pH
7.4) for 30 min at 4 °C. The restored peptide was dialyzed extensively to remove excess hydroxylamine hydrochloride, and its
ability to aggregate at pH 7.4 and 6.6 with and without
Cu2+, Fe3+ and Zn2+ was
retested.
Western Blot Analysis of the Aggregation of Nanomolar
Concentrations of A--
Stock A
1-40 or
A
1-42 (1 mg/ml) was diluted to a final concentration of
200 ng in 1 ml (44 nM) of buffer containing 150 mM NaCl, 20 mM Tris (pH 7.4 or 6.6), 0.1 µM BSA and CuCl2 (0, 0.1, 0.5, 1 or 5 µM), and the samples were mixed and then incubated (30 min, 37 °C). The reaction mixtures were then centrifuged at
12,000 × g for 10 min in a fixed angle rotor
centrifuge, and the supernatant was carefully removed leaving a pellet
of aggregated A
on the side of the tube. Sample buffer (30 µl;
containing 4% SDS, 5%
-mercaptoethanol) was added to the tubes
which were vigorously mixed, then heated to 95 °C for 5 min, and
spun briefly. The SDS-extracted samples and a standard containing 50 ng
of A
in sample buffer were loaded and analyzed by polyacrylamide gel
electrophoresis (Tricine gels, 10-20%; Novex, San Diego, CA),
transferred to polyvinylidene difluoride membranes (Bio-Rad), fixed,
blocked, and then probed with the anti-A
monoclonal antibody 6E10
(Senetek, Maryland Heights, MI) overnight at 4 °C. The blot was then
incubated with anti-mouse horseradish peroxidase conjugate (Pierce) for
2 h at 22 °C and incubated (5 min) with Supersignal Ultra
(Pierce) according to the manufacturer's instructions. The
chemiluminescent signal was captured for 10 min at maximum sensitivity
using the Fluoro-S Image Analysis System (Bio-Rad), and the electronic
images were analyzed using Multi-Analyst Software (Bio-Rad). This
chemiluminescent image analysis system is linear over 2 orders of
magnitude and has comparable sensitivity to film.
Immunofiltration Detection of Nanomolar Concentrations of A
Aggregate--
A
1-40 or A
1-42 (~1
mg/ml) stock solutions were diluted to 20 nM in buffer
containing 150 mM NaCl, 20 mM Tris (pH 7.4 or
6.6), 0.1 µM BSA and CuCl2 (0, 0.1, 0.2, 0.3, 0.5, 1, or 2 µM) added, and the samples were incubated
(15 min, 37 °C). The reaction mixtures (200 µl) were then placed
into the 96-well Easy-Titer ELIFA system (Pierce) and filtered through
a 0.22-µm cellulose acetate filter (MSI, Westboro, MA). Aggregated
particles were fixed to the membrane (0.1% glutaraldehyde, 15 min),
washed thoroughly with Tris-buffered saline, and then probed with the anti-A
monoclonal antibody 6E10 (Senetek, Maryland Heights, MI) overnight at 4 °C. Blots were then processed as described above.
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RESULTS |
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To determine the effect of mild acidity on the behavior of A in
the presence of different metals, A
1-40 was incubated with different bioessential metal ions at pH 6.6, 6.8, and 7.4 at total
metal ion concentrations observed in serum (Fig.
1, A and B).
Peptide aggregation was measured by sedimentation (Fig. 1A)
and turbidometry (Fig. 1B), which generally gave
confirmatory results. Incubation of A
in the absence of metal ions
induced no detectable aggregation over the pH range tested. Only
incubation with Zn2+ or Cu2+ induced >30%
aggregation under these conditions (Fig. 1A). As the
[H+] was increased, all other metal ions induced an
observable increase in sedimented A
(Fig. 1A), but
Cu2+ induced the most striking increase in A
aggregation
as measured by both sedimentation and turbidometry. Fe3+
induced a marked increase in turbidity (Fig. 1B), but this
aggregate was not as readily sedimented as the Cu2+- or
Zn2+-induced A
aggregates (Fig. 1A), probably
due to decreased density of the assembly. As expected, Zn2+
induced ~50% of the soluble peptide to sediment over the pH range tested, but increasing the [H+] caused a decrease in
Zn2+-induced turbidity (Fig. 1B) which may be
due to an alteration in the size or light scattering properties of the
Zn2+-induced aggregates. Incubation of the mixtures for
16 h did not induce any appreciable change in the turbidometry
readings compared with the original readings (data not shown),
suggesting that the aggregation reactions had reached equilibrium
within 30 min.
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The effects of higher metal ion concentrations were also compared at
equimolar (30 µM) concentrations (Fig. 1C).
Under these conditions, as the [H+] increased,
Ni2+ induced A aggregation (~85%) which was
comparable to the effect of incubation with the same concentration of
Cu2+. Co2+ induced a comparable level of A
aggregation to Zn2+ (~70%) and, like Zn2+,
Co2+-induced aggregation was independent of
[H+]. At 30 µM, Fe3+,
Al3+, and Pb2+ were observed to induce partial
(~30%) A
aggregation at pH 6.6 but no significant aggregation at
pH 7.4.
To quantify the binding affinity of Cu2+ for A, spectral
analysis was performed. The half-maximal binding of Cu2+
for A
1-40 and A
1-42 based upon shift in
absorbance was estimated as 4.0 and 0.3 µM, respectively
(Fig. 2, A and B). The stoichiometry of Cu2+ binding to A
1-40
in the Cu2+-induced A
aggregate was 3.4 and 3.0 at pH
7.0 and 6.6, respectively. The stoichiometry of Cu2+
binding to A
1-40 required for precipitation is in
agreement with that required for the saturation of the spectral shift
(Fig. 2A). Cu2+ supplied as a
Cu2+-(glycine)2 complex did not alter the
amount of A
aggregation or binding stoichiometry compared with that
induced by non-complexed Cu2+ in the same buffer (data not
shown).
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Next, we tested other proteins to determine whether
Cu2+-induced protein aggregation under mildly acidic
conditions was specific for A (Fig.
3A). In the absence of metal
ions, BSA aggregation was low (<15%) at both pH 7.4 and 6.6. However,
in contrast to A
1-40, the amount of BSA aggregation
increased only marginally in the presence of Cu2+ at both
pH 7.4 and 6.6. Aprotinin was significantly aggregated (~30%) by
mildly acidic conditions, but unlike A
its aggregation at pH 6.6 was
not potentiated by the presence of Cu2+. In contrast,
insulin was not aggregated by pH 6.6 or by Cu2+ at pH 7.4. However, there was a marked increase in insulin aggregation at pH 6.6 in the presence of Cu2+, resembling that seen with
A
1-40.
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Since higher concentrations of Zn2+ are required to induce
the aggregation of rat A1-40 compared with human
A
1-40, we examined whether rat A
1-40
also was resistant to Cu2+-induced aggregation (Fig.
3B). At pH 7.4, there was a similar slight increase in the
aggregation of both human A
1-40 and rat
A
1-40 as the [Cu2+] increased. However,
the amount of aggregation of rat A
1-40 as
[Cu2+] increased at pH 6.6 was less than half that of
human A
1-40 under the same conditions.
We next proceeded to elaborate the effects of [H+] upon
Cu2+-mediated precipitation of A over a broader pH range
(Fig. 4A). [H+]
alone was sufficient to precipitate A
1-40 (2.5 µM) dramatically once the pH was brought below 6.3 (Fig.
4A). At pH 5.0, 80% of the peptide was precipitated, but
the peptide was less aggregated by acidic environments below pH 5.0, in
agreement with previous reports on the effect of pH on A
solubility
(reviewed in Ref. 63). Zn2+ (30 µM) induced a
constant level (~50%) of aggregation between pH 6.2 and 8.5, whereas
below pH 6.0, aggregation could be explained predominantly by the
effect of [H+].
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In the presence of Cu2+ (30 µM), a decrease
in pH from 8.8 to 7.4 induced a marked drop in A1-40
solubility, whereas a slight decrease in pH below pH 7.4 strikingly
potentiated the effect of Cu2+ on the aggregation of the
peptide (Fig. 4A). Surprisingly, Cu2+ caused
>85% of the available peptide to aggregate by pH 6.8, a pH which
plausibly represents a mildly acidotic environment. We hypothesize that
conformational changes in A
brought about by small increases in
[H+] result in the unmasking of a second metal
interaction site that leads to its rapid
Cu2+-dependent aggregation. Below pH 5.0, the
ability of both Zn2+ and Cu2+ to aggregate A
was diminished, consistent with the observation that Zn2+
binding to A
is abolished below pH 6.0 (42), probably due to
protonation of histidine residues.
Further elaboration of the relationship between pH and Cu2+
on A1-40 solubility (Fig. 4B) confirmed that
there was potentiation between [H+] and
[Cu2+] in producing A
aggregation; as the pH fell,
less Cu2+ was required to induce the same level of
aggregation, suggesting that [H+] controls
Cu2+-induced A
1-40 aggregation. At pH 7.4, Cu2+-induced A
aggregation was far less than that
induced by Zn2+ at similar concentrations, consistent with
our earlier report (43).
To determine if this reaction occurred at lower concentrations of A
and to compare the effects of Cu2+-induced aggregation of
A
1-40 with A
1-42 (which is not stable
in solution at micromolar concentrations in aqueous buffers), we
incubated both peptides (50 nM) with various
Cu2+ concentrations (0-5 µM), sedimented the
aggregated peptide, resolubilized the A
pellets with SDS, and
visualized the A
on Western blots. Increased aggregation of
A
1-40 was apparent at Cu2+ concentrations
as low as 500 nM (Fig.
5A). As previously observed at
higher A
1-40 concentrations, a decrease in pH from 7.4 to 6.6 potentiated the effect of Cu2+ on the aggregation of
A
1-40 in this system. A
1-42 aggregation
followed a similar pattern to that of A
1-40, but 500 nM Cu2+ appeared to induce more aggregation of
A
1-42 than A
1-40 at pH 7.4 (Fig.
5B).
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To study this reaction at even lower concentrations of
A1-40, A
1-42 (20 nM), and
Cu2+ (
1 µM), such as those found in
cerebrospinal fluid (6, 11, 13, 14), we employed a novel filtration
immunodetection system2 (Fig.
6, A and B). This
sensitive technique confirmed that a decrease in pH from 7.4 to 6.6 potentiated the effect of
Cu2+ (
200 nM) on the aggregation of
A
1-40 (Fig. 6A). An increase in
A
1-42 aggregation also was observed with increasing [Cu2+] which also was potentiated by pH 6.6 (Fig.
6B). At pH 7.4, A
1-42 was more sensitive
than A
1-40 to aggregation induced by increasing
Cu2+ concentrations in this range.
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Since transition metals often coordinate to histidine residues, and
since rat A1-40 (His
Arg substitution at position 13) exhibits attenuated aggregation by both Cu2+ and
Zn2+, we tested whether modifying histidine residues with
DEPC (61) affected metal ion-induced aggregation of human
A
1-40. Treatment of A
1-40 with DEPC
resulted in an increase in absorbance at 240 nm compared with the
unmodified protein, confirming the formation of
N-carbethoxyhistidine residues (data not shown), whereas the
formation of O-carbethoxytyrosine, characterized by a
decrease in the absorption spectrum at 278 nm, was not observed. N-Carbethoxyhistidine modification completely abolished A
aggregation in the presence of Zn2+ (Fig.
7). Reversal of the modifications with
hydroxylamine resulted in almost complete restoration of
Zn2+-induced aggregation of A
, indicating the absolute
requirement for the coordination of Zn2+ to the
histidine(s) of A
1-40 in order to induce aggregation. Likewise, the requirement for Fe3+-histidine coordination
for A
aggregation was demonstrated by the abolition of
Fe3+-induced A
aggregation at pH 6.6 following
N-carbethoxyhistidine modification. As with
Zn2+-mediated A
aggregation, reversal of the
modifications restored Fe3+-mediated A
aggregation (Fig.
7). A marked decrease in Cu2+-induced aggregation of the
modified protein at both pH 7.4 and 6.6 also was observed, and reversal
of the modifications restored Cu2+-induced aggregation,
indicating that Cu2+-histidine coordination also was
required for A
1-40 aggregation (Fig. 7).
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We have recently reported that Zn2+-mediated
A1-40 aggregation is totally reversible by chelation,
whereas A
1-40 aggregation induced by pH 5.5 is
irreversible with alkalinization (45). Therefore, using turbidometry,
we examined whether Cu2+/pH-mediated A
1-40
aggregation was also reversible. We observed that
Cu2+-induced A
1-40 aggregation at pH 7.4 was reversible following EDTA chelation, although for each new
aggregation cycle, complete resolubilization of the aggregates required
a longer incubation (Fig.
8A).
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The reversibility of pH-potentiated, Cu2+-induced
A1-40 aggregation in buffers cycled between pH 7.4 and
6.6 was studied by turbidometry (Fig. 8B). Unlike the
irreversible aggregation of A
1-40 observed at pH 5.5 (45), Cu2+-induced A
1-40 aggregation was
fully reversible as the pH oscillated between pH 7.4 and 6.6.
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DISCUSSION |
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These results indicate that subtle conformational changes mediated
by [H+] alter the interaction of A with transition
metal ions, modulating peptide aggregation induced particularly by
Cu2+ and Ni2+ over a narrow pH range
(6.6-7.4). This reaction resembles the pH-potentiated, metal
ion-induced aggregation that is observed for insulin, in agreement with
previous reports (64-66), but was not a significant feature of the
behavior of albumin, aprotinin, or rat A
1-40 (Fig. 3,
A and B). The inability of Cu2+ to
precipitate rat A
is of interest because the rat does not develop
cerebral A
deposits with age (32, 33), and in the absence of
Zn2+ (43) or Cu2+ (Fig. 3B) the
solubility of rat A
1-40 is indistinguishable from human
A
1-40. Interestingly, apolipoprotein E, a protein that
is closely associated with A
aggregation and implicated in the
pathogenesis of AD (67-69), also binds Cu2+ (70), but its
relationship with the reactions described here remains to be
determined.
Evidence for an interaction between Cu2+ and
A1-40 has been previously observed. Cu2+
stabilized an apparent A
1-40 dimer on gel
chromatography (42), and, in one study, 65Zn2+
binding to A
was displaced by co-incubation with excess
Cu2+ (71). Although Clements and co-workers (71) in their
study were unable to detect the high affinity binding site for
Zn2+, this may be explained by two major procedural
differences in their metal binding analyses compared with ours as
follows: first, the absence of a competing transition metal ion in
their assay system (Mn2+ in our assay (42)) to abolish low
affinity metal binding, and second, the absence of physiological (150 mM) NaCl in their assays which is a factor that we have
found profoundly alters the interaction of metal ions with
A
1-40 (45). Therefore, we suspect that the displacement
of 65Zn2+ by Cu2+ binding to A
described by Clements et al. (71) reflects competition for
the less specific, lower affinity Zn2+-binding site on the
peptide. We have previously shown that high affinity Zn2+
binding to A
1-40, appreciated when Mn2+ is
used to create stringent conditions that eliminate lower affinity interactions (42), cannot be displaced by other transition metals. Since Cu2+ has been shown to compete for low affinity metal
binding under conditions where high affinity Zn2+ binding
would be preserved, we suspect that A
1-40 may be
capable of binding Cu2+ and Zn2+
simultaneously, a possibility that may be contingent upon the peptide
subunits dimerizing in solution, as we have previously observed (45,
72).
Our results support the likelihood of at least two classes of
metal-binding sites on A as follows: the
Cu2+/Ni2+ site that is responsible for
significant A
assembly only under mildly acidic (pH 6.6-7.0)
conditions, and the Zn2+/Co2+ site that
mediates significant A
assembly at pH 7.4 (Fig. 1). The finding that
A
1-40 associates with Co2+ in a similar
manner to Zn2+ is of interest since Co2+,
unlike Zn2+, is paramagnetic and therefore might be used to
substitute for Zn2+ in structural biology studies (73). The
interactions of Ni2+ and Co2+ with A
were
achieved with metal ion concentrations that are supraphysiological for
these metal ions, although they are physiologically possible for
Cu2+ and Zn2+. Therefore, we believe that
Cu2+ and Zn2+ would be the most relevant
interacting metal ions with A
in biological systems.
A exhibits spectral alterations in the presence of Cu2+
(Fig. 2), which appear to be due to saturable binding of the metal ion.
Like the prion protein (74), we found that A
binds multiple copper
ions. By using the half-maximal binding of Cu2+ for A
as
an indicator of affinity, we found that A
1-42 has a
higher affinity for Cu2+ than A
1-40, which
is in agreement with our current findings that A
1-42 is
more readily precipitated by Cu2+ at pH 7.4 (Fig. 5).
Although the spectral alterations that we observed saturated in
response to micromolar Cu2+ concentrations, these changes
may correspond to relatively low affinity Cu2+ binding.
Since three to four Cu2+ ions bind to each A
subunit
when the peptide is precipitated by Cu2+, it is possible
that Cu2+-binding sites of submicromolar affinity exist but
could not be appreciated by the current aggregation and spectral
assays.
Total Cu2+ has been measured at ~15 µM in
the synaptic cleft and up to 100 µM in the normal
neocortex (46, 75-78). However, the concentrations of
Cu2+, Fe3+, and Zn2+ are each more
than doubled (~300, ~700, and ~790 µM,
respectively) in the neuropil of the cortical and accessory basal
nuclei of the amygdala of AD brains compared with the neuropil of
normal age-matched brains (46). These metal ions are even further
concentrated within the core and periphery of senile plaque deposits
(~400, ~950, and ~1100 µM, respectively) (46). Our
present and previous findings would explain a basis for
Cu2+, Fe3+, and Zn2+ enrichment in
cerebral A deposits, which would, in turn become a sink for these
metal ions.
The lower levels of A1-40 turbidity seen at pH 7.4 in
the presence of all metals combined (Fig. 1B) compared with
that induced by Zn2+ alone may reflect a protective effect
of one of the metal ions in the mixture, possibly Mg2+,
which is the only metal ion not to exhibit a precipitating effect even
at millimolar concentrations. This protective effect is, however, not
evident in incubations performed at lower pH, indicating that
Mg2+ may block the pH-insensitive binding site for
Zn2+/Co2+. This possibility awaits experimental
verification.
We have previously described the rapid aggregation of
A1-40 by Zn2+ (42-45), as well as the
abolition of zinc binding to A
at pH <6.0, consistent with
histidine-mediated interaction. In the current study, modification of
A
with DEPC abolished Zn2+-induced A
aggregation
(Fig. 7), supporting the possibility that the aggregation of
A
1-40 by Zn2+ is dependent upon
coordination with histidine. Although Fe3+ induced
considerably less aggregation of A
than Zn2+ or
Cu2+, even this aggregation reaction appeared to be
coordinated by the histidine residues of A
, suggesting that the
association of redox active iron with senile plaques from AD brains
recently reported by Smith et al. (79) may be due to the
metal ion binding directly to A
.
Histidine modification did not completely abolish
Cu2+-induced A aggregation, suggesting that a proportion
of that form of aggregate is assembled by interaction with
non-histidine residues on the peptide. Histidine modification of human
A
1-40 reduced the amount of Cu2+-induced
A
aggregation to that observed following the incubation of rat
A
1-40 with the same Cu2+ concentrations.
Taken together, these data indicate that Cu2+ coordination
to histidine at position 13 and tyrosine at position 10 may coordinate
the aggregation of A
1-40. Access to these residues by
Cu2+ therefore appears to be restricted at pH 7.4.
The relationship of A1-40 solubility with pH alone
mirrors the conformational changes observed by CD spectra within the
N-terminal fragment (residues 1-28) of A
(63) as follows:
-helical and soluble between pH 1-4 and >7, but
-sheet and
aggregated between pH 4-7. Thus, the altered interaction of
Cu2+ with soluble A
below pH 7.0 may be due to changes
in peptide conformation. A
1-40 aggregation by
Cu2+ was observed to be rapidly reversible by
alkalinization. Since increasing pH above 7.0 promotes the
-helical
conformation (63), this conformation may be unfavorable for the
Cu2+ coordination that mediates peptide assembly.
Cu2+-induced A1-40 aggregation at pH 7.4 also was reversible by chelation; however, less peptide was
resolubilized during each cycle perhaps because a denser aggregate was
formed during each aggregation cycle which retarded chelator access to
the peptide mass. This possibility is supported by the observation that
complete resolubilization of the Cu2+-induced A
aggregates by EDTA eventually occurred with additional incubation time.
The modulation of A
solubility by Cu2+ through a number
of aggregation/resolubilization cycles suggests that its interaction
does not induce the amyloid
-sheet conformation, which is not easily
reversible.
Although the pathogenic nature of A in AD is well described (4, 5),
the function of A
remains unclear. Taken together, our results
emphasize three physiologically plausible environments that could
aggregate A
as follows: lowered pH (Fig. 4; Refs. 80-86), elevated
[Zn2+] (Figs. 1, 4, and 7; Refs. 42-45 and 86), and
under mildly acidic conditions, elevated [Cu2+] (Figs.
1-7). Inflammation generally induces locally acidic conditions (87,
88) and the mobilization of both Zn2+ and Cu2+
(89-94), which are both found in high concentrations in the neocortex (Zn2+ ~150 µM, Ref. 95; Cu2+ ~100
µM, Refs. 77 and 78). Inflammatory mechanisms also have been suggested as participating in the pathophysiology of AD (96), and
acute phase inflammatory proteins such as
1-antichymotrypsin and
C-reactive protein, elements of the complement system, and activated
microglial and astroglial cells are observed in AD-affected brains (see
Refs. 50 and 51 for reviews). A sustained increase in regional
Zn2+, Cu2+, and H+ concentrations
induced by a cortical inflammatory response is predicted by our data to
contribute to the deposition of A
in AD.
Systemic amyloidoses are often associated with chronic inflammation
(97-99). An example is the amyloidosis caused by serum amyloid A, an
acute phase-reactant protein (100). Serum copper levels increase during
inflammation, associated with increases in ceruloplasmin, a
Cu2+-transporting protein that transfers Cu2+
to enzymes active in processes of basic metabolism and wound healing
such as cytochrome oxidase and lysyl oxidase (101, 102), and whose
metabolism is abnormal in AD-affected neocortex (103). Whereas the
neocortex contains high levels of Cu2+, the availability of
Cu2+ is normally restricted by its binding to
metalloproteins such as albumin and ceruloplasmin (104). However, the
release of reduced copper from ceruloplasmin is greatly facilitated by
acidic environments (52), and periods of mild acidosis may promote an
environment of increased "free" Cu2+ and therefore
promote its exchange to other regional proteins. Such an exchange of
Cu2+ at low pH has been described as mediating the binding
of serum amyloid P component, an acute phase reactant that also forms
an amyloid, to the cell wall polysaccharide zymosan (105). A similar exchange of Cu2+ within a low pH milieu may be responsible
for precipitating a fraction of the brain collections of A in
AD.
A further mechanism by which Zn2+, Cu2+, and
H+ levels may be elevated in the cortical interstitium is
through abnormal energy metabolism. Intracellular concentrations of
Zn2+ and Cu2+ are approximately 1000- and
100-fold higher than extracellular concentrations, respectively (77,
78, 95). This large gradient between intracellular and extracellular
compartments is sustained by highly energy-dependent
mechanisms (95, 106). Therefore, alterations in energy metabolism, such
as those seen in AD (49), trauma, or vascular compromise, may affect
the compartmentalization of these metal ions and possibly promote their
pooling in the A compartment. A
precipitation by Cu2+
in this case may be potentiated by the acidosis that is associated with
abnormal energy metabolism. These mechanisms may contribute to the
rapid appearance of A
deposits reported to occur following head
injury (16, 107). The reversible aggregation properties of A
at
local sites of injury may, in this context, be compatible with a role
for the peptide in maintaining regional structural integrity.
If the involvement of Cu2+ in A deposition in AD is
confirmed, then the reversibility of this pH-mediated,
Cu2+-induced interaction presents the potential for
therapeutic intervention. Thus, cerebral alkalinization or metal ion
chelation may be explored as potential therapies for the reversal of
A
deposition in vivo.
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ACKNOWLEDGEMENTS |
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We are grateful to C. Glabe (University of California, Irvine), M. J. Shields (NCI, National Institutes of Health, Bethesda), and Eva Migdal for helpful discussions.
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FOOTNOTES |
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* This work was supported by funds from National Institutes of Health Grant 1R29AG1268601, Alliance for Aging Research (Paul Beeson Award to A. I. B.), Alzheimer's Association Grant IIRG-94110, International Life Sciences Institute, Prana Corporation, and the Commonwealth of Massachusetts Research Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Genetics and Aging Unit, Neuroscience Center, Massachusetts General Hospital East, Bldg. 149, 13th St., Charlestown, MA 02129-9142. Tel.: 617-726-8244; Fax: 617-724-9610; E-mail: bush{at}helix.mgh.harvard.edu.
1 The abbreviations used are: AD, Alzheimer's disease; DS, Down's syndrome; DEPC, diethyl pyrocarbonate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MES, 4-morpholineethanesulfonic acid; BSA, bovine serum albumin.
2 R. D. Moir, A. I. Bush, D. M. Romano, C. S. Atwood, X. Huang, and R. E. Tanzi, personal communication.
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