Note: Descriptions are shown in the official language in which they were submitted.
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Identification of Agents for Use in the Treatment of
Alzheimer's Disease
Backg, round of the Invention
Statement as to Rights to In entions Made Under
Federally-Sponsored Research and Development
Part of the work performed during the development of this invention
utilized U.S. Government F'unds under Grant No. R29AG12686 from the
National Institutes of Health. The government may have certain rights in this
invention.
Field of the Invention
This invention is in the field of medicinal chemistry. In particular, the
invention is related to the detection of drugs useful in the treatment of
Alzheimer's disease. The invention is also related to compositions for
treatment
of Alzheimer's disease.
Related Art
Polymers of Abeta (A13), the 4.3 kD, 39-43 amino acid peptide product of
the transmembrane protein, amyloid protein precursor (APP), are the main
components extracted from the neuritic and vascular amyloid of Alzheimer's
disease (AD) brains. A(3 deposits are usually most concentrated in regions of
high neuronal cell death, and may be present in various morphologies,
including
amorphous deposits, neurophil plaque amyloid, and amyloid congophilic
angiopathy (Mastersõ C.L., el al., EMBO J. 4:2757 (1985); Masters, C.L. et
al.,
Proc. Natl. Acad. Sci. USA 8.2: 4245 (1985)). Growing evidence suggests that
amyloid deposits are intimate;ly associated with the neuronal demise that
leads to
dementia in the disorder.
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The presence of an enrichment of the 42 residue species of A(3 in these
deposits suggests that this species is more pathogenic. The 42 residue form
ofA(3
(AP,.42), while a minor component of biological fluids, is highly enriched in
amyloid, and genetic studies strongly implicate this protein in the
etiopathogenesis of AD. Amyloid deposits are decorated with inflammatory
response proteins, but biochemical markers of severe oxidative stress such as
peroxidation adducts, advanced glycation end-products, and protein cross-
linking
are seen in proximity to the lesions. To date, the cause of Ap deposits is
unknown, although it is believed that preventing these deposits may be a means
of treating the disorder.
When polymers of A(3 are placed into culture with rat hippocampal
neurons, they are neurotoxic (Kuo, Y-M., et al., J. Biol. Chem. 271:4077-81
(1996); Roher, A.E., el al., Journal of Biological Chemistry 271:20631-20635
(1996)). The mechanism underlying the formation of these neurotoxic polymeric
Ap species remains unresolved. The overexpression of A(3 alone cannot
sufficiently explain amyloid formation, since the concentration of A(3
required for
precipitation is not physiologically plausible. That alterations in the
neurochemical environment are required for amyloid formation is indicated by
its
solubility in neural phosphate buffer at concentrations of up to 16 mg/ml
(Tomski, S. & Murphy, R.M. Archives of Biochemistry and Biophysics 294:630
(1992)), biological fluids such as cerebrospinal fluid (CSF) (Shoji, M., et
al.,
Science 258:126 (1992); Golde et al. Science, 255(5045):728-730 (1992);
Seubert, P., et al., Nature 359:325 (1992); Haass, C., et al., Nature 359:322
(1992)) and in the plaque-free brains of Down's syndrome patients (Teller,
J.K.,
et al., Nature Medicine 2:93-95 (1996)).
Studies into the neurochemical vulnerability of Ap to form amyloid have
suggested altered zinc and [H+] homeostasis as the most likely explanations
for
amyloid deposition. A(3 is rapidly precipitated under mildly acidic conditions
in
vitro (pH 3.5-6.5) (Barrow, C.J. & Zagorski, M.G., Science 253:179-182 (1991);
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Fraser, P.E., et al., Biophys. .i: 60:1190-1201 (1991); Barrow, C.J., et al.,
J. Mol.
Biol. 225:1075-1093 (1992); Burdick, D., J. Biol. Chem. 267:546-554 (1992);
Zagorski, M.G. & Barrow, C.J., Biochemistry 31:5621-5631 (1992);
Kirshenbaum, K. & Daggett, V., Biochemistry 34:7629-7639 (1995); Wood, S.J.,
et al., J. Mol. Biol. 256:870-877 (1996)). Recently, it has been shown that
the
presence of certain biometals, in particular redox inactive Zn'-' and, to a
lesser
extent, redox active Cu'-' and Fe3+, markedly increases the precipitation of
soluble
Ap (Bush, A.I., et al., J. Biol. Chem. 268:16109 (1993); Bush, A.I., et al.,
J. Biol.
Chem. 269:12152 (1994); Bush, A.I., el al., Science 265:1464 (1994); Bush,
A.I.,
et al., Science 268:1921 (1995)). At physiological pH, AP1_40 specifically and
saturably binds Zn", manifesting high affinity binding (Kp = 107 nM) with a
1:1
(Znz+:Ap) stoichiornetry, and low affinity binding (KD = 5.2 M) with a 2:1
stoichiometry.
The reduction by APP of copper (II) to copper (I) may lead to irreversible
Ap aggregation and crosslinking. This reaction may promote an environment that
would enhance the production of hydroxyl radicals, which may contribute to
oxidative stress in A:D (Multl:iaup, G., et al.. Science 271:1406-1409
(1996)). A
precedence for abnormal Cu rnetabolism already exists in the neurodegenerative
disorders of Wilson's disease, Menkes' syndrome and possibly familial
amyotrophic lateral sclerosis (Tanzi, R.E. et al., Nature Genetics 5:344
(1993);
Bull, P.C., et al., Nature Genetics 5:327 (1993); Vulpe, C., et al., Nature
Genetics
3:7 (1993); Yamaguchi, Y., et al., Biochem. Biophys. Res. Commun. 197:271
(1993); Chelly, J., et al., Nature Genetics 3:14 (1993); Wang, D. & Munoz,
D.G.,
J. Neuropathol. Exp. Neurol. 54:548 (1995); Beckman, J.S., et al., Nature
364:584 (1993); Har-tmann, H.A. & Evenson, M.A., Med Hypotheses 38:75
(1992)).
Although much fundarnental pathology, genetic susceptibility and biology
associated with AD is becoming clearer, a rational chemical and structural
basis
for developing effective drugs to prevent or cure the disease remains elusive.
While the genetics of the disorder indicates that the metabolism of A(3 is
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intimately associated with the etiopatholgenesis of the disease, drugs for the
treatment of AD have so far focused on "cognition enhancers" which do not
address the underlying disease processes.
Summary of the Invention
An object of the present invention is to provide identification of agents for
use in
the treatment of Alzheimer's Disease.
In one aspect, the invention relates to a method for the identification of an
agent to be used in the treatment of AD, wherein the agent is capable of
altering
the production of Cu+ by A(3, the method comprising:
(a) adding Cu2=' to a first Ap sample;
(b) allowing the first sample to incubate for an amount of time
sufficient to allow said first sample to generate Cu';
(c) adding Cu'-+ to a second Ap sample, the second sample
additionally comprising a candidate pharmacological agent;
(d) allowing the second sample to incubate for the same
amount of time as the first sample;
(e) determining the amount of Cu+ produced by the first
sample and the second sample; and
(f) comparing the amount of Cu+ produced by the first sample
to the amount of Cu' produced by the second sample;
whereby a difference in the amount of Cu+ produced by the first sample as
compared to the second sample indicates that the candidate pharmacological
agent has altered the production of Cu' by A.
In a preferred embodiment, the amount of Cu+ present in said first and
said second sample is determined by
(a) adding a complexing agent to said first and said second
sample, wherein said complexing agent is capable of combining with Cu+ to form
a complex compound, wherein said complex compound has an optimal visible
absorption wavelength;
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(b) measuring the absorbancy of said first and said second
sample; and
(c) calculating the concentration of Cu+ in said first and said
second sample using the absorbancy obtained in step (b).
In a more: preferred embodiment, the complexing agent is
bathocuproinedisulfonic (BC) anion. The concentration of Cu+ produced by AP
may then be calculated on the basis of the absorbance of the sample at about
478 nm to about 488 nm, more preferable about 480 to about 486 nm, and most
preferably about 483 nm. 1[n another preferred embodiment, the method is
performed in a microtiter plate, and the absorbancy measurement is performed
by
a plate reader. Most preferrably, two or more different test candidate agents
are
simultaneously evaltiated for an ability to alter the production of Cu+ by Ap.
In
another preferred embodiment, said Ap samples of step 1(a) and step 1(c) are
biological samples. Most preferrably, said biological samples are CSF.
In another aspect, the invention relates to a method for the identification
of an agent to be used in the treatment of AD, wherein said agent is capable
of
altering the production of Fe' by A(3, said method comprising:
(a) adding Fe3+ to a first Ap sample;
(b) allowirig said first sample to incubate for an amount of
time sufficient to allow said first sample to generate Fe'+;
(c) adding Fe3+ to a second Ap sample, said second sample
additionally comprising a candidate pharmacological agent;
(d) allowirig said second sample to incubate for the same
amount of time as said first sample;
(e) determining the amount of Fe'+ produced by said first
sample and said second sample; and
(f) comparing the amount of Fe'+ present in said first sample
to the amount of Fe'+ present in said second sample;
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whereby a difference in the amount of Fez+ present in said first sample as
compared to said second sample indicates that said candidate pharmacological
agent has altered the production of Fez+ by Ap.
In a preferred embodiment, the amount of Fe'-" present is determined by
using a spectrophotometric method analogous to that used for the determination
of Cu', above. In this method, the complexing agent is batho-
phenanthrolinedisulfonic (BP) anion. The concentration of Fe'-+-BP produced by
A(3 may then be calculated on the basis of the absorbance of the sample at
about
530 to about 540 nm, more preferably about 533 nm to about 538 nm, and most
preferably about 535 nm. In another preferred embodiment, said method is
performed in a microtiter plate, and the absorbancy measurement is performed
by
a plate reader. Most preferrably, two or more different test candidate agents
are
simultaneously evaluated for an ability to alter the production of FeZ+ by AP.
In another preferred embodiment, said A(3 samples of step 1(a) and step
1(c) are biological samples. Most preferrably, the biological sample is CSF.
In yet another aspect, the invention relates to a method for the
identification of an agent to be used in the treatment of AD, wherein said
agent
is capable of altering the production of H,O2 by Ap, said method comprising:
(a) adding Cu2' or Fe3 to a first A(3 sample;
(b) allowing said first sample to incubate for an amount of
time sufficient to allow said first sample to generate H2O2;
(c) adding Cu'-+ or Fe3+ to a second Ap sample, said second
sample additionally comprising a candidate pharmacological agent;
(d) allowing said second sample to incubate for the same
amount of time as said first sample;
(e) determining the amount of HZO, produced by said first
sample and said second sample; and
(f) comparing the amount of H202 present in said first sample
to the amount of H202 present in said second sample;
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whereby a difference in the amount of H,OZ present in said first sample as
compared to said second sanlple indicates that said candidate pharmacological
agent has altered the production of H,OZ by Ap. In a preferred embodiment, the
determination of the amount of H,OZ present in said first and said second
sample
is determined by
(a) adding catalase to a first aliquot of said first sample
obtained in step (a) above in an amount sufficient to break down all of the
H2O2
generated by said sample;
(b) adding TCEP, in an amount sufficient to capture all of the
H7O2 present in said samples, to
(i) said first aliquot
(ii) a second aliquot of said first sample obtained in
step (a) above; and
(iii) said second sample obtained in step (b) above;
(c) incubating the samples obtained in step (b) for an amount
of time sufficient to allow the TCEP to capture all of the H202;
(d) adding; DTNB to said samples obtained in step (c);
(e) incubating said samples obtained in step (d) for an amount
of time sufficient to generate TMB;
(f) measuring the absorbancy at about 407 to about 417 nm of
said samples obtained in step (e); and
(g) calculating the concentration of H202in said first and said
second sample using the absorbancies obtained in step (f). In a preferred
embodiment, the absorbancy of TMB is measured at about 412 nm. In preferred
embodiment, said method is performed in a microtiter plate, and the absorbancy
measurement is performed by a plate reader. Most preferrably, two or more
different test candidate agents are simultaneously evaluated for an ability to
alter
the production of H,Oz by Af4.
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In another aspect, the invention relates to a method for the identification
of an agent to be used in the treatment of AD, wherein said agent is capable
of
decreasing the production of O; by Ap, said method comprising:
(a) adding A(3 and to a first buffer sample having an O2 tension
greater than 0;
(b) allowing said first sample to incubate for an amount of
time sufficient to allow said first sample to generate Oz;
(c) adding Ap and a candidate pharmacological agent to a
second buffer sample having an O, tension greater than 0;
(d) allowing said second sample to incubate for the same
amount of time as said first sample;
(e) determining the amount of 02 produced by said first
sample and said second sample; and
(f) comparing the amount of 02 present in said first sample to
the amount of OZ present in said second sample;
whereby a difference in the amount of O2 present in said first sample as
compared
to said second sample indicates that said candidate pharmacological agent has
altered the production of O; by Ap. In a preferred embodiment, the Ap used is
AF'I-42=
In a preferred embodiment, the determination of the amount of Oz present
in said samples is accomplished by measuring the absorbancy of the sample at
about 250 nm.
Because the ability of A(3 to generate H,O, from O; may in many
instances be beneficial. Therefore, in a preferred embodiment, the invention
also
relates to a method for the identification of an agent to be used in the
treatment
of AD, wherein said agent is capable of interfering with the interaction of O,
and
A(3 to produce 02, without interfering with the SOD-like activity of AP, said
method comprising:
(a) identifying an agent capable of decreasing the production
of 02 by A(3; and
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(b) determining the ability of said agent to alter the SOD-like
activity of Ap. In a preferred embodiment, the determination of the ability of
said
agent to alter the SOD-like activity of Ap is made by determining whether A(3
is
capable of catalytically producing Cu+, Fez' or HzOZ.
In another aspect the invention relates to a method for the identification
of agents useful in the treatment of Alzheimer's disease (AD) because they are
capable of reducing the toxiciity of Ap.
In one aspect the invention relates to a method for the identification of an
agent to be used in the treatment of AD, wherein said agent is capable of
reducing
the toxicity of Ap, said method comprising:
(a) adding Ap to a first cell culture;
(b) adding Ap to a second cell culture, said second cell culture
additionally containing a candidate pharmacological agent;
(c) determining the level of neurotoxicity of A(3 in said first
and said second samples; and
(d) comparing the level of neurotoxicity of Ap in said first and
said second samplesõ
whereby a lower neurotoxicity level in said second sample as compared to said
first sample indicates that saici candidate pharmacological agent has reduced
the
neurotoxicity of A(3, and is thereby capable of being used to treat AD. In a
preferred embodiment, the neurotoxicity of A(3 is determined by using an MTT
assay. In another preferred embodiment, the neurotoxicity of Ap is determined
by
using an LDH release assay. In still another preferred embodiment, the
neurotoxicity of A(3 is determined by using a Live/Dead assay. Preferrably
said
cells utilized in the assays are rat cancer cells. Even more preferrably said
cells
are rat primary frontal neuronal cells.
Yet another aspect of the invention relates to a kit for determining whether
an agent is capable of altering the production of Cu' by A(3 which comprises a
carrier means being compartmentalized to receive in close confinement therein
one or more container means wherein
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(a) the first container means contains a peptide comprising Ap
peptide;
(b) a second container means contains a Cuz+ salt; and
(c) a third container means contains BC anion.
Preferrably, said Ap peptide is present as a solution in an aqueous buffer or
a
physiological solution, at a concentration above about 10 M.
In another aspect the invention relates to a kit for determining whether an
agent is capable of altering the production of Fe2+ by Ap which comprises a
carrier means being compartmentalized to receive in close confinement therein
one or more container means wherein
(a) the first container means contains a peptide comprising AP
peptide;
(b) a second container means contains an Fe3+ salt; and
(c) a third container means contains BP anion.
Preferrably, said Ap peptide is present as a solution in an aqueous buffer or
a
physiological solution, at a concentration above about 10 M..
In another aspect, the invention relates to a kit for determining whether an
agent is capable of altering the production of H202 by A(3 which comprises a
carrier means being compartmentalized to receive in close confinement therein
one or more container means wherein
(a) the first container means contains a peptide comprising AP
peptide;
(b) a second container means contains a CuZ+ salt;
(c) a third container means contains TCEP; and
(d) a fourth container means contains DTNB.
Preferably, said Ap peptide is present as a solution in an aqueous buffer or a
physiological solution, at a concentration above about 10 M.
In yet another aspect the invention relates to a method for the
identification of an agent to be used in the treatment of AD, wherein said
agent
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is capable of inhibitirig redox-reactive metal-mediated polymerization of A(3,
said
method comprising:
(a) adding a redox-reactive metal to a first A(3 sample;
(b) allowiiig said first sample to incubate for an amount of
time sufficient to allow Ap polymerization;
(c) adding said redox-reactive metal to a second Ap sample,
said second sample additionally comprising a candidate pharmacological agent;
(d) allowing said second sample to incubate for the same
amount of time as said first sample;
(e) removing an aliquot from each of said first and said second
sample; and
(f) determining presence or absence of polymerization in said
first and second samples,
whereby an absence of A(3 po lymerization in said second sample as compared to
said first sample indicates that said candidate pharmacological agent has
inhibited
Ap polymerization. Preferrably, at step (f), a western blot analysis is
performed
to determine the presence or absence of polymerization in the first and the
second
sample.
Another aspect of the present invention contemplates a method for
treating AD in a subject, said method comprising administering to said subject
an
effective amount of an agent which is capable of inhibiting or otherwise
reducing
metal-mediated production of free radicals.
The present irivention provides a method for treating AD in a subject, said
method comprising administering to said subject an effective amount of an
agent
comprising a metal clielator arid/or a metal complexing compound for a time
and
under conditions sufficient to inhibit or otherwise reduce metal-mediated
production of free radicals by A(3. In one aspect, the free radicals are
reactive
oxygen species such as O', or OH-. In another aspect, the free radicals
include
forms of Ap.
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Still another aspect of the present invention relates to a method of treating
AD in a subject comprising administering to said subject an agent capable of
preventing, reducing or otherwise inhibiting ROS production by Ap deposits in
the brain for a time and under conditions to effect said treatment.
In one aspect, the invention relates to a method of treating amyloidosis
in a subject, said method comprising administering to said subject an
effective
amount of (a) a metal chelator selected from the group consisting of:
bathocuproine, bathophenanthroline, penacillamine, TETA, TPEN or
hydrophobic derivatives thereof; and (b) one or more pharmaceutically
acceptable
carriers or diluents; for a time and under conditions to bring about said
treatment;
and wherein said chelator reduces, inhibits or otherwise interferes with Ap-
mediated production of radical oxygen species. The invention also relates to
said
method further comprising administering to the subject an effective amount of
a
compound selected from the group consisting of: rifampicin, disulfiram, and
indomethacin, or a pharmaceutically acceptable salt thereof.
In another aspect, the invention relates to a method of treating amyloidosis
in a subject, said method comprising administering to said subject a
combination
of (a) a metal chelator selected from the group consisting of: bathocuproine,
bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN,
or hydrophobic derivatives thereof; and (b) a supplement selected from the
group
consisting of: ammonium salt, calcium salt, magnesium salt, and sodium salt,
for
a time and under conditions to bring about said treatment; and wherein said
chelator reduces, inhibits or otherwise interferes with A(3-mediated
production of
radical oxygen species. In a preferred embodiment, the metal chelator is EGTA.
In another preferred embodiment, the metal chelator is TPEN. In yet another
preferred embodiment, the supplement is magnesium salt.
In yet another aspect, the invention relates to said method further
comprising administering to the subject an effective amount of a compound
selected from the group consisting of: rifampicin, disulfiram, and
indomethacin,
or a pharmaceutically acceptable salt thereof.
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In yet another aspect, the invention relates to a method of treating
amyloidosis in a subject, said method comprising administering to said subject
an effective amount of a salt of a metal chelator, wherein said chelator is
selected
from the group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA,
EGTA, penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof;
wherein said salt is selected from the group consisting of: ammonium, calcium,
magnesium, and sodium; and wherein said salt of a metal chelator reduces,
inhibits or otherwise interfe:res with Ap-mediated production of radical
oxygen
species. In a preferred embodiment, the metal chelator is EGTA. In another
preferred embodiment, the the metal chelator is TPEN. In yet another preferred
embodiment, the salt of a nletal chelator is a magnesium salt. In yet another
aspect, the invention relates to said method further comprising administering
to
said subject a compound selected from the group consisting of: rifampicin,
disulfiram, and indomethacin, or a pharmaceutically acceptable salt thereof.
In another aspect, the invention relates to a method of treating amyloidosis
in a subject, said method comprising administering to said subject an
effective
amount of a chelator specific for copper; wherein said chelator reduces,
inhibits
or otherwise interferes with Ap-mediated production of radical oxygen species.
In a preferred embodiment, the chelator specific for copper is specific for
the
reduced form of copper. Most preferreably, the chelator is bathocuproine or a
hydrophobic derivative thereof.
In yet another aspect, the invention relates to a method of treating
amyloidosis in a subject, saiid method comprising administering to said
subject
an effective amount of an alkalinizing agent, wherein said alkalinizing agent
reduces, inhibits or otherwise interferes with A(3-mediated production of
radical
oxygen species. In a preferred embodiment, the alkalinizing agent is magnesium
citrate. In another p:referred embodiment, the alkalinizing agent is calcium
citrate.
Still another aspect of the present invention contemplates a method of
treating AD in a subject comprising administering to said subject an agent
capable
of preventing formation oiE Ap amyloid, promoting, inducing or otherwise
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facilitating resolubilization of A(3 deposits in the brain, or both, for a
time and
under conditions to effect said treatment.
In one aspect, the invention relates to a method of treating amyloidosis
in a subject, said method comprising administering to said subject an
effective
amount of (a) a metal chelator selected from the group consisting of:
bathocuproine, bathophenanthroline, penacillamine, TETA, TPEN or
hydrophobic derivatives thereof; and (b) one or more pharmaceutically
acceptable
carriers or diluents; for a time and under conditions to bring about said
treatment;
and
wherein said chelator prevents formation of Ap amyloid, promotes, induces or
otherwise facilitates resolubilization of Ap deposits, or both. In another
aspect,
the invention relates to said method further comprising administering to the
subject an effective amount of a compound selected from the group consisting
of:
rifampicin, disulfiram, and indomethacin, or a pharmaceutically acceptable
salt
thereof.
In yet another aspect, the invention relates to a method of treating
amyloidosis in a subject, said method comprising administering to said subject
a combination of (a) a metal chelator selected from the following group:
bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine,
TETA, and TPEN, or hydrophobic derivatives thereof; and (b) a supplement
selected from the group consisting of: ammonium salt, calcium salt, magnesium
salt, and sodium salt, for a time and under conditions to bring about said
treatment; and wherein said combination prevents formation of A(3 amyloid,
promotes, induces or otherwise facilitates resolubilization of AP deposits, or
both.
In a preferred embodiment, the metal chelator is EGTA. In another
preferred embodiment, the the metal chelator is TPEN. In yet another preferred
embodiment, the supplement is a magnesium salt. In another aspect, the
invention relates to said method further comprising administering to the
subject
an effective amount of a compound selected from the group consisting of:
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rifampicin, disulfiram, and itidomethacin, or a pharmaceutically acceptable
salt
thereof.
In another as;pect, the invention relates to a method of treating amyloidosis
in a subject, said method cornprising administering to said subject an
effective
amount of a salt of a metal chelator, wherein said chelator is selected from
the
group consisting of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA,
penacillamine, TETA, and TPEN, or hydrophobic derivatives thereof; wherein
said salt is selected from the group consisting of: ammonium, calcium,
magnesium, and sodium; and wherein said salt of a metal chelator prevents
formation of A(3 amyloidl, promotes, induces or otherwise facilitates
resolubilization of A.P deposits, or both. In a preferred embodiment, the
metal
chelator is EGTA. ][n another preferred embodiment, the the metal chelator is
TPEN. In yet another preferred embodiment, the salt of a metal chelator is a
magnesium salt. In another a.spect, the invention relates to said method
further
comprising adminisitefing to the subject an effective amount of a compound
selected from the group consisting of: rifampicin, disulfiram, and
indomethacin,
or a pharmaceutically acceptable salt thereof.
In another aspect, the invention relates to a method of treating amyloidosis
in a subject, said method comprising administering to said subject an
effective
amount of a chelator specific for copper; wherein said chelator prevents
formation
of Ap amyloid, prornotes, induces or otherwise facilitates resolubilization of
A(3
deposits, or both. In a preferred embodiment, the chelator specific for copper
is
specific for the reduced form of copper. Most preferrably, the chelator is
bathocuproine or a hydrophobic derivative thereof.
In yet another aspect, the invention relates to a method of treating
amyloidosis in a subject, said method comprising administering to said subject
an effective amount of an alkalinizing agent, wherein said alkalinizing agent
prevents formation of Ap aniyloid, promotes, induces or otherwise facilitates
resolubilization of A(3 deposits, or both. In a preferred embodiment, the
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alkalinizing agent is magnesium citrate. In another preferred embodiment, the
alkalinizing agent is calcium citrate.
Still another aspect contemplates pharmaceutical compositions for the
prevention, reduction or inhibition of ROS production bv Ap deposits, or the
prevention of formation of A(3 amyloid, promoting, inducing or otherwise
facilitating the resolubilization of Ap deposits, or both, in the brain.
In accordance with another aspect of the invention, there is provided a
pharmaceutical composition for treatment of conditions caused by amyloidosis,
Ap-
mediated ROS formation, orboth, comprising: (a) a metal chelator selected from
the
group consisting of: bathocuproine, bathophenanthroline, penacillamine, TETA,
and
TPEN, or hydrophobic derivatives thereof; and (b) one or more pharmaceutically
acceptable carriers or diluents.
In one aspect, the invention relates to a pharmaceutical composition for
treatment of conditions caused by amyloidosis, Ap-mediated ROS formation, or
both, comprising: (a) a metal chelator selected from the group consisting of:
bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine,
TETA, and TPEN, or hydrophobic derivatives thereof; and (b) a supplement
selected from the group consisting of: ammonium salt, calcium salt, magnesium
salt, and sodium salt, together with one or more pharmaceutically acceptable
carriers or diluents.
In a preferred embodiment, the metal chelator is EGTA. In another
preferred embodiment, the metal chelator is TPEN. In yet another preferred
embodiment, the supplement is a magnesium salt.
In another aspect, the invention relates to a pharmaceutical composition
for treatment of conditions caused by amyloidosis, A(3-mediated ROS formation,
or both, comprising a salt of a metal chelator selected from the group
consisting
of: bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine,
TETA, and TPEN, or hydrophobic derivatives thereof; and wherein said salt is
selected from the group consisting of: ammonium, calcium, magnesium, and
sodium, together with one or more pharmaceutically acceptable carriers or
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diluents. In a preferred embodiment, the metal chelator is EGTA. In another
preferred embodiment, the the metal chelator is TPEN. In yet another preferred
embodiment, the salt of a metal chelator is a magnesium salt.
In yet another aspect, the invention relates to pharmaceutical composition
for treatment of conditions caused by amyloidosis, Ap-mediated ROS formation,
or both, comprising a chelator specific for copper, with one or more
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pharmaceutically acceptable carriers or diluents. In a preferred embodiment,
the
chelator is specific for the reduced form of copper. Most preferrably, the
chelator
specific for the reduced form. of copper is bathocuproine.
In another aspect, the invention relates to a pharmaceutical composition
for treatment of conditions caused by amyloidosis, Ap-mediated ROS formation,
or both, comprising an alkalinizing agent, with one or more pharmaceutically
acceptable carriers or diluents. In a preferred embodiment, the alkalinizing
agent
is magnesium citrate. In another preferred embodiment, the alkalinizing agent
is
calcium citrate.
In yet another aspect, the invention relates to a composition of matter
comprising: (a) a metal c:helator selected from the group consisting of:
bathocuproine, bathophenarithroline, penacillamine, TETA, and TPEN, or
hydrophobic derivatives thei=eof; and (b) a compound selected from the group
consisting of: rifampicin, disulfiram, and indomethacin.
In still anotfier aspect, the invention relates to a composition of matter
comprising: (a) a metal chelator selected from the group consisting of:
bathocuproine, bathophenanthroline, DTPA, EDTA, EGTA, penacillamine,
TETA, and TPEN, or hydrophobic derivatives thereof; and (b) a supplement
selected from the group consisting of: ammonium salt, calcium salt, magnesium
salt, and sodium salt. In a preferred embodiment, the metal chelator is EGTA.
In
another preferred embodiment, the metal chelator is TPEN. In yet another
preferred embodiment, the supplement is a magnesium salt.
Still another aspect, the invention relates to a method for determining
which metal chelators used in the treatment of amyloidosis, should be
supplemented with, ammonium, calcium, magnesium, or sodium salts,
comprising:
(a) contacting A(3 aggregates with solutions containing a range
of concentrations of' said met:al chelators;
(b) prepar=ing a dilution curve from data obtained in step (a);
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(c) selecting chelators which solubilize less A(3 aggregates at
higher concentrations than at lower or intermiate concentrations;
(d) contacting Ap aggregates with chelators selected in step(c),
in the presence of an ammonium, calcium, magnesium or sodium salt; and
(e) determining if resolubilization is increased in the presence
of said salt; thereby determining whether a metal chelator used in the
treatment
of amyloidosis should be supplemented with ammonium, calcium, magnesium,
or sodium salts.
Brief Description of the Figures
Figure 1 is a graph showing the proportion of soluble AP1_40 remaining
following centrifugation of reaction mixtures.
Figures 2A-2C: Figure 2A is a graph showing the proportion of soluble
AP,_qo remaining in the supernatant after incubation with various metal ions.
Figure 2B is a graph showing a turbidometric analysis of pH effect on metal
ion-
induced AP1_40 aggregation. Figure 2C is a graph showing the proportion of
soluble Ap 1-40 remaining in the supernatant after incubation with various
metal
ions, where high metal ion concentrations were used.
Figure 3 is a graph showing a competition analysis of AP,_40 binding to
Cu2+.
Figures 4A-4C: Figure 4A is a graph showing the proportion of soluble
AP1_40 remaining in the supematant following incubation at various pHs in PBS
Zn2+ or Cu'-+. Figure 4B is a graph showing the proportion of soluble AP,-40
remaining in the supernatant following incubation at various pHs with
different
Cu'-+concentrations. Figure 4C is a graph showing the relative aggregation of
nM
concentrations of AP 1_40 at pH 7.4 and 6.6 with different Cu'-
concentrations.
Figures 5A and 5B: Figure 5A is a graph showing a turbidometric
analysis of Cu2i-induced Ap,_40 aggregation at pH 7.4 reversed by successive
cycles of chelator. Figure 5B is a graph showing a turbidometric analysis of
the
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reversibility of Cu'-+-induced Ap,_qo aggregation as the pH cycles between 7.4
and 6.6.
Figure 6 shows the aniino acid sequence of APP669_7,6 near Ap,.4,. Rat Ap
is mutated (R5G, 'Y'IOF, H13R; bold). Possible metal-binding residues are
underlined.
Figure 7 is a graph showing the effects of pH, Zn'i or Cu2' upon AP
deposit formation.
Figure 8 is a western blot showing the extraction of Ap from post-mortem
brain tissue.
Figure 9 is a western blot showing Ap SDS-resistant polymerization by
copper.
Figure 10 is a graph showing Cu+ generation by Ap.
Figure 11 is a graph showing H,O, production by Ap.
Figure 12 is a graphical representation showing a model for the
generation of reduceil metal ions, OZ, H,O,, and OH= by A(3 peptides. Note
that
A(3 facilitates two consecutive steps in the pathway: the reduction of metal
ions,
and the reaction of O, with reduced metal ions. The peptide does not appear to
be consumed or modified in a one hour time frame by participation in these
reactions.
Figures 13A and 13B are graphical representations showing Fe3+ or Cu'-+
reduction by A(3 peptides. Figure 13A illustrates the reducing capacity of A(3
species (10 M), compared to Vitamin C and insulin (Sigma) (all 10 M) towards
Fe3+ or Cu'-' (10 M) in PBS, pH 7.4, after 1 hour co-incubation, 37 C. Data
indicate concentration of reduced metal ions generated. Figure 13B shows the
effect of oxygen tension and chelation upon A(31_4, metal reduction. AD,42 was
incubated as in Figure 13A under various buffer gas conditions. "Ambient" = no
efforts were made to adjust the gas tension in the bench preparations of the
buffer
vehicle, "O," = 100%' 02 was continuously bubbled through the PBS vehicle for
2 hours (at 20 C), before the remainder of the incubation components were
added, "Ar" = 100% Ar was continuously bubbled through the PBS vehicle for
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2 hours (at 20 C), before the remainder of the incubation components were
added. "+DFO or TETA" = Desferrioxamine (DFO, Sigma, 200 M) was added
to the A(3,42 incubation in the presence of Fe3+ 10 M, or
triethylenetetramine
dihydrochloride (TETA, Sigma, 200 M) was added to the Ap 1-42 incubation in
the presence of Cu2+ 10 M, under ambient oxygen conditions. All data points
are
means fSD, n = 3.
Figures 14A-14E are graphical representations showing production of
H,O, from the incubation of Ap in the presence of substoichiometric amounts of
Fe'; or Cu''. Figure 14A shows H1O, produced by Ap_4, (in PBS, pH 7.4, under
ambient gas conditions, 1 hour, 37 C) following co-incubation with various
concentrations of catalase in the presence of 1 M Fe3+. Figure 14B shows a
comparison of H202 generation by variant A(3 species: A(31_42, AP1-40, rat
Ap1_40,
A(340_1 and AP,.28 (vehicle conditions as in Figure 14A). Figure 14C shows the
effect of metal chelators (200 M) on H202 production from AP1_4, when
incubated in the presence of Fe31 or Cu''+ (1 M) (vehicle conditions as in
Figure 14A). BC = Bathocuproinedisulfonate, BP = Bathophenanthroline-
disulfonate. The effects of DFO were assessed in the presence of Fe3-, and
TETA
was assessed in the presence of Cu'', as indicated. Figure 14D shows H20z
produced by AP1-42, A(31_40, and Vitamin C in the presence of Fe3+ (1 M) (in
PBS,
pH 7.4 buffer, 1 hr, 37 C) under various dissolved gas conditions (described
in
Figure 13B): ambient air, O, enrichment, and anaerobic (Ar) conditions, as
indicated. Figure 14E shows H202 produced by A(31_42, A(31-40, and Vitamin C
in
the presence of Cu2+ (1 M) (in PBS, pH 7.4 buffer, 1 hr, 37 C) under various
dissolved gas conditions (as in Figure 14D). All data points are means fSD, n
= 3.
Figure 15A and 15B are graphical representations showing superoxide
anion detection. Figure 15A shows the spectrophotometric absorbance at 250 nm
(after subtracting buffer blanks) for A(31_42 (10 M, in PBS, pH 7.4, with I
M
Fe3+, incubated 1 hr, 3 7 C) under ambient air (+ 100 U/mL superoxide
dismutase,
SOD), 02 enrichment, and anaerobic (Ar) buffer gas conditions (described in
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Figure 13B). Figwre 15B shows the spectrophotometric absorbance at 250 nm
(after subtracting buffer blanks) for variant A(3 peptides: Ap 1-42, Ap 1_40,
rat Ap ,-ao~
A(340-1, and Ap 1-28 (10 M in PBS, pH 7.4, with I M Fe3+, incubated I hr, 37
C,
under ambient buffer gas conditions). All data points are means SD, n = 3.
Figure 16A and 16B are graphical representations showing production
of the hydroxyl radical (OH=) from the incubation of Ap in the presence of
substoichiometric amounts of Fe3+ or Cu2+. Figure 16A shows the signal from
the
TBARS assay of OfI= produced from Vitamin C (100 M) and variant Ap species
(10 M): AP,-42, AP,140, rat AP,-40, AP40-1, and A(3t-28 (in PBS, pH 7.4, with
1 M
Fe3" or Cuz' as indicated, incubated I hr, 37 C, under ambient buffer gas
conditions). Figure 16B illustrates the effect of OH=-specific scavengers upon
OH= generation by'Vitamin C and AP1_42. Mannitol (5 mM, Sigma) or dimethyl
sulfoxide (DMSO, 5 mM, Siigma), was co-incubated with Vitamin C (10 M +
500 M H20,) or A(31-42 ( I 0 M) (conditions as for Figure 16A). All data
points
are means SD, n = 3.
Figure 17 shows the reversibility of zinc-induced Ap-40aggregation with
EDTA. Aggregation induced by pH 5.5 was not reversable in the same manner
(data not shown).
Figure 18 shows the: reversibility of zinc-induced aggregation of AP1.40
mixed with 5% A(3,_42.
Figures 19A-19C shows dilution curves for TPEN, EGTA, and
bathocuproine, respectively, used in extracting a representative AD brain
sample.
Figures 19A-19C show that metal chelators promote the solubilization of AP
from human brain sample homogenates.
Figures 20A and 20B - Figure 20A shows a western blot of chelation
response in a typical AD brain. Figure 20B shows a western blot comparing
extracted Ap from an AD brain (AD) to that of sedimentable deposits from
healthy brain tissue (young control - C). In the experiments of Figure 20B,
TBS
buffer was used rather than PBS.
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Figure 21 shows an indicative blot from AD brain extract. The blot
shows that chelation treatment results in disproportionate solubilization of
A(3
dimers, while PBS alone does not.
Figure 22 shows that recovery of total soluble protein is not affected by
the presence of chelators in the homogenization step.
Figure 23 shows that extraction volume affects A(3 solubilization.
Figures 24A and 24B - Figure 24A shows the effect of metals upon the
solubility of brain-derived A(3: copper and zinc can inhibit the
solubilization of
Ap. Figure 24B shows that Ap solubility in metal-depleted tissue is restored
by
the addition of magnesium.
Figures 25A and 25B - Figure 25A shows that patterns of chelator-
promoted solubilization of Ap differ in AD and aged-matched, non-AD tissue.
Upper panel: representative blot from AD specimen.
Lower panel: representative blot from aged non-AD tissue bearing a
similar total A(3 load.
Figure 25B shows soluble Ap resulting from chelation treatment for AD
and aged-matched, non-AD tissue, expressed as a percentage of the PBS-only
treatment group.
Figure 26 shows that chelation promotes the solubilization of Ap1_40 and
AP1_4, from AD and non-AD tissue. Representative AD (left panels) and aged-
matched control specimens (right panels) were prepared as described in PBS or
5 mM BC. Identical gels were run and Western blots were probed with mAbs
W02 (raised against residues 5-16, recognizes Ap1_40 and Ap 1_42) G2 10
(raised
against residues 35-40, recognizes AP1_40) or G211 (raised against residues 35-
42,
recognizes A(3i42) (See Ida, N. et al., J. Biol. Chem. 271:22908 1996).
Figure 27A and 27B - Figure 27A shows SDS-resistant polymerization
of human AP, _40 versus human A(3, _4, with Cuz+or Fe3+. Figure 27B shows SDS-
resistant polymerization of rat A(31_40 with CuZ+ or Fe3+
Figures 28A - 28C - Figure 28A shows Hz02/Cu induced SDS-resistant
polymerization of AP1_42 (2.5 M). Figure 28B shows HZOZ/Fe induced SDS-
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resistant polymerization of A(31_42 (2.5 uM). Figure 28C shows that BC
attenuates SDS-resistant polymerization of Ap , _42 (2.5 M).
Figures 29A and 2913 show that H,02 generation is required for SDS-
resistant polymerization of human A(31_42. Solution concentrations of metal
ion
and H202 were 30 M and 100 M, respectively. Figure 29A shows that TCEP
(Tris(2-Carboxyethy;l)-Phosphine Hydrochloride) attenuates SDS-resistant A(3,
_ 42
polymerization. Ap , _42 (2.5 ,uM), H,O, (100 M), ascorbic acid (100 ,uM),
TCEP
(100 M). Figure 29B shows that anoxic conditions prevent SDS-resistant AP
polymerization. Ap , _4, (2.5 A.cM) was incubated with no metal or CuZ' at
either
pH 7.4 or 6.6 and incubated i:or 60 min. at 25 C under normal or argon purged
conditions. Argon was continuously bubbled through the buffer for 2 h (at 20
C)
before the remainder of the incubation components were added.
Figures 30A-=30E shoiv dissolution of SDS-resistant A(3 polymers. Figure
30A shows that chaotrophic agents are unable to disrupt polymerization. Figure
30B shows that metal ion chelators disrupt SDS-resistant AP, _ao polymers.
Figure
30C shows that metal ion chelators disrupt SDS-resistant AP, _42 polymers. The
chelators, their log stability constant, and their molecular weight,
respectively, are
as follows: TETA (te-traethyleiiediamine), 20.4,146; EDTA
(ethylenediaminetetra
acetic acid), 18.1, 292; DTPA (diethylenetriaminopenta acetic acid), 21.1,
393;
CDTA (trans-l,2-diaminocyclohexanetetra acetic acid), 22.0, 346; and NTA
(nitrilotriacetic acid), 13.1, 191. Figure 30D shows that a-helical promoting
solvents and low pH disrupt polymers. Aliquots of Ap_4, were incubated at pH
1 or with DMSO/HF'IP (75%:25%) for 2 h (30 min., 37 C). Figure 30E shows
that metal ion chelators disrupt SDS-resistant Ap polymers extracted from AD
brains. Aliquots of SDS-resistant A(3 polymers extracted from AD brains were
incubated with no chelator, T'ETA (1 mM or 5 mM) or BC (1 mM or 5 mM) for
2 h (30 min., 37 C) and aliquots collected for analysis. Monomer AP,_40 is
indicated.
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Detailed Description of the Preferred Embodiments
Definitions
In the description that follows, a number of terms are utilized extensively.
In order to provide a clear and consistent understanding of the specification
and
claims, including the scope to be given such terms, the following definitions
are
provided.
Ap peptide is also known in the art as Ap, P protein, P-A4 and A4. In the
present invention, the A(3 peptide may be comprised of peptides API-39, AI'I-
40,
AP,-4,, AP, -4,, and AV43. The most preferred embodiment of the invention
makes
use of A(3,-4Q. However, any of the Ap peptides may be employed according to
the present invention. The sequence of Ap peptide is found in Hilbich, C., et
al.,
J. Mol. Biol. 228:460-473 (1992).
Amyloid as is commonly known in the art, and as is intended in the
present specification, is a form of aggregated protein.
Amyloidosis is any disease characterized by the extracellular
accumulation of amyloid in various organs and tissues of the body.
Ap Amyloid is an aggregated A(3 peptide. It is found in the brains of
patients afflicted with AD and DS and may accumulate following head injuries.
Biological fluid means fluid obtained from a person or animal which is
produced by said person or animal. Examples of biological fluids include but
are
not limited to cerebrospinal fluid (CSF), blood, serum, and plasma. In the
present
invention, biological fluid includes whole or any fraction of such fluids
derived
by purification by any means, e.g., by ultrafiltration or chromatography.
Copper(II), unless otherwise indicated, means salts of Cu2+, i.e., Cu'-+ in
any form, soluble or insoluble.
Copper(I), unless otherwise indicated, means salts of Cu+, i.e., Cu+in any
form, soluble or insoluble.
Metal chelators include metal-binding molecules characterized by two
or more polar groups which participate in forming a complex with a metal ion,
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and are generally well-known in the art for their ability to bind metals
competitively.
Physiological solution as used in the present specification means a
solution which cornprises compounds at physiological pH, about 7.4, which
closely represents a bodily or biological fluid, such as CSF, blood, plasma,
et cetera.
Treatment: delay or prevention of onset, slowing down or stopping the
progression, aggravation, or deterioration of the symptoms and signs of
Alzheimer's disease, as well as amelioration of the symptoms and signs, or
curing
the disease by reversing the physiological and anatomical damage.
Zinc, unless otherwise indicated, means salts of zinc, i.e., Zn'1 in any
form, soluble or insoluble.
Methods for ldent fying Agents Useful in the Treatment of AD
The aim of the present invention is to clarify both the factors which
contribute to the neurotoxicity of Ap polymers and the mechanism which
underlies their formation. These findings can then be used to (i) identify
agents
that can be used to dlecrease the neurotoxicity of Ap, as well as the
formation of
Ap polymers, and (ii) utilize such agents to develop methods of preventing,
treating or alleviating the sytnptoms of AD and related disorders.
The present invention relates to the unexpected discovery that Ap peptides
directly produce oxidative stress through the generation of abundant reactive
oxygen species (ROS), which include hydroxyl radical (OH-) and hydrogen
peroxide (H202). The production of ROS occurs by a metal (Cu, Fe) dependant,
pH mediated mechanism, wherein the reduction of Cu'-+ to Cu+, or Fe3i to Fe'-
+,
is catalyzed by Ap. Ap is highly efficient at reducing Cu'-+ and Fe3T
All the redox properties of Ap 1-40 (the most abundant form of soluble AP)
are exaggerated in A(31_4,. Additionally, A(31_42, but not AP,-40, recruits O,
into
spontaneous generation of another ROS, O;, which also occurs in a metal-
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dependent manner. The exaggerated redox activity of A(31_4, and its enhanced
ability to generate ROS are likely to be the explanation for its neurotoxic
properties. Interestingly, the rat homologue of Ap, which has 3 substitutions
that
have been shown to attenuate zinc binding and zinc-mediated precipitation,
also
exhibits less redox activity than its human counterpart. This may explain why
the rat is exceptional in that it is the only manunal that does not exhibit
amyloid
pathology with age. All other mammals analyzed to date possess the human Ap
sequence.
The sequence of ROS generation by A(3 follows the pathway of
superoxide-dismutation, which leads to hydrogen peroxide production in a Cu/Fe-
dependent manner. After forming H,O,, the hydroxyl radical (OH=) is rapidly
formed by a Fenton reaction with the Fe or Cu that is present, even when these
metals are only at trace concentrations. The OH= radical is very reactive and
rapidly attacks the Ap peptide, causing it to cross-link and polymerize. This
is
very likely to be the chemical mechanism that causes the covalent cross-
linking
that is seen in mature plaque amyloid. Importantly, the redox activity ofAD is
not
attenuated by precipitation of the peptide, suggesting that, in vivo, amyloid
deposits could be capable of generating ROS in situ on an enduring basis. This
suggests that the major source of the oxidative stress in an AD-affected brain
are
amyloid deposits.
A model for free radical and amyloid formation in AD is shown in
Figure 12. The proposed mechanism is explained as follows.
(1) Soluble and precipitated A(3 species possess superoxide dismutase
(SOD)-like activity. Superoxide (O2), the substrate for the dismutation, is
generated both by spillover from mitochondrial respiratory metabolism, and by
Ap1_4, itself. Ap-mediated dismutation produces hydrogen peroxide (H20,)(see
Figure 11), requiring Cu2+ or Fe3+, which are reduced during the reaction.
Since
H+ is required for H2O2 production, an acidotic environment will increase the
reaction.
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(2) HZO., is relatively stable, and freely permeable across cell
membranes. Nornially, it will be broken down by intercellular catalase or
glutathione peroxidase.
(3) In a;ging and AD, levels of H2O2 are high, and catalase and
peroxidase activities are low. If H,Oz is not completely catalyzed, it will
react
with reduced Cu+ and Fe'+ im the vicinity of Ap to generate the highly
reactive
hydroxyl radical (OH=) by Fenton chemistry.
(4) OH= engenders a non-specific stress and inflammatory response
in local tissue. Among the neurochemicals that are released from microglia and
possibly neurons in the response are Zn'-+, Cu'' and soluble Ap. Familial AD
increases the likelihood that A(31_4, will be released at this point. Local
acidosis
is also part of the stress/inflammatory response. These factors combine to
make
Ap precipitate and accumulate, presumably so that it may function in situ as
an
SOD, since these factors induce reversible aggregation. Hence, more soluble
A(3
species decorate the perimeter of the accumulating plaque deposits.
(5) If A',P encounters OH=, it will covalently cross-link during the
oligomerization process, malcing it a more difficult accumulation to
resolubilize,
and leading to the formation of SDS-resistant oligomers characteristic of
plaque
amyloid.
(6) If Al~1_42 accumulates, it has the property of recruiting 0, as a
substrate for the abundant production of O. by a process that is still not
understood. Since O2 is abundant in the brain, A(3,_42 is responsible for
setting off
a vicious cycle in wl:iich the accumulation of covalently linked Ap is a
product of
the unusual ability of A(3 to reduce O,, and feed an abundant substrate (02)
to
itself for dismutation, leading to OH= formation. The production of abundant
free
radicals by the accumulating amyloid may further damage many systems
including metal regulatory proteins, thus compounding the problem. This
suggests that the major source of the oxidative stress in an AD-affected brain
are
amyloid deposits.
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The metal-dependent chemistry of Ap-mediated superoxide dismutation
is reminiscent of the activity of superoxide dismutase (SOD). Interestingly,
mutations of SOD cause amyotrophic lateral sclerosis, another
neurodegenerative
disorder. SOD is predominantly intracellular, whereas Ap is constitutively
found
in the extracellular spaces where it accumulates. Investigation of A(3 by
laser
flash photolysis confirmed the peptide's SOD-like activity, suggesting that
A(3
may be an anti-oxidant under physiological circumstances. Since H,O, has been
shown to induce the production of Ap, the accumulation of Ap in AD may reflect
a response to an oxidant stress paradoxically caused by A(3 excess. This may
cause and, in turn, be compounded by, damage to the biometal homeostatic
mechanisms in the brain environment.
Thus, it has recently been discovered (i) that much of the AP aggregate
in AD-affected brain is held together by zinc and copper, (ii) that A(3
peptides
exhibit Fe/Cu-dependent redox activity similar to that of SOD, (iii) that
A(3,42 is
especially redox reactive and has the unusual property of reducing O, to Oz,
and
(iv) that deregulation of Ap redox reactivity causes the peptide to
conveniently
polymerize. Since these reactions must be strongly implicated in the
pathogenetic
events of AD, they offer promising targets for therapeutic drug design.
The discovery that Ap can generate H2O2 and Cu+, both of which are
associated with neurotoxic effects, offers an explanation for the
neurotoxicity of
Ap polymers. These findings suggest that it may be possible to lessen the
neurotoxicity of Ap by controlling factors which alter the concentrations of
Cu+
and ROS, including hydrogen peroxide, being generated by accumulated and
soluble A. It has been discovered that manipulation of factors such as zinc,
copper, and pH can result in altered Cu+ and H,OZ production by A. Therefore,
agents identified as being useful for the adjustment of the pH and levels of
zinc
and copper of the brain interstitium can be used to adjust the concentration
of Cu+
and H202, and can therefore be used to reduce the neurotoxic burden. Such
agents will thus be a means of treating Alzheimer's disease.
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Thus, one object of the present invention is to provide a method for the
identification of agerits to be used in the treatment of AD. As may be
understood
by reference to the Examples below, agents to be used in the treatment of AD
include:
(a) agents that reduce the amount of Cu' or Fe2+ produced by A(3;
(b) agents that promote or inhibit the production of hydrogen peroxide
by Af3;
(c) agents that inhibit the production of O; by AP;
(d) agents that inhibit the production of OH=.
Of course, as aggregation and especially crosslinking of AP contributes
to the neurotoxic burden, agents which have been identified to have the
activities
listed above may then also be subjected to tests which determine if an agent
is
capable of inhibiting Ap plaqiie deposition or facilitating plaque
resolubilization
(see Example 1).
Agents identified as having the above-listed activities may then be tested
for their ability to reduce the neurotoxicity of both soluble and crosslinked
Ap.
Thus, in one aspect, the invention relates to a method for the identification
of an agent to be used in the treatment of AD, wherein the agent is capable of
altering, and preferably decre;asing, the production of Cu+ by A(3, the method
comprising:
(a) adding Cu2+ to a first Ap sample;
(b) allowirig the first sample to incubate for an amount of time
sufficient to allow sa.id first sample to generate Cu';
(c) adding Cu2+ to a second A(3 sample, the second sample
additionally comprising a candidate pharmacological agent;
(d) allowirig the second sample to incubate for the same
amount of time as the first sainple;
(e) determining the amount of Cu+ produced by the first
sample and the secorid sample; and
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(f) comparing the amount of Cu+ produced by the first sample
to the amount of Cu+ produced by the second sample;
whereby a difference in the amount of Cu- produced by the first sample as
compared to the second sample indicates that the candidate pharmacological
agent has altered the production of Cu+ by A. Of course, where the amount of
Cu+ is lower in the second sample than in the first sample, this will indicate
that
the agent has decreased Cu' production.
In a preferred embodiment, the amount of Cu+ present in said first and
said second sample is determined by
(a) adding a complexing agent to said first and said second
sample, wherein said complexing agent is capable of combining with Cu+ to form
a complex compound, wherein said complex compound has an optimal visible
absorption wavelength;
(b) measuring the absorbancy of said first and said second
sample; and
(c) calculating the concentratioh of Cu' in said first and said
second sample using the absorbancy obtained in step (b).
In a more preferred embodiment, the complexing agent is
bathocuproinedisulfonic (BC) anion. The concentration of Cu' produced by A(3
may then be calculated on the basis of the absorbance of the sample at about
478 nm to about 488 nm, more preferable about 480 to about 486 nm, and most
preferably about 483 nm.
In an even more preferred embodiment, the above-described method may
be performed in a microtiter plate, and the absorbancy measurement is
performed
by a plate reader, thus allowing large numbers of candidate pharmacological
compounds to be tested simultaneously.
In another aspect, the invention relates to a method for the identification
of an agent to be used in the treatment of AD, wherein said agent is capable
of
altering, and preferably decreasing, the production of Fe2+ by Ap, said method
comprising:
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(a) adding, Fe3i to a first Ap sample;
(b) allowing said first sample to incubate for an amount of
time sufficient to allow said first sample to generate CuT;
(c) adding Fe3i to a second Ap sample, said second sample
additionally comprising a carididate pharmacological agent;
(d) allowing said second sample to incubate for the same
amount of time as said first sample;
(e) determining the amount of Fe'-T produced by said first
sample and said second sample; and
(f) comparing the amount of Fe'-" present in said first sample
to the amount of Fe'-- present in said second sample;
whereby a difference in the amount of Fe''+ present in said first sample as
compared to said second sample indicates that said candidate pharmacological
agent has altered the production of Fez+ by Ap. Of course, where the amount of
Fe'' is lower in the second sample than in the first sample, this will
indicate that
the agent has decreased Fe' 1 production.
In a preferred. embodiment, the amount of Fe'-' present is determined by
using a spectrophoto metric method analogous to that used for the
determination
of Cu', above. In this method, the complexing agent is batho-
phenanthrolinedisulfonic (BP) anion. The concentration of Fe2+-BP produced
by Ap may then be calculated on the basis of the absorbance of the sample at
about 530 to about 540 run, more preferably about 533 nm to about 538 nm, and
most preferably about 535 nm.
In an even more preferred embodiment, the above-described method may
be performed in a microtiter plate, and the absorbancy measurement is
performed
by a plate reader, thus allowing large numbers of candidate pharmacological
compounds to be tested simultaneously.
In yet another aspect, the invention relates to a method for the
identification of an agent to be used in the treatment of AD, wherein said
agent
is capable of altering the production of H202 by A(3, said method comprising:
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(a) adding Cu'+ or Fe3+ to a first A(3 sample;
(b) allowing said first sample to incubate for an amount of
time sufficient to allow said first sample to generate H2O2;
(c) adding Cu'-+ or Fe3+ to a second Ap sample, said second
sample additionally comprising a candidate pharmacological agent;
(d) allowing said second sample to incubate for the same
amount of time as said first sample;
(e) determining the amount of H202 produced by said first
sample and said second sample; and
(f) comparing the amount of HZO, present in said first sample
to the amount of H202 present in said second sample;
whereby a difference in the amount of H202 present in said first sample as
compared to said second sample indicates that said candidate pharmacological
agent has altered the production of H2O, by A(3. As will be understood by one
of
ordinary skill in the art, this method may be used to detect agents which
decrease
the amount of H2O2 produced (in which case the amount of H202 will be lower
in the second sample than in the first sample), or to increase the amount of
H,O,
produced (in which case the amount of H2O2 will be lower in the first sample
than
in the second sample).
In a preferred embodiment, the determination of the amount of H,O7
present in said first and said second sample is determined by
(a) adding catalase to a first aliquot of said first sample
obtained in step (a) of claim I in an amount sufficient to break down all of
the
H2O2 generated by said sample;
(b) adding TCEP, in an amount sufficient to capture all of the
H,02 generated by said samples, to
(i) said first aliquot
(ii) a second aliquot of said first sample obtained in
step (a) of claim 1; and
(iii) said second sample obtained in step (b) of claim 1;
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(c) incubating the samples obtained in step (b) for an amount
of time sufficient to allow the TCEP to capture all of the H202;
(d) adding DTNB to said samples obtained in step (c);
(e) incubating said samples obtained in step (d) for an amount
of time sufficient to generate TMB;
(f) measuring the absorbancy at about 407 to about 417 nm of
said samples obtained in step (e); and
(g) calculating the concentration of H2O2 in said first and said
second sample using the absorbancies obtained in step (f).
In a preferred embodiment, the absorbancy of TMB is measured at about
412 nm.
In a preferreci embodiment, the above-described method is performed in
a microtiter plate, and the absorbancy measurement is performed by a plate
reader, thus makirig it possible to screen large numbers of candidate
pharmacological age;nt simultaneously.
In another embodiment, the invention provides a method for the
identification of an agent to be used in the treatment of AD, wherein said
agent
is capable of decreasing the production of Oz by A(3, said method comprising:
(a) adding, Ap and to a first buffer sample having an O, tension
greater than 0;
(b) allowing said first sample to incubate for an amount of
time sufficient to allow said first sample to generate Oz;
(c) adding, Ap and a candidate pharmacological agent to a
second buffer sample having an O, tension greater than 0;
(d) allowing said second sample to incubate for the same
amount of time as said first sample;
(e) determining the amount of 02 produced by said first
sample and said second sample; and
(f) comparing the amount of OZ present in said first sample to
the amount of 02 present in said second sample;
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whereby a difference in the amount of O2 present in said first sample as
compared
to said second sample indicates that said candidate pharmacological agent has
altered the production of Oz by A. In a preferred embodiment, the Ap used is
AF'1-42=
Of course, the amount of 0, produced by A(3 may be measured by any
method known to those of ordinary skill in the art. In a preferred embodiment,
the determination of the amount of 02 present in said samples is accomplished
by
measuring the absorbancy of the sample at about 250 nm.
Because the ability of A(3 to generate H,O2 from O-, may in many
instances be beneficial, in a preferred embodiment, the invention also relates
to
a method for the identification of an agent to be used in the treatment of AD,
wherein said agent is capable of interfering with the interaction of O, and AP
to
produce O2, without interfering with the SOD-like activity of AP, said method
comprising:
(a) identifying an agent capable of decreasing the production
of Oz by AP; and
(b) determining the ability of said agent to alter the SOD-like
activity of Ap. In a preferred embodiment, the determination of the ability of
said
agent to alter the SOD-like activity of A(3 is made by determining whether Ap
is
capable of catalytically producing Cu+, Fe=' or H202. Methods, besides those
which are disclosed elsewhere in this application, for determining if Ap is
capable
of catalytically producing Cu+, Fe2+ or H202 are well known to those of
ordinary
skill in the art. In particular, the catalytic production of H202 may be
determined
by using laser flash photolysis or pulse radiolysis (Peters, G. & Rodgers,
M.A. J.,
Biochim. Biophys. Acta 637:43-52 (1981).
In another aspect, candidate pharmacological agents which have been
identified by one or more of the above screening assays can undergo further
screening to determine if the agents are capable of altering, and preferably
reducing or eliminating, A(3-mediated toxicity in cell culture. Such assays
include the MTT assay, which measures the reduction of 3-(4,5-dimethylthiazol-
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2-yl)-2,5, diphenyl tetrazolium bromide (MTT) to a colored formazon (Hansen
et al., Jlmmunol Methods, 119:203-210 (1989)). Although alternatives have not
been ruled out (see: Burdon. et al., Free Radic Res Commun.,18(6):369-380
(1993)), the major site of MT'T reduction is thought to be at two stages of
electron
transport, the cytochrome ox:idase and ubiquinone of mitochondria (Slater et
al.,
1963). A second cytotoxic assay is the release of lactic dehydrogenase (LDH)
from cells, a measurement routinely used to quantitate cytotoxicity in
cultured
CNS cells (Koh, J.Y. and D.W. Choi, J. Neurosci. Meth. 20:83-90 (1987). While
MTT measures priinarily early redox changes within the cell reflecting the
integrity of the electron transport chain, the release of LDH is thought to be
through cell lysis. .A third assay is visual counting in conjunction with
trypan
blue exclusion. Other commercially available assays for neurotoxicity,
including
the Live-Dead assay, may a;lso be used to determine if a candidate compound
which alters Cu', Fe2+, H20,, OH-, and OZ production, or alters copper-
induced,
pH dependent aggregation and crosslinking of Ap, is also capable of reducing
the
neurotoxicity of Ap.
Thus, in another preferred embodiment, the invention relates to a method
for the identificatiori of an agent to be used in the treatment of AD, wherein
said
agent is capable of reducing the toxicity of A(3, said method comprising:
(a) adding Ap to a first cell culture;
(b) adding Ap to a second cell culture, said second cell culture
additionally containing a candidate pharmacological agent;
(c) determining the level of neurotoxicity of Ap in said first
and said second samples; and
(d) comparing the level of neurotoxicity of Ap in said first and
said second samples,
whereby a lower neurotoxicity level in said second sample as compared to said
first sample indicates that said candidate pharmacological agent has reduced
the
neurotoxicity of Ap, and is thereby capable of being used to treat AD.
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Assays which can be used to determine the neurotoxicity of a candidate
agent include, but are not limited to, the MTT assay and the LDH release
assay,
as described in Behl et al. , Cell 77:817-827 (1994), and the Live/Dead
EukoLight
Viability/Cytotoxicity Assay, commercially available from Molecular Probes,
Inc.
(Eugene, OR).
Cells types which may be used for these neurotoxicity assays include both
cancer cells and primary cells, such as rat primary frontal neuronal cells.
Candidate pharmacological agents to be tested in any of the above-
described methods will be broad-ranging but can be classified as follows:
Candidate pharmacological agents for the alteration of the SOD-like activity
of
A(3 will be broad-ranging but can be classified as follows:
Agents Which Modify the Availability of Zn or Cu for Interaction witl: A,6
They include chelating agents such as desferrioxamine, but also include
amino acids histidine and cysteine which bind free zinc, and are thought to be
involved in bringing zinc from the plasma across the blood-brain barrier
(BBB).
These agents include all classes of specific zinc chelating agents, and
combinations of non-specific chelating agents capable of chelating zinc such
as
EDTA (Edetic acid, N,N'-1,2-Ethane diylbis[N-(carboxymethyl)glycine] or
(ethylenedinitrilo)tetraacetic acid, entry 3490 in Merck Index 10th edition)
and
all salts of EDTA, and/or phytic acid [myo-Inositol hexakis(dihydrogen
phosphate), entry 7269 in the Merck Index 10th edition] and phytate salts.
Preferred candidate agents within this class include bathocuproine and
bathophenanthroline.
Miscellaneous
Because there is no precedent for an effective anti-amyloidotic
pharmaceutical, it is reasonable to serendipitously try out compounds which
may
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have access to the brain compartment for their ability to inhibit either Cu+
or HZOZ
production by Ap. 'These compounds include dye compounds, heparin, heparin
sulfate, and anti-oxidants, e.g., ascorbate, trolox and tocopherols.
In the present invention, the Ap used may be any form of Ap. In a
preferred embodiment, the Ap used is selected from the group consisting of
ARl-39, ANl-40, AP,-419 ARl42, and Ap1-43. Even more preferably, the Ap used
is
AP1_40 or Ap,_4,. The most preferred embodiment of the invention makes use of
AP,-40= The sequence of Ap peptide is found in Hilbich, C., et al., J. Mol.
Biol.
228:460-473 (1992).
The pH of the various reaction mixtures are preferably close to neutral
(about 7.4). The pF[, therefore, may range from about 6.6 to about 8,
preferably
from about 6.6 to about 7.8, and most preferably about 7.4.
Buffers which can be used in the methods of the present invention include,
but are not limiteci to, PBS, Tris-chloride and Tris-base, MOPS, HEPES,
bicarbonate, Krebs, and Tyrode's. The concentration of the buffers may be
between about 10 n>.M and about 500 mM. Because of the nature of the assays
which are included in the methods of the claimed invention, when choosing a
buffer, it must be borne in mind that spontaneous free radical production
within
a given buffer miglit interfere with the reactions. For this reason, PBS is
the
preferred buffer for use in the methods of the invention, although other
buffers
may be used provided that proper controls are used to correct for the above-
mentioned free radical formation of a given buffer.
Cu2+ must be present in the reaction mixture for Ap to produce Cu+. Any
salt of CuZ' may be used to satisfy this requirement, including, but not
limited to,
CuC12 Cu(NO3)2, etc. Concentrations of copper from at least about I M may be
used; most preferable, a copper concentration of about 10 M is to be included
in the reaction mixture.
Similarly, a redox active metal such as CuZ+ or Fe3+ must be present in the
reaction mixture foi- Ap to catalytically produce HZO,. Any salt of Cu2+ may
be
used to satisfy this :requirement, including, but not limited to,'CuCI,
Cu(N03)21
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etc. Similarly, and salt of Fe" may be used in accordance with the invention,
such as FeC13. Concentrations of copper or iron from at least about I M may
be
used; most preferably, a copper or iron concentration of about 10 M is to be
included in the reaction mixture.
The present invention may be practiced at temperatures ranging from
about 25 C to about 40 C. The preferred temperature range is from about 30 C
to about 40 C. The most preferred temperature for the practice of the present
invention is about 37 C, i.e., human body temperature.
The production of Cu" and H202 by A(3 peptide occurs at near-
instantaneous rate. Hence, the measurement of the concentration of Cu' or HZOz
produced may be performed by the present methods substantially immediately
after the addition of Cu2+ to the AD peptide. However, if desired, the
reaction
may be allowed to proceed longer. In a preferred embodiment of the invention,
the reaction is carried out for about 30 minutes.
The invention may also be carried out in the presence of biological fluids,
such as the preferred biological fluid, CSF, to closely simulate actual
physiological conditions. Of course, such fluids will already contain A(3, so
that
where the methods of the invention are to be carried out utilizing a
biological
fluid such as CSF, no further Ap peptide will be added to the sample. The
biological fluid may be used directly or diluted from about 1:1,000 to about
1:5
fold.
The amount of H1O2, Cu+ or Fe2+ produced by a sample may be measured
by any standard assay for H,OZ, Cu+ or Fe2+. For example, the PeroXOquant
Quantitative Peroxide Assay (Pierce, Rockford, IL) may be used to determine
the
amount of H202 produced. FeZ+ may be determined using the spectrophotometric
method of Linert et al., Biochim. Biophys. Acta 1316:160-168 (1996). Other
such
methods will be readily apparent to those of ordinary skill in the art.
In a preferred embodiment, the H202 or Cu+ produced by the sample is
complexed with a complexing agent having an optimal visible absorption
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wavelength. The amount of H,O, or Cu+ produced by a sample is then detected
using optical spectrophotometry (see Example 2).
In a preferred embodiment, the complexing agent to be used for the
determination of the amount of Cu+ produced is bathocuproinedisulfonic anion
(BC), (see Example 2); the complex Cu+-BC has an optimal visible absorption
wavelength of about 483 nm. As is mentioned above, Ap will produce H,02 and
Cu+ almost immediately following the addition of Cuz+ and Zn'-+ to the
reaction
mixture. Thus, BC may be added to the reaction immediately following the
addition of Cu'' and Zn2+ to the Ap samples. The concentration of BC to be
achieved in a sample is between about 10 M to about 400 gM, more preferably
about 75 M to about 300 M, and still more preferably about 150 gM to about
275 gM. In the most preferred embodiment, the concentration of BC to be
achieved in a sample is about 200 M. Of course, one of ordinary skill in the
art
can easily optimize the concentration of BC to be added with no more than
routine experimentation.
Where the anaount of Fez+ produced is to be determined, the complexing
agent to be used for the determination of the amount of Fe2+ produced is
bathophenanthrolinedisulfoni,c (BP) anion, (see Example 2); the complex Fe2+-
BP
has an optimal visible absorption wavelength of about 535 nm. As is mentioned
above, A(3 will produce H202 and Fe2+ almost immediately following the
addition
of Fe3+ and Zn'-+ to the reaction mixture. Thus, BP may be added to the
reaction
immediately followiing the addition of Fe3 and ZnZ+ to the A(3 samples. The
concentration of BP to be achieved in a sample is between about 10 M to about
400 M, more preferably about 75 M to about 300 M, and still more preferably
about 150 M to about 275 M. In the most preferred embodiment, the
concentration of BP to be achieved in a sample is about 200 M. Of course, one
of ordinary skill in the art can easily optimize the concentration of BP to be
added
with no more than routine experimentation.
The above-dE:scribed spectrophotometric assays may be used to determine
the concentration of Cu' or Fe2', as is described in Example 2.
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Each of the assays of the present invention is ideally suited for the
preparation of a kit. Such a kit may comprise a carrier means being
compartmentalized to receive in close confinement therein one or more
container
means, such as vials, tubes, and the like, each of said container means
comprising
one of the separate elements of the assay to be used in the method. For
example,
there may be provided a container means containing standard solutions of the
A(3
peptide or lyophilized AD peptide and a container means containing a standard
solution or varying amounts of a salt of redox active metal, such as Cu'+ or
Fe3+
in any form, i.e., in solution or dried, soluble or insoluble, in addition to
further
carrier means containing varying amounts and/or concentrations of reagents
used
in the present methods. For example, solutions to be used for the
determination
of Cu+ or Fe2+ as described in Example 2 will include BC anion and BP anion,
respectively. Similarly, solutions to be used for the determination of HZO2 as
described in Example 2 include TCEP and DTNB, as well as catalase (1 OU/ml).
Standard solutions of A{3 peptide preferably have concentrations above about
10
M, more preferably from about 10 to about 25 gM or if the peptide is provided
in its lyophilized form, it is provided in an amount which can be solubilized
to
said concentrations by adding an aqueous buffer or physiological solution. The
standard solutions of analytes may be used to prepare control and test
reaction
mixtures for comparison, according to the methods of the present invention.
Agents Useful in the Treatment ofAD
A further aspect of the present invention is predicted in part on the
elucidation of mechanisms of neurotoxicity in the brain in AD subjects. One
mechanism involves a novel O; and biometal-dependent pathway of free radical
generation by Ap peptides. The radicals of this aspect of the present
invention
may comprise reactive oxygen species (ROS) such as but not limited to O2 and
OH as well as radicalized A(3 peptides. It is proposed, according to the
present
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invention, that by ir.iterfering; in the radical generating pathway, the
neurotoxicity
of the A(3 peptides is reduced.
Accordingly, one aspect of the present invention contemplates a method
for treating Alzheimer's disease (AD) in a subject, said method comprising
administering to said subject, an effective amount of an agent which is
capable of
inhibiting or otherviise reducing metal-mediated production of free radicals.
The preferred agents according to this aspect are metal chelators, metal
complexing compounds, anti.oxidants and compounds capable of reducing radical
formation of Ap peptides or mediated by Ap peptides. Particularly preferred
metal chelators and metal complexors are capable of interacting with metals
(M)
having either a reduced charge state (M"+') or an oxidized state of (M-')+.
Even
more particularly, M is Fe and/or Cu.
It is proposed that interactions of A(3 with Fe and Cu are of significance
to the genesis of the oxidation insults that are observed in the AD-affected
brain.
This is due to redox-active inetal ions being concentrated in brain neurons
and
participating in the generation of ROS or other radicals by transferring
electrons
in their reduced staie and described in the following reactions:
Reduced Fe/Cu reacts with lnolecular oxygen to generate the superoxide anion.
M"+ + O, _ M("+' O2 Reaction (1)
The 02 generated undergoes dismutation to H202 either catalyzed by SOD or
spontaneously.
Oz + 02 + 2]:i+ - H2O, + 02
Reaction (2)
The reaction of reduced metals with H202 generates the highly reactive
hydroxyl
radical by the Fenton reaction.
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M"' + H,O2 - Ml"+'1' + OH + OH- Reaction (3)
Additionally, the Haber-Weiss reaction can form OH in a reaction catalyzed by
M("+')+/M"+ (Miller et al., 1990).
OZ + H202 - OH + OH- + O, Reaction (4)
Still more preferably, the agent comprises one or more of bathocuproine
and/or bathophenanthroline or compounds related thereto at the structural
and/or
functional levels. Reference to compounds such as bathocuproine and
bathophenanthroline include functional derivatives, homologues and analogues
thereof.
Accordingly, another aspect of the present invention provides a method
for treating AD in a subject said method comprising administering to said
subject
an effective amount of an agent comprising at least one metal chelator and/or
metal complexing compound for a time and under conditions sufficient to
inhibit
or otherwise reduce metal-mediated production of free radicals.
In one aspect, the free radicals are reactive oxygen species such as O2 or
OH=. In another aspect, the free radicals include forms of Ap. In another
aspect,
the free radicals include forms of A. However, in a broader sense, it has been
found that the metal-mediated Ap reactions in the brain of AD patients results
in
the generation of reduced metals and hydrogen peroxide, as well as superoxide
and hydroxyl radicals. Furthermore, formation of any other radical or reactive
oxygen species by interaction of any of these products with any other
metabolic
substrate (e.g., superoxide + nitric acid = peroxynitrite) contributes to the
pathology observed in AD and Down's syndrome patients. Cu'' reaction with A(3
generates Cu+, Ap-, Oz, H202, and OH=, all of which not only directly damage
the
cells, but also react with biochemical substrates like nitric oxide.
Yet a further aspect of the present invention is directed to a method for
treating AD in a subject, said method comprising administering to said subject
an
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effective amount of an agerit, said agent comprising a metal chelator, metal
complexing compound or a compound capable of interfering with metal mediated
free radical formation mediated by A(3 peptides for a time and under
conditions
sufficient to inhibit or othervrise reduce production of radicals.
The preferred metals according to these aspects of the present invention
include Cu and Fe and their various oxidation states. Most preferred are
reduced
forms of copper (Cu') and iron (FeZ" ).
Another mechanism elucidated in accordance with the present invention
concerns the formation of aggregates of Ap, as in conditions involving
amyloidosis. In a preferred embodiment, the aggregates are those of amyloid
plaques occurring in, the brains of AD-affected subjects.
The aggrega es according to this aspect of the present invention are non-
fibrillary and fibrillary aggregates and are held together by the presence of
a metal
such as zinc and copper. A method of treatment involves resolubilizing these
AP
aggregates.
The data indicate that Zn-induced AP,_40 aggregation is completely
reversible in the presence of divalent metal ion chelating agents. This
suggests
that zinc binding may be a reversible, normal function of Ap and implicates
other
neurochemical meclianisms in the formation of amyloid. A process involving
irreversible Ap aggregation, such as the polymerization of A(3 monomers, in
the
formation of polymesric species of Ap that are present in amyloid plaques is
thus
a more plausible explanation for the formation of neurotoxic polymeric AP
species.
According to this aspect of the present invention, there is provided a
method of treating AD in a subject comprising administering to said subject an
agent capable of promoting, inducing or otherwise facilitating
resolubilization of
amyloid deposits for a time and under conditions to effect said treatment.
With respect to this aspect of the present invention, it is proposed that a
metal chelator or metal complexing agent be administered. AP deposits which
are composed of fibi=illary and non-fibrillary aggregates may be resolubilized
by
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the metal chelating or metal complexing agents, according to this aspect.
While
fibrile aggregations per se, may not be fully disassociated by administration
of
such agents, overall deposit resolubilization approaches 70%.
In addition, the agent of this aspect of the present invention may comprise
a metal chelator or metal complexing agent alone or in combination with
another
active ingredient such as but not limited to rifampicin, disulfiram,
indomethacin
or related compounds. Preferred metal chelators are bathocuproine,
bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN,
or hydrophobic derivatives thereof.
A "related" compound according to these and other aspects of the present
invention are compounds related to the levels of structure or function and
include
derivatives, homologues and analogues thereof.
Accordingly, the present invention contemplates compositions such as
pharmaceutical compositions comprising an active agent and one or more
pharmaceutically, acceptable carriers and/or diluents. The active agent may be
a single compound such as a metal chelator or metal complexing agent or may be
a combination of compounds such as a metal chelating or complexing compound
and another compound. Preferred active agents include, for reducing radical
formation and for promoting resolubilization, bathocuproine,
bathophenanthroline, DTPA, EDTA, EGTA, penacillamine, TETA, and TPEN,
or hydrophobic derivatives thereof, or any combination thereof.
It has been found that for some chelators there is an optimal concentration
"window" within which the Ap aggregates are dissolved (see Example 5 below).
Increasing the concentration of chelators above the concentration window may
not only be toxic to the patient, but also can sharply decrease the
dissolution
effect of chelators on the A(3 amyloid. Similarly, amounts below the optimal
concentration window are too small to result in significant dissolution.
Although the data indicate that higher concentrations of chelators may be
effective in dissolution of Ap aggregates when supplemented by certain
substances which favor dissolution, e.g. magnesium, it is expected that there
will
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still be an optimallv effective window of chelator concentration. Within the
optimal dissolution window, it will be important to balance optimal
dissolution
against possible sicle effects or toxicity inherent in the use of chelators as
pharmaceutical compositions.
Therefore, for each given patient, the attending physician need be mindful
of the window effect and attend to varying the dosages of chelator
compsositions
so that during the course of administration, chelator concentrations will be
varied
frequently to randoimly allow achieving the most effective concentration for
dissolving Ap amyloid deposits in the given patient.
It is, therefore, desired that the plasma levels of chelators not be steady
state, but be kept fluctuating, so that transiently optimal concentrations
occur in
the patient. The best way to dose the patient is no more often than every
three
hours, preferably every six hours or eight hours, but as infrequently as once
every
day or once every two days are expected to be therapeutic.
For the treatment of moderately affected or severely affected patients,
where risking the neurological side effects is less of a concern since the
quality
of their life is very poor, the patient may be put on a program of treatment
consisting of high dose chelator compositions for 1 to 21 days, but preferably
no
more than 14 days, followed by a period of low dose therapy for seven days to
three months. A convenient schedule would be two weeks of high dose therapy
followed by two weeks of low dose therapy, oscillating between high and low
dose periods for up to 12 months. If after 12 months the patient has made no
clinical gains on high/low chelator therapy, the treatment should be
discontinued.
Another typical case would be the treatment of a mildly affected
individual. Such a patient would be treated with low dose chelators for up to
12
months. If after 6 rnonths no clinical gains have been made, the patient could
then be placed on the high/low alternation regimen for up to another 12
months.
Accordingly, the present invention contemplates compositions such as
pharmaceutical compositions comprising an active agent and one or more
pharmaceutically, acceptable carriers and/or diluents. The active agent may be
a
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metal chelator or a combination of a metal chelator and another active agent,
e.g.
an antioxidant or an alkalinizing agent
Most preferrably, the invention involves the co-administration hydrophobic
and hydrophillic derivatives of chelators. Also most preferrably, the
invention
involves the co-administration of chelators of oxidized metals and chelators
of
reduced metals. Various permutations of both classes of chelators may be
administered to achieve optimal results.
The pharmaceutical forms containing the active agents may be
administered in any convenient manner either orally or parenteraly, such as by
intravenous, intraperitoneal, subcutaneous, rectal, implant, transdermal, slow
release, intrabuccal, intracerebral or intranasal administration. Generally,
the
active agents need to pass the blood brain barrier and may have to be
chemically
modified, e.g. made hydrophobic, to facilitate this or be administered
directly to
the brain or via other suitable routes. For injectable use, sterile aqueous
solutions
(where water soluble) are generally used or alternatively sterile powders for
the
extemporaneous preparation of sterile injectable solutions may be used. It
must
be stable under the conditions of manufacture and storage and must be
preserved
against the contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for example,
water,
ethanol, polyol (for example, glycerol, propylene glycol and liquid
polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable oils. The
preventions of the action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol,
sorbic acid, thirmerosal and the like. In many cases, it will be preferable to
include
isotonic agents, for example, sugars or sodium chloride. Prolonged absorption
of
the injectable compositions can be brought about by the use in the
compositions
of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active agents
in the required amount in the appropriate solvent with various of the other
ingredients enumerated above, as required, followed by sterilization by, for
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example, filtration or irradiation. In the case of sterile powders for the
preparation
of sterile injectable solutions, the preferred methods of preparation are
vacuum
drying and the freeze-drying technique which yield a powder of the active
ingredient plus any additional desired ingredient from previously sterile-
filtered
solution thereof. Pre:ferred compositions or preparations according to the
present
invention are prepared so that an injectable dosage unit contains between
about
0.25 g and 500 mg of active compound.
When the active agents are suitably protected they may be orally
administered, for example, with an inert diluent or with an assimilable edible
carrier, or it may be enclosecl in hard or soft shell gelatin capsule, or it
may be
compressed into tabllets, or it may be incorporated directly with the food of
the
diet. For oral therapeutic administration, the active compound may be
incorporated with excipients and used in the form of ingestible tablets,
buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the
like. Such
compositions and preparations should contain at least 1% by weight of active
compound. The percentage of the compositions and preparations may, of course,
be varied and may conveniently be between about 5 to about 80% of the weight
of the unit. The arnount of active compound in such therapeutically useful
compositions is such that a suitable dosage will be obtained. Preferred
compositions or preparations according to the present invention are prepared
so
that an oral dosage unit folm contains between about 1 g and 2000 mg of
active
compound.
The tablets, t.roches, pills, capsules and the like may also contain other
components such as l isted hereafter: A binder such as gum, acacia, corn
starch or
gelatin; excipients such as dicEdcium phosphate; a disintegrating agent such
as corn
starch, potato starch, alginic acid and the like; a lubricant such as
magnesium
stearate; and a sweetening agent such a sucrose, lactose or saccharin may be
added
or a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring.
When the dosage unit form is a capsule, it may contain, in addition to
materials of
the above type, a liquid carrier. Various other materials may be present as
coatings
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or to otherwise modify the physical form of the dosage unit. For instance,
tablets,
pills, or capsules may be coated with shellac, sugar or both. A syrup or
elixir may
contain the active compound, sucrose as a sweetening agent, methyl and
propylparabens as preservatives, a dye and flavoring such as cherry or orange
flavor. Of course, any material used in preparing any dosage unit form should
be
pharmaceutically pure and substantially non-toxic in the amounts employed. In
addition, the active compound(s) may be incorporated into sustained-release
preparations and formulations.
Pharmaceutically acceptable carriers and/or diluents include any and all
solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic
and absorption delaying agents and the like. The use of such media and agents
for
pharmaceutical active substances is well known in the art. Except insofar as
any
conventional media or agent is incompatible with the active ingredient, use
thereof
in the therapeutic compositions is contemplated. Supplementary active
ingredients
can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in
dosage unit form for ease of administration and uniformity of dosage. Dosage
unit
form as used herein refers to physically discrete units suited as unitary
dosages for
the mammalian subjects to be treated; each unit containing a predetermined
quantity of active material calculated to produce the desired therapeutic
effect in
association with the required pharmaceutical carrier. The specification for
the
novel dosage unit forms of the invention are dictated by and directly
dependent on
(a) the unique characteristics of the active material and the particular
therapeutic
effect to be achieved, and (b) the limitations inherent in the art of
compounding
such an active material for the treatment of disease in living subjects having
a
diseased condition in which bodily health is impaired as herein disclosed in
detail.
The principal active ingredient is compounded for convenient and effective
administration in effective amounts with a suitable pharmaceutically
acceptable
carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can,
for
example, contain the principal active compound in amounts ranging from 0.5 g
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to about 2000 mg. Alternatively, amounts ranging from 200 ng/kg/body weight
to above 10 mg/kg/body weight may be administered. The amounts may be for
individual active agents or for the combined total of active agents.
Compositions of the present invention include all compositions wherein
the compounds of the present invention are contained in an amount which is
effective to achieve their intended purpose. They may be administered by any
means that achieve their intended purpose. The dosage administered will depend
on the age, health, and weight of the recipient, kind of concurrent treatment,
if
any, frequency of the treatment, and the nature of the effect desired. The
dosage
of the various compositions can be modified by comparing the relative in vivo
potencies of the drugs and the bioavailability using no more than routine
experimentation.
The pharmaceutical ecimpositions of the invention may be administered to
any animal which may experience the beneficial effects of the compounds of the
invention. Foremost among such animals are mammals, e.g., humans, although
the invention is not i ntended to be so limited.
The following examples are provided by way of illustration to further
describe certain preferred embodiments of the invention, and are not intended
to
be limiting of the present invention, unless specified.
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Examples
Example 1
Copper-Induced, pH Dependent Aggregation of A,(3
Materials and Methods
a) Preparation of A/3 Stock
Human AP1-40 peptide was synthesized, purified and characterized by
HPLC analysis. amino acid analysis and mass spectroscopy by W.M. Keck
Foundation Biotechnology Resource Laboratory (Yale University, New Haven,
CT). Synthetic A(3 peptide solutions were dissolved in trifluoroethanol (30 %
in
Milli-Q water (Millipore Corporation, Milford, MA)) or 20 mM HEPES (pH 8.5)
at a concentration of 0.5-1.0 g/ml, centrifuged for 20 min. at 10,000g and the
supernatant (stock A(31-40) used for subsequent aggregation assays on the day
of the
experiment. The concentration of stock Ap,-4o was determined by UV
spectroscopy at 214 nm or by Micro BCA protein assay (Pierce, Rockford, IL).
The Micro BCA assay was performed by adding 104l of stock AP1-40 (or bovine
serum albumin standard) to 140 l of distilled water, and then adding an equal
volume of supernatant (15041) to a 96-well plate and measuring the absorbance
at
562 nm. The concentration of Ap,-ao was determined from the BSA standard
curve. Prior to use all buffers and stock solutions of metal ions were
filtered
though a 0.22 m filter (Gelan Sciences, Ann Arbor, MI) to remove any
particulate
matter. All metal ions were the chloride salt, except lead nitrate.
b) Aggregation Assays
A(31-40 stock was diluted to 2.5 M in 150 mM NaCI and 20 mM glycine
(pH 3-4.5), MES (pH 5-6.2) or HEPES (pH 6.4-8.8), with or without metal ions,
incubated (30 min., 37 C), centrifuged (20 min., 10,000g). The amount of
protein
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in the supernatant was determined by the Micro BCA protein assay as described
above.
c) Turbidometric Assay,s
Turbidity measurements were performed as described by Huang, X., et al.,
J. Biol. Chem. 272:26464-26470 (1997), except Ap 1-40 stock was brought to 10
M
(300 l) in 20 mM HEPES buffer, 150 mM NaC1 (pH 6.6. 6.8 or 7.4) with or
without metal ions prior to incubation (30 min., 37 C). To investigate the pH
reversibility of Cu'-+-induced .Ap aggregation, 25 M AP1_40 and 25 M Cu'-
were
mixed in 67 mM phosphate buffer, 150 mM NaCI (pH 7.4) and turbidity
measurements were taken at four 1 min. intervals. Subsequently, 20 l aliquots
of 10 mM EDTA or 10 mM Cu'-' were added into the wells alternatively, and,
following a 2 min. delay, a further four readings were taken at 1 min.
intervals.
After the final EDTA addition and turbidity reading, the mixtures were
incubated
for an additional 30 min. before taking final readings. To investigate the
reversibility of pH mediated Cu2+-induced Ap1_40 aggregation, 10 M Ap 1_40
and
30 M Cu'-; were mixed in 67 mM phosphate buffer, 150 mM NaCI (pH 7.4) and
an initial turbidity measurement taken. Subsequently, the pH of the solution
was
successively decreased to 6.6 and then increased back to 7.5. The pH of the
reaction was monitored with a microprobe (Lazar Research Laboratories Inc.,
Los
Angeles, CA) and the turbidiity read at 5 min. intervals for up to 30 min.
This
cycle was repeated three times.
d) Immunofiltnation Detection of Low Concentrations ofAA_40Aggregate
Physiological concentrations of Ap1-40 (8 nM) were brought to 150 mM
NaCI, 20 mM HEPES (pH 6.6 or 7.4), 100 nM BSA with CuC1z (0, 0.1, 0.2, 0.5
and 2 M) and incubated (30 min., 37 C). The reaction mixtures (200 l) were
then placed into the 96-well Easy-Titer ELIFA system (Pierce, Rockford, IL)
and
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filtered through a 0.22 m cellulose acetate filter (MSI, Westboro, MA).
Aggregated particles were fixed to the membrane (0.1 % glutaraldehyde, 15
min.),
washed thoroughly and then probed with the anti-A(3 mAB 6E10 (Senetek,
Maryland Heights, MI). Blots were washed and exposed to film in the presence
of ECL chemiluminescence reagents (Amersham, Buckinghamshire, England).
Immunoreactivity was quantified by transmitance analysis of ECL film from the
immunoblots.
e) A,6 Metal-capture ELISA
A(31-40 (1.5 ng/well) was incubated (37 C, 2 hr) in the wells of Cu'-+ coated
microtiter plates (Xenopore, Hawthorne. NJ) with increasing concentrations of
Cu2+ (1-100 nM) as described by Moir et al., Journal of Biological Chemistry
(submitted). Remaining ligand binding sites on well surfaces were blocked with
2% gelatin in tris-buffered saline (TBS) (3 hr at 37 C) prior to overnight
incubation at room temperature with the anti-A(3 mAb 6E10 (Senetek, Maryland
Heights, MI). Anti-mouse IgG coupled to horseradish peroxidase was then added
to each well and incubated for 3 hr at 37 C. Bound antibodies were detected by
a 30 minute incubation with stable peroxidase substrate buffer/3,3',5,5'-
Tetramethyl benzidine (SPSB/TMB) buffer, followed by the addition of 2 M
sulfuric acid and measurement of the increase in absorbance at 450 nm.
J) Extraction of A,6from Post-mortem Brain Tissue
Identical regions of frontal cortex (0.5g) from post-mortem brains of
individuals with AD, as well as non-AD conditions, were homogenized in TBS,
pH 4.7 metal chelators. The homogenate was centrifuged and samples of the
soluble supernatant as well as the pellet were extracted into SDS sample
buffer and
assayed for A(3 content by western blotting using monoclonal antibody (mAb)
W02. The data shows a typical (of n=12 comparisons) result comparing the
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amount of A(3 extracted into -the supernatant phase in AD compared to control
(young adult) samples. N,N,N',N'-tetrakis [2-pyridyl-methyl] ethylenediamine
(TPEN) (5 M) allows the visualization of a population of pelletable Ap that
had
not previously been recognized in unaffected brain samples ( Figure 8).
g) A# Cross-linking by Copper
Cu', -induced SDS-resi.stant oligomerization of A(3: A(31_40 (2.5 M), 150
mM NaCI, 20 mM :hepes (pH 6.6, 7.4, 9) with or without ZnC12 or CuCI,.
Following incubatioti (37 C), aliquots of each reaction (2 ng peptide) were
collected at 0 d, 1 d, :3 d and 5 d and western blotted using anti-A[3
monoclonal
antibody 8E 10 ( Figure 9). Miigration of the molecular size markers are
indicated
(kDa). The dimer formed ur.ider these conditions has been found to be SDS-
resistant. Cu'-' (2-30 ~uM) induced SDS-resistant polymerization of peptide.
Co-
incubation with similar-concentrations of Zn'-- accelerates the
polymerization, but
zinc alone has no effect. The antioxidant sodium metabisulfite moderately
attenuates the reaction, while ascorbic acid dramatically accelerates A(3
polymerization. This suggests reduction of Cu2+ to Cu+ with the latter
mediating
SDS-resistant polymerization of A[3. Mannitol also abolishes the
polymerization,
suggesting that the bridging is mediated by the generation of the hydroxyl
radical
by a Fenton reaction that reci=uits Cu'. It should be noted that other means
of
visualizing and/or determining the presence or absence of polymerization other
than western blot analysis may be used. Such other means include but are not
limited to density sedimentation by centrifugation of the samples.
Results
It has previously been reported that Zn'-+ induces rapid precipitation of A(3
in vitro ( Bush, A.I., et al., J. Biol. Chenz. 269:12152 (1994)). This metal
has an
abnormal metabolism in AD atld is highly concentrated in brain regions where
A[3
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precipitates. The present data indicate that under very slightly acidic
conditions,
such as in the lactic acidotic AD brain, Cuz strikingly induces the
precipitation of
Ap through an unknown conformational shift. pH alone dramatically affects AP
solubility, inducing precipitation when the pH of the incubation approaches
the pl
of the peptide (pH 5-6). Zinc induces 40-50% of the peptide to precipitate at
pH
> 6.2, below pH 6.2 the precipitating effects of Zn'-' and acid are not
summative.
At pH <_ 5, Zn'' has little effect upon Ap solubility. Cu'-+ is more effective
than
Zn'" in precipitating A(3 and even induces precipitation at the
physiologically
relevant pH 6-7. Copper-induced precipitation of A(3 occurs as the pH falls
below
7.0, comparable with conditions of acidosis (Yates, C.M., el al., J.
Neurochem.
55:1624 (1990)) in the AD brain. Investigation of the precipitating effects of
a
host or other metal ions in this system indicated that metal ion precipitation
of Ap
was limited to copper and zinc, as illustrated, although Fe'-' possesses a
partial
capacity to induce precipitation (Bush, A.I., et al., Science 268:1921
(1995)).
On the basis these in vitro findings, the possibility that A(3 deposits in the
AD-affected brain may be held in assembly by zinc and copper ions was
investigated. Roher and colleagues have recently shown that much of the AP
that
deposits in AD-affected cortex can be solubilized in water (Roher, A.E, et
al., J.
Biol. Chem. 271:20631 (1996)). Supporting the clinical relevance of in vitro
findings, it has recently been demonstrated that metal chelators increase the
amount of Ap extracted by Roher's technique (in neutral saline buffer), and
that
the extraction of Ap is increased as the chelator employed has a higher
affinity for
zinc or copper. Hence TPEN is highly efficient in extracting AP, as are TETA,
and bathocuproine, EGTA and EDTA are less efficient, requiring higher
concentrations 91 mm) to achieve the same level of recovery as say, TPEN (5
M).
Zinc and copper ions (5-50 M) added back to the extracting solution abolish
the
recovery of Ap (which is subsequently extracted by the SDS sample buffer in
the
pellet fraction of the centrifuged brain homogenate suspension), but Ca'-+ and
Mg'+
added back to the chelator-mediated extracts of Ap cannot abolish A(3
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resolubilization frorn AD-affected tissue even when these metal ions are
present
in millimolar concentrations.
Importantly, atomic absorption spectrophotometry assays of the metal
content of the chelator-mediated extracts confirms that Cu and Zn are co-
released
with Ap by the chelators, along with lower concentrations of Fe. These data
strongly indicate that Ap deposits (probably of the amorphous type) are held
together by Cu and Zn and may also contain Fe. Interestingly. A(3 is not
extractable from control brairi without the use of chelators. This suggests
that
metal-assembled Ap deposits may be the earliest step in the evolution of A(3
plaque pathology.
These findings propelled further inquiries into chemistry of metal ion- Ap
interaction. The precipitating effects upon Ap of Zn2* and Cu21 were found to
be
qualitatively different. Zn-mediated aggregation is reversible with chelation
and
is not associated with neurotoxicity in primary neuronal cell cultures,
whereas Cu-
mediated aggregatiori is acconlpanied by the slow formation of covalently-
bonded
SDS-resistant dimers and induction of neurotoxicity. These neurotoxic SDS-
resistant dimers are similar to 1:hose described by Roher (Roher, A.E, et al.,
J. Biol.
Chem. 271:20631 (1996)).
To accuratelv quantitate the effects of different metals and pH on AP
solubility, synthetic liuman Ap 1-40 (2.5 M) was incubated (37 C) in the
presence
of metal ions at various pH for 30 min. The resultant aggregated particles
were
sedimented by centrifugatiori to permit determination of soluble AP1_40 in the
supernatant. To determine the centrifugation time required to completely
sediment
the aggregated particl es generated under these conditions, A(3, _40 was
incubated for
30 min at 37 C with no meital, Zn'-+ (100 M), Cu'-+ (100 M) and pH (5.5).
Reaction mixtures were centrifuged at 10 000g for different times, or
ultracentrifuged at 100 000g for I h. ( Figure 1). Figure 1 shows the
proportion
of soluble AP1_40 remaining following centrifugation of reaction mixtures.
A(3,_0
was incubated (30 m.in., 37 C) with no metal, under acidic conditions (pH
5.5),
ZnZ+ (100 M) or Cuz+ (100 M), and centrifuged at 10 000g for different time
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intervals, or at 100,000g (ultracentrifuged) for I h for comparison. All data
points
are means SD, n = 3.
Given that conformational changes within the N-terminal domain of AP
are induced by modulating [H] (Soto, C., et al., J. Neurochem. 63:1191-1198
(1994)), and that there is a metal (Zn'-+) binding domain in the same region,
experiments were designed to determine whether there was a synergistic effect
of
pH on metal ion-induced A(3 aggregation. A(31_40 was incubated with different
bioessential metal ions at pH 6.6, 6.8 and 7.4. The results are show in Figure
2A,
where "all metals" indicates incubation with a combination containing each
metal
ion at the nominated concentrations, concurrently. Figure 2A shows the
proportion of soluble AD_40 remaining in the supernatant after incubation (30
min.,
37 C) with various metals ions at pH 6.6, 6.8 or 7.4 after centrifugation
(10,000g,
min.).
The [H+] chosen represented the most extreme, yet physiologically
15 plausible [H+] that A(3,:40 would be likely to encounter in vivo. The
ability of
different bioessential metal ions to aggregate Ap 1_40 a't increasing W
concentrations
fell into two groups; Mg'', Ca'', Al3+, Co-', Hg2+, Fe', Pb-' and Cu-' showed
increasing sensitivity to induce APi_40aggregation, while Fe', MnZT, Ni'-",
and Zn''+
were insensitive to alterations in [H+] in their ability to aggregate A[31_40.
Cu21 and
20 Hg2- induced most aggregation as the [H+] increased, although the [H+]
insensitive
Zn2+-induced aggregation produced a similar amount of aggregation. Fe2+, but
not
Fe', also induced considerable aggregation as the [H+] increased, possibly
reflecting increased aggregation as a result of increased crosslinking of the
peptide.
Similar results were obtained when these experiments were repeated using
turbidometry as an index of aggregation (Figure 2B). The data indicate the
absorbance changes between reaction mixtures with and without metal ions at pH
6.6, 6.8 or 7.4. Thus, A[i,_ao has both a pH insensitive and a pH sensitive
metal
binding site. At higher concentrations of metal ions this pattern was
repeated,
except Co2+ and Al3"-induced A[3 aggregation became pH insensitive, and Mn
became sensitive (Figure 2C).
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Since 64Cu is impractically short-lived (t1/2 = 13 h), a novel metal-capture
ELISA assay was used to perform competition analysis of A[31_40 binding to a
microtiter plate impregnated with CuZ', as described in Materials and Methods.
Results are shown in Figure 3. All assays were performed in triplicate and are
means SD, n=3. Ccimpetition analysis revealed that A(31_40 has at least one
high
affinity, saturable Cu'+ binding site with a Kd = 900 pM at pH 7.4 (Figure 3).
The
affinity of Ap for Cu2' is higheir than that for Znz+ (Bush, A.I., et al., J.
Biol. Chem.
269:12152 (1994)). Since CuZ+ does not decrease Zn2+-induced aggregation
(Bush,
A.I., et al., J. Biol. Chem. 269:12152 (1994)), indicating Cu'-' does not
displace
bound ZnZ', there are likely to be two separate metal binding sites. This is
supported by the fact that therE: is both a pH sensitive and insensitive
interaction
with different metal ions.
Since the conformatior.ial state and solubility of A[3 is altered at different
pH (Soto, C., et al., J. Neurochem. 63:1191-1198 (1994)), the effects of [H+]
on
Zn'+- and Cu+-induced AP1_40 aggregation were studied. Results are shown in
Figures 4A, 4B and 4C. Figure 4A shows the proportion of soluble A[i,-4o
remaining in the supe:rnatant following incubation (30 min., 37 C) at pH 3.0-
8.8
in buffered saline Zn2+ (30 pM) or Cu2' (30 M) and centrifugation (10 000g,
min.), expressed as; a percentage of starting peptide. All data points are
means
20 .= SD, n=3. [H+] alone precipitates AP1_40 (2.5 M) as the solution is
lowered
below pH 7.4, and dramatically once the pH falls below 6.3 ( Figure 4A). At pH
5.0, 80% of the peptide is precipitated, but the peptide is not aggregated by
acidic
environments below pH 5, confirming and extending earlier reports on the
effect
ofpH on A(3 solubility (Burdick, D.,J. Biol. Chem. 267:546-554 (1992)). Zn2+
(30
M) induced a constant level (-50%) of aggregation between pH 6.2-8.5, while
below pH 6.0, aggregation could be explained solely by the effect of [H+].
In the presence of Cu2+ (30 M), a decrease in pH from 8.8 to 7.4 induced
a marked drop in A(31_qo solubiliity, while a slight decrease below pH 7.4
strikingly
potentiated the effect of CuZ+ on the peptide's aggregation. Surprisingly,
Cu'-+caused >85 % of the available peptide to aggregate by pH 6.8, a pH which
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plausibly represents amildly acidotic environment. Thus, conformational
changes
in A(3 brought about by small increases in [Hi ] result in the unmasking of a
second
metal binding site that leads to its rapid self-aggregation. Below pH 5.0, the
ability of both ZnZ+ and Cu2+to aggregate A(3 was diminished, consistent with
the
fact that Zn binding to Ap is abolished below pH 6.0 (Bush, A.I., et al., J.
Biol.
Chem. 269:12152 (1994)), probably due to protonation of histidine residues.
The relationship between pH and Cu'+ on A(31_40solubility was then further
defined by the following experiments (Figure 4B). The proportion of soluble
A(3,_
;o remaining in the supernatant after incubation (30 min., 37 C) at pH 5.4-7.8
with
different Cu2+ concentrations (0, 5, 10, 20, 30 M), and centrifugation
(10,000g,
min.), was measured and expressed as a percentage of starting peptide. All
data
points are means SD, n=3. At pH 7.4, Cu'-+-induced A[3 aggregation was 50%
less than that induced by Zn2+ over the same concentration range, consistent
with
earlier reports (Bush, A.I., et al., J. Biol. Chem. 269:12152 (1994)). There
was a
15 potentiating relationship between [H+] and [Cu2+] in producing A(3
aggregation;
as the pH fell, less Cuz+was required to induce the same level of aggregation,
suggesting that [H+] is controlling Cu'+ induced Ap 1_40 aggregation.
To confirm that this reaction occurs at physiological concentrations of Ap,_
4. and Cuz+, a novel filtration immunodetection system was employed. This
20 technique enabled the determination of the relative amount of AP1-40
aggregation
in the presence of different concentrations of H+ and Cu'' (Figure 4C). The
relative aggregation of nM concentrations of Ap 1_40 at pH 7.4 and pH 6.6 in
the
presence of different CuZ+ concentrations (0, 0.1, 0.2, 0.5 M) were
determined by
this method. Data represent mean reflectance values of immunoblot densitometry
expressed as a ratio of the signal obtained when the peptide is treated in the
absence of CuZ+. All data points are means SD, n = 2.
This sensitive technique confirmed that physiological concentrations of
AP,-4o are aggregated under mildly acidic conditions and that aggregation was
greatly enhanced by the presence of CuZ' at concentrations as low as 200 nM.
Furthermore, as previously observed at higher Ap 1_40 concentrations, a
decrease in
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pH from 7.4 to 6.6 potentiatecl the effect of Cu'-+ on aggregation of
physiological
concentrations of A[31_40. Thu,s, A[3i.40 aggregation is concentration
independent
down to 8 nM where Cu'-+ is available.
It has recentl.y been shown that Znz+ mediated AP,4o aggregation is
reversible whereas 41_40 aggregation induced by pH 5.5 was irreversible.
Therefore, experiments were performed to determine whether Cuzi/pH-mediated
A[3, _40 aggregation wa.s reversible. Cu2+-induced AP1_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. This
result suggested that a more complex aggregate is formed during each
subsequent
aggregation cycle, preventing the chelator access to remove Cu'-- from the
peptide.
This is supported by the fact that complete resolubilization occurs with time,
and
indicates that the peptide is not adopting a structural conformation that is
insensitive to Cu'-+-induced aggregation/EDTA-resolubilization.
The reversibility of pH potentiated Cu'-+-induced A(31-40 aggregation was
studied by turbidometry between pH 7.5 to 6.6, representing H+ concentration
extremes that might be found in vivo (Figures 5A and 5B). Unlike the
irreversible
aggregation of A(3,_40 observed at pH 5.5. Cu'-+-induced AP,_,,o aggregation
was
fully reversible as the pH oscillated between p17.4 and 6.6. Figure 5A shows
the
turbidometric analysis of Cu'+-induced A[3,40 aggregation at pH 7.4 reversed
by
successive cycles of chelator (EDTA), as indicated. Figure 5B shows
turbidometric
analysis of the reversiibility of Cu"-induced A[31_40 as the pH cycles between
7.4
and 6.6. Thus, subtle conformational changes within the peptide induced by
changing [H+] within a narrovr pH window, that corresponds to physiologically
plausible [H'], allows the aggregation or resolubilization of the peptide in
the
presence of Cu'-.
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Discussion
These results suggest that subtle conformational changes in A[3 induced by
[Ht} promote the interaction of A(3,_40with metal ions, in particular Cu'' and
Hg''
allowing self-aggregate or resolubilize depending on the [H+] (Figures 2A-2C,
4A-
4C). A decrease in pH below 7.0 increases the [3-sheet conformation (Soto, C.,
et
al., J. Neurochem. 63:1191-1198 (1994)), and this may allow the binding of Cu-
'+
to soluble A[3 that could further alter the conformation of the peptide
allowing for
self aggregation, or simply help coordinate adjacent A(3 molecules in the
assembly
of the peptides into aggregates. Conversely, increasing pH above 7.0 promotes
the
a-helical conformation (Soto, C., et al., J. Neurochem. 63:1191-1198 (1994)),
which may alter the conformational state of the dimeric aggregated peptide,
releasing Cu and thereby destabilizing the aggregate with the resultant
release of
A(3 into solution. Thus, in the presence of Cu2+, A(3,_40 oscillates between
an
aggregated and soluble state dependent upon the [H+].
AP1_40 aggregation by Co''+, like Zn2+, was pH insensitive and per mole
induced a similar level of aggregation. Unlike Zn'-', AP,_40 binding of Co2+
may
be employed for the structural determination of the pH insensitive binding
site
given its nuclear magnetic capabilities (See Figure 2C).
The biphasic relationship of A[3 solubility with pH mirrors the
conformational changes previously observed by CD spectra within the N-terminal
fragment (residues 1-28) of A(3 (reviewed in (Soto, C., et al., J. Neurochem.
63:1191-1198 (1994)); a-helical between pH 1-4 and >7, but [3-sheet between pH
4-7. The irreversible aggregates of Ap formed at pH 5.5 supports the
hypothesis
that the (3-sheet conformation is a pathway for A[i aggregation into amyloid
fibrils.
Since aggregates produced by Zn'-' and Cu'-" under mildly acidic conditions
(Figures 5A and 5B) are chelator/pH reversible, their conformation may be the
higher energy a-helical conformation.
These results now indicate that there are three physiologically plausible
conditions which could aggregate A(3: pH (Figures 1, 4A-4C; Fraser, P.E., et
al.,
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Biophys. J. 60:1190==1201 (1991); Barrow, C.J. and Zagorski, M.G., Science
253:179-182 (1991); Burdick, D., J. Biol. Chenz. 267:546-554 (1992); Barrow,
C.J., et al., J. Mol. Biol. 225:1075-1093 (1992); Zagorski, M.G. and Barrow,
C.J.,
Biochemistry 31:5621-5631 (1992); Kirshenbaum, K. and Daggett, V.,
Biochemistry 34:7629-7639 (1995); Wood, S.J., et al., J. Mol. Biol. 256:870-
877
(1996), [Zn'-+] ( Figures 1, 2A, and 2B, 4A-4C; Bush, A.I., el al., J. Biol.
Chenz.
269:12152 (1994); Bush, A.I., et al., Science 265:1464 (1994); Bush, A.I., et
al.,
Science 268:1921 (1995); Wood, S.J., et al., J. Mol. Biol. 256:870-877
(1996))and
under mildly acidic conditions, [Cu'''] (Figures 2A, 4A-4C, 5B).
Interestingly,
changes in metal ion concentrations and pH are common features of the
inflammatory response to injury. Therefore, the binding of Cu'' and Zn'-' to
A[3
may be of particular importance during inflammatory processes, since local
sites
of inflammation can become acidic (Trehauf, P.S. & McCarty, D.J., Arthr.
Rheunz.
14:475-484 (1971); Menkin, V., Ani. J. Pathol. 10:193-210 (1934)) and both Zn'-
'
and Cu'+ are rapidly rnobilizecl in response to inflammation (Lindeman, R.D.,
et
al., J. Lab. Clin. Med. 81:194-204 (1973); Terhune, M.W. & Sandstead, H.H.,
Science 177:68-69 (1972); Hsu, J.M., et al., J. Nutrition 99:425-432 (1969);
Haley, J.V., J. Surg. Res. 27:168-174 (1979); Milaninio, R., et al., Advances
in
Inflammation Research 1:281-291 (1979); Frieden, E., in Inflammatory Diseases
and Copper, Sorenson, J.R.J., ed, Humana Press, New Jersey (1980), pp. 159-
169).
Serum copper levels increase during inflammation, associated with
increases in ceruloplasmin, a Cu2* transporting protein that may donate Cu2+
to
enzymes active in processes of basic metabolism and wound healing such as
cytochrome oxidase and lysyl oxidase (Giampaolo, V., et al., in Inflammatory
Diseases and Copper, Sorensom, J.R.J., ed, Humana Press, New Jersey (1980),
pp.
329-345; Peacock, E.E. and vanWinkle, W., in WoundRepair, W.B. Saunders Co.,
Philadelphia, pp. 145-155) (1976)). Since the release of Cu2- from
ceruloplasmin
is greatly facilitated by acidic environments where the cupric ion is reduced
to its
cuprous form (Owen, C.A., Jr., Proc. Soc. Exp. Biol. Med. 149:681-682 (1975)),
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periods of mild acidosis may promote an environment of increased free Cu'-.
Similarily, aggregation of another amyloid protein, the acute phase reactant
serum
amyloid P component (SAP) to the cell wall polysaccharide, zymosan, has been
observed with Cu2+ at acidic pH (Potempa, L.A., et al., Journal of Biological
Chemistry 260:12142-12147 (1985)). Thus, exchange of Cu2+ to A[i1_40 during
times of decreased pH may provide a mechanism for altering the biochemical
reactivity of the protein required by the cell under mildly acidic conditions.
Such
a function may involve alterations in the aggregation/adhesive properties
(Figures
1-5B) or oxidative functions of A[3 at local sites of inflammation.
While the pathogenic nature of AP1_4, in AD is well described (Maury,
C.P.J., Lab. Investig. 72:4-16 (1995); Multhaup, G., et al., Nature 325:733-
736
(1987)), the function of the smaller A(3,_40 remains unclear. The present data
suggest that Cuz+-binding and aggregation of A[3 will occur when the pH of the
microenvironment rises. This conclusion can be based on the finding that the
reaction is [H] and [Cu'-+] dependent and reversible within a narrow,
physiologically plausible, pH window. This is further supported by the
specificity
and high affinity of Cu'+ binding under mildly acidic conditions compared to
the
constant Zn'-'-induced aggregation (and binding) of A[31_40 over a wide pH
range
(6.2-8.5). The brain contains high levels of both Zn2+ (- 150 M;
Frederickson,
C.J. International Review of Neurobiology 31:145-237 (1989)) and Cu'- (-100
M; Warren, P.J., et al., Brain 83:709-717 (1960); Owen, C.A., Physiological
Aspects of Copper, Noyes Publications, Park Ridge, New Jersey (1982), pp 160-
191). Intracellular concentrations are approximately 1000 and 100 fold higher
than extracellular concentrations. This large gradient between intracellular
and
extracellular compartments suggests a highly energy dependent mechanism is
required in order to sequester these metals within neurons. Therefore, any
alterations in energy metabolism, or injury, may affect the reuptake of these
metal
ions and promote their release into the extracellular space, and together with
the
synergistic affects of decreased pH (see above) induce membrane bound A[31_40
to
aggregate. Since increased concentrations of Znz- and Cu2+, and decreased pH,
are
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common features of all forms of cellular insult, the initiation of AP,-41
function
likely occurs in a coordinated fashion to alter adhesive and/or oxidative
properties
of this membrane pi-otein essential for maintaining cell integrity and
viability.
That Ap , _40 has such a high affinity for these metal ions, indicates a
protein that has
evolved to respond to slight changes in the concentration of extracellular
metal
ions. This is supported by the fact that aggregation in the presence of Cu is
approx. 30% at pH 7.1, the pH of the brain (Yates, C.M., et al., J. Neurochem.
55:1624-1630 (1990)), but 85% at pH 6.8. Taken together, our present results
indicate that AP1_40 may have evolved to respond to biochemical changes
associated with neuironal darnage as part of the locally mediated response to
inflammation or cell injury. Thus. it is possible that Cu21 mediated A(31_40
binding
and aggregation might be a purposive cellular response to an environment of
mild
acidosis.
The deposition of anlyloid systemically is usually associated with an
inflammatory response (Pepys, M.B. & Baltz, M.L., Adv. Immunol. 34:141-212
(1983); Cohen, A.S.,, in Arthritis and Allied Conditions, D.J. McCarty, ed.,
Lea
and Febiger, Philadelphia, pp. 1273-1293 (1989); Kisilevsky, R., Lab.
Investig.
49:381-390 (1983)). For exaniple, serum amyloid A, one ofthe major acute phase
reactant proteins that is elevated during inflammation, is the precursor of
amyloid
A protein that is deposited in various tissues during chronic inflammation,
leading
to secondary amyloidosis (Gorevic, P.D., et al., Ann. NY Acad. Sci. 380:393
(1982)). An involve;ment of inflammatory mechanisms has been suggested as
contributing to plaque formation in AD (Kisilevsky, R., Mol. Neurobiol. 49:65-
66
(1994)). Acute-phase protein,s such as alpha 1-antichymotrypsin and c-reactive
protein, elements of the complement system and activated microglial and
astroglial
cells are consistently found in AD brains.
The rapid appearance, within days of Ap deposits and APP
immunoreactivity following head injury (Roberts, G.W., et al., Lancet.
338:1422-
1423 (1991); Pierce, J.E.S., et al., Journal ofNeuroscience 16:1083-1090
(1996)),
rather than the more gradual accumulation of Ap into more dense core amyloid
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plaques over months or years in AD may be compatible with the release of Zn-',
Cu'-+ and mild acidosis in this time frame. Thus, pH/metal ion mediated
aggregation may form the basis for the amorphous Ap deposits observed in the
aging brain and following head injury, allowing the maintenance of endothelial
and neuronal integrity while limiting the oxidative stress associated with
injury
that may lead to a diminishment of structural function.
Since decreased cerebral pH is a complication of aging (Yates, C.M., et al.,
J. Neurochem. 55:1624-1630 (1990)), these data indicate that Cu and Zn
mediated
Ap aggregation may be a normal cellular response to an environment of mild
acidosis. However, prolonged exposure of A(3 to an environment of lowered
cerebral pH may promote increased concentrations of free metal ions and
reactive
oxygen species, and the inappropriate interaction of A(3, 42 over time
promoting the
formation of irreversible Ap oligomers and it's subsequent deposition as
amyloid
in AD. The reversibility of this pH mediated Cu2+ aggregation does however
present the potential for therapeutic intervention. Thus, cerebral
alkalinization
may be explored as a therapeutic modality for the reversibility of ainyloid
deposition in vivo.
Example 2
Free Radical Formation and SOD-like activity ofAlzheimer's A,8 Peptides
Materials and Methods
a) Determination of Cu+ and Fe2+
This method is modified from a protocol assaying serum copper and iron
(Landers, J.W., et al., Amer. J. Clin. Path. 29:590 (1958)). It is based on
the fact
that there are optimal visible absorption wavelengths of 483 nm and 535 nm for
Cu+ complexed with bathocuproinedisulfonic (BC) anion and Fe'-+ coordinated by
bathophenanthrolinedisulfonic (BP) anion, respectively.
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Determination of miolar absorption of these two complexes was
accomplished essentially as follows. An aliquot of 500 l of each complex (500
M, in PBS pH 7.4, with ligands in excess) was pipetted into 1 cm-pathlength
quartz cuvette, and their absorbances were measured. Their molar absorbancy
was
determined based ori Beer-Lambert's Law. Cu+-BC has a molar absorbancy of
2762 M-' cm- ', while Fe'+-BP has a molar absorbancy of 7124 M-' cm-'.
Determination of the equivalent vertical pathlength for Cu+-BC and
Fe''+-BP in a 96-well plate was carried out essentially as follows.
Absorbances of
the two complexes with a 500 M, 100 M, 50 M, and 10 M concentration of
relevant metal ions (Cu', Fe',) were determined both by 96-well plate readers
(300
L) and UV-vis spectrometer (500 L). with PBS, pH 7.4, as the control blank.
The resulting absorbancies from the plate reader regress against absorbancies
by
a UV-vis spectromeber. The slope k from the linear regression line is
equivalent
to the vertical pathlerigth if the measurement is carried out on a plate. The
results
are:
k(cm) r'
Cu'-BC 1.049 0.998
Fe'+-B1P 0.856 0.999
With molar absorbancy and equivalent vertical pathlength in hand, the
concentrations ( M) of Cu' or Fe2+ can be deduced based on Beer-Lambert's Law,
using proper buffers as controls.
for F'e 2': [Fe 2 ] ( 111) AA (535nm) x 106
(7124 x 0.856)
for Cu ': [Cu '] ( M) _ AA(483nm) x 106
(2762 x 1.049)
where AA is absorbancy difference between sample and control blank.
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b) Determination of H101
This method is modified from a H2O, assay reported recently (Han, J.C.,
et al., Anal. Biochem. 234:107 (1996)). The advantages of this modified H,O,
assay on 96-well plate include high throughput, excellent sensitivity (-1 M),
and
the elimination of the need for a standard curve of H,O2 , which is
problematic due
to the labile chemical property of H7O2.
A(3 peptides were co-incubated with a H,O,-trapping reagent (Tris(2-
carboxyethyl)-phosphine hydrochloride, TCEP, 100 M) in PBS (pH 7.4 or 7.0)
at 37 C for 30 mins. Then 5,5'-dithio-bis(2-nitrobenzoic acid) (DBTNB, 100
M)
was added to react with remaining TCEP. The product of this reaction has a
characteristic absorbance maximum of 412 nm [18]. The assay was adapted to a
96-well format using a standard absorbance range (see Figure 11).
The chemical scheme for this novel method is:
Scheme I:
CHZ CH2 COO' CH2 CHZ COO-
I
-OOCCH2CH2-P + H202 --~ -OOCCH2CH2-P=O + H20
CHZ CHZ COO CHZ CHZ COO
(TCEP) [Tris (2-carboxethyl) phosphine]
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Scheme II:
70'
CHZ CH2 COO" S O NOZ
-OOCCHZCHZ-P + + H20 CHZCH2COO
S NO2
remaining TCEP COO'
'
CHZ C:H2 COO" c00
-OOCCH2CH2-P=0 + 2S" O NOZ + 2H+
CH2 CH2 COO'
has characteristic optimal
absorption peak at 412 nm
with 14,150 M-I cm-1
molar extinction coefficient
TCEP=HC1 was synthesized by hydrolyzing tris (2-cyno-ethyl) phosphine
(purchased from Johnson-Mathey (Waydhill, MA)), in refluxing aqueous HC1
(Burns, J.A.. et al., J. Org. Cliem. 56:2648 (1991)) as shown below.
aq HCI +
(NCCH>CH2)3P ---> (HO2CCH2CH2)3 PH Cl
reflux TCEP= HCl
In order to carry out the above-described assay in a 96-well plate, it was
necessary to calculate the equivalent vertical pathlength of 2-nitro-5-
thiobenzoic
acid (TMB) in a 96-well plate. This determination was carried out essentially
as
described for Cu+-B(: and Fe'+-BP in Example 2. The resulting absorbancies
from the plate reader regress against absorbancies by a UV-vis spectrometer.
The
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slope k from the linear regression line is equivalent to the vertical
pathlength if
the measurement is carried out on a plate. The results are:
k r2
0.875 1.00
The concentration of H,O, can then be deduced from the difference in
absorbance between the sample and the control (sample plus 1000 U/ l catalase)
DA (412nm)
[HzOz) { tl~ (2 x 0.875 x 14150)
c) Determination of OH=
Determination of OH- was performed as described in Gutteridge et al.
Biochini. Biophys. Acta 759: 38-41 (1983).
d) Cu+ Generation by Afl: Influence of ZiTZ+ and pH
Ap (10 M in PBS, pH 7.4 or 6.8, as shown) was incubated for 30
minutes (37 C) in the presence of Cuz+ 10 M Zn2+ 10 M). Cu+ levels (n=3,
-SD) were assayed against a standard curve. These data indicate that the
presence of Zn2+ can mediate the reduction of Cu2+ in a mildly acidic
environment. The effects of zinc upon these reactions are strongly in evidence
but complex. Since the presence of 10 M zinc will precipitate the peptide, it
is
clear that the peptide possesses redox activity even when it is not in the
soluble
phase, suggesting that cortical A(3 deposits will not be inert in terms of
generating
these highly reactive products. Cerebral zinc metabolism is deregulated in AD,
and therefore levels of interstitial zinc may play an important role in
adjusting the
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Cu+ and H202 production generated by A. The rat homologue of Ap 1_40 does not
manifest the redox i-eactivity of the human equivalent. Insulin, a histidine-
containing peptide that can bind copper and zinc, exhibits no Cu'+ reduction.
e) Hydrogen Peroxide Production by A,6species
A(3,_4, (10 M) was incubated for 1 hr at 37 C, pH 7.4 in ambient air (first
bar), continuous argon purging (Ar), continuous oxygen enrichment (O,) at pH
7.0 (7.0), or in the presence of the iron chelator desferioxamine (220 M;
DFO).
Variant Ap species (10 M) were tested: A(31_40 (Ap,_40, rat A(3,_40
(rAp1_40), and
scrambled Ap 1_40 (sAP 1_40) were incubated for 1 hr at 37 C, pH 7.4 in
ambient air.
Values (mean fSD, n=3) represent triplicate samples minus values derived from
control samples run under identical conditions in the presence of catalase (10
U/ml). The details of the experiment are as follows: A(3 peptides were co-
incubated with a H,O,-trapping reagent (Tris(2-carboxyethyl)-phosphine
hydrochloride, TCEP, 100 M) in PBS (pH 7.4 or 7.0) at 37 C for 30 mins.
Then 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB, 100 M) was added to react
with remaining TCEF', the product has a characteristic absorbance maximum of
412 nm. The assay was adapted to a 96-well format using a standard absorbance
range.
Results and Discussion
A,6 exhibits metal-dependent and independent redox activity
Because Ap was observed to be covalently linked by Cu, the ability of the
peptide to reduce metals and generate hydroxyl radicals was studied. The
bathocuproine and bathophenarithroline reduced metal assay technique employed
by Multhaup et al. was used in order to determine that APP itself possesses a
Cu2+
reducing site on its ectodomain (Multhaup, G., et al., Science 271:1406
(1996)).
It has been discovered. that Ap possesses a striking ability to reduce both
Fe3+ to
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Fe'-+, and Cu'-' to Cu+, modulated by Zn'-+ and pH (6.6-7.4) (See Figure 10).
It
is of great interest that the relative redox activity of the peptides studied
correlates
so well with their relative pathogenicity viz AP1_42>AP1_40>ratAp in all redox
assays studied. Since one of the caveats in using the reduced metals assay is
that
the detection agents can exaggerate the oxidation potential of Cu2+ or Fe
(III),
other redox products were explored by assays where no metal ion indicators
were
necessary. It was discovered that hydrogen peroxide was rapidly formed by AP
species (Figure 11). Thus, Ap produces both H,O, and reduced metals whilst
also
binding zinc. Structurally, this is difficult to envisage for a small peptide,
but we
have recently shown that Ap is dimeric in physiological buffers. Since H1O2
and
reduced metal species are produced in the same vicinity, these reaction
products
are liable to produce the highly toxic hydroxyl radical by Fenton chemistry,
and
the formation of hydroxyl radicals from these peptides has now been shown with
the thiobarbituric acid assay. The formation of hydroxyl radicals correlates
with
the covalent polymerization of the peptide (Figure 9) and can be blocked by
hydroxyl scavengers. Thus the concentrations of Fe, Cu, Zn & H+ in the brain
interstitial milieu could be important in facilitating precipitation and
neurotoxicity
for Ap by direct (dimer formation) and indirect (Fe'-+/Cu' and H,O, formation)
mechanisms.
H,O, production by Ap explains the mechanism by which H202 has been
described to mediate neurotoxicity (Behl, C. et al., Cell 77:827 (1994)),
previously thought to be the product of cellular overproduction alone.
Interestingly, the scrambled A(3 peptide (same size and residue content as
Figure
6) produces appreciable H,O, but no hydroxyl radicals. This is because the
scrambled Ap peptide is unable to reduce metal ions. This leads to the
conclusion that what makes Ap such a potent neurotoxin is its capacity to
produce
both reduced metals and H20, at the same time. This "double whammy" can then
produce hydroxyl radicals by the Fenton reaction, especially if the H2O, is
not
rapidly removed from the vicinity of the peptide. Catalase and glutathione
peroxidase are the principal means of catabolizing H,Oõ and their levels are
low
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in the brain, especially in AD, perhaps explaining the propensity of AR to
accumulate in brain tissue.
Figure 11 shows that the production of H202 is oxygen dependent, and
further investigatiori has indicated that Ap can spontaneously produce the
superoxide radical (O;) in the absence of metal ions. This property of Ap is
particularly exaggerated in the case of AP1_42, probably explaining why this
peptide is more neurotoxic and more enriched than AP1_40 in amyloid. 02
generation will be subject to spontaneous dismutation to generate H,O,,
however,
this is a relatively slow reaction, although it may account for the majority
of the
HO, detected in our Ap assays. O; is reactive, and the function of superoxide
dismutase (SOD) is to accelerate the dismutation to produce H,O, which is then
catabolized by catalase and peroxidases into oxygen and water. The most
abundant form of SOD is Cu/Zn SOD, mutations of which cause another
neurodegenerative disease, arnyotrophic lateral sclerosis (Rosen, D., et al. ,
Nature
364:362 (1993)). Sirice Ap, like Cu/Zn SOD, is a dimeric protein that binds Cu
and Zn and reduces Cu' and Fe3', we studied the 02 dismutation behavior of A(3
in the sec time-scale using laser pulse photolysis. These experiments have
shown that Ap exhibits Fe/Cu-dependent SOD-like activity with rate constants
of dismutation at z l eg M'sec", which are strikingly similar to SOD. Hence,
AP
appears to be a good candiclate to possess the same function as SOD, and
therefore may function as an antioxidant. This may explain why oxidative
stresses cause it to be released by cells (Frederikse, P.H., et al., Journal
of
Biological Chemistry 271: 10169 (1996)). However, if Ap,4, is involved in the
reaction to oxidative! stress, or if the HZO, clearance is compromised at the
cellular level, A(3 will accumulate, recruiting more O, and producing more O;
leading to a vicious cycle and localizing tissue peroxidation damage and
protein
cross-linking.
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Example 3
Cell Culture and Cytotoxic Assays
Several different assays may be utilized to determine whether a candidate
pharmacological agent identified by any of the above-summarized assays is
capable of altering the neurotoxicity of Ap. The first is the MTT assay, which
measures the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5, diphenyl
tetrazolium
bromide (MTT) to a colored formazon (Hansen et al., J Immunol Methods,
119:203-210 (1989)). A second cytotoxic assay is the release of lactic
dehydrogenase (LDH) from cells, a measurement routinely used to quantitate
cytotoxicity in cultured CNS cells (Koh, J.Y. and D.W. Choi. J. Neurosci.
Meth.
20:83-90 (1987). While MTT measures primarily early redox changes within the
cell reflecting the integrity of the electron transport chain, the release of
LDH is
thought to be through cell lysis. A third assay is visual counting in
conjunction
with trypan blue exclusion. Yet another assay is the Live/Dead EukoLight
Viability/Cytotoxicity Assay (Molecular Probes, Inc., Eugene, OR).
Example 4
Therapeutic Agents for Inhibition of
Metal-Mediated Production of Reactive Oxygen Species
Materials and Methods
a) Synthesis of Peptides
Synthetic Ap peptides Ap,,o and Ap1_4, were synthesized by the W. Keck
Laboratory, Yale, CT. In order to verify the reproducibility of the data
obtained
with these peptides, confirmatory data were obtained by reproducing
experiments
with these Ap peptides synthesized and obtained from other sources: Glabe
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laboratory, University of California, Irvine, CA, Multhaup Laboratory,
University
of Heidelberg, U.S. Peptides, Bachem, and Sigma. Rat Ap was synthesized and
characterized by the 1vlulthaup Laboratory, University of Heidelberg. AP1_28
was
purchased from U.S. Peptides, Bachem, and Sigma. A(3 peptide stock solutions
were prepared in chelex-100 resin (BioRad) treated water and quantified.
b) Metal Reduction Assay
The metal reduction assay was performed using a 96-well microtiter plate
(Costar) based upon a modification of established protocols (Landers, J.W., et
al.,
Amer. Clin. Path. 29:590 (1958); Landers, J.W., et al., Clinica Chimica Acta
3:329 (1958)). Polypeptides (10 M) or Vitamin C (100 M), metal ions (10
M, Fe(N03); or Cu(N03)2), and reduced metal ion indicators,
bathophenanthrolinedisulfonic acid (BP, for Fe' , Sigma, 200 M) or
bathocuproinedisulfonic acid,(BC, for Cu+, Sigma, 200 RM), were coincubated
in phosphate buffered saline (PBS), pH 7.4, for 1 hr at 37 C. The metal ion
solutions were prepared by direct dilution in the buffer from their aqueous
stocks
purchased from National Institute of Standards and Technology (NIST).
Absorbances were then measured at 536 nm (Fe'-+-BP complex) and 483 nm
(Cu'-BC complex), respectively, using a 96-well plate reader (SPECTRAmax
250, Molecular Devices, CA). In control samples, both metal ion and indicator
were present to determine the background buffer signal. As a further control,
both metal ion and peptide were present in the absence of indicator to
estimate the
contribution of light scattering due to turbidity to the absorbance reading at
these
wavelengths. The net absorbances (DA) at 536 nm or 483 nm were obtained by
deducting the absorbances from these controls from the absorbances generated
by
the peptide and metal in the pi=esence of the respective indicator.
The concentrations of reduced metal ions (Fe'-+ or* Cu+) were quantified
based on the formula: Fe'-+ or Cu'(-M) = A* 106/(L*M), where L is the measured
equivalent vertical pathlength for a well of 300 L volume as described in the
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instrument's specifications manual (0.856 cm for Fez+; 1.049 cm for Cu+); M is
the known molecular absorbance (M-' cm-' ) which is 7124 (for Fe'+-BP complex)
or 2762 (for Cu+-BC complex).
c) HZO, Assay
The H2O2 assay was performed in a UV-transparent 96-well microtiter
plate (Molecular Devices, CA), according to a modification of an existing
protocol (Han, J.C., et al., Anal. Biochem. 234:107 (1996); Han et al., Anal.
Biochem. 220: 5-10 (1994)). Polypeptides (10 M) or Vitamin C (10 M), Fe'"'
or Cu21 (1 M) and a H,O, trapping agent- Tris(2-Carboxvethyl)Phosphine
Hydrochloride (TCEP, Pierce, 50 LM)- were co-incubated in PBS buffer (300
L), pH 7.4, for 1 hour at 37 C. Under identical conditions, catalase (Sigma,
100
U/mL) was substituted for the polypeptides, to serve as a control signal
representing 0 M H2O2. Following incubation, the unreacted TCEP was
detected by 5,5-Dithio-bis(2-Nitrobenzoic acid) (DTNB, Sigma, 50 KLM) which
generates 2 moles of the coloured product. The reactions are:
TCEP + H,O, -TCEP=O + H,O,
then the remaining TCEP is reacted with DTNB:
TCEP + DTNB + H20 - TCEP=O + 2NTB (2-nitro-5-thiobenzoate).
The amount of H,O, produced was quantified based on the formula: H,O,
( M) = hA* 106/(2*L*M), where hA is the absolute absorbance difference
between a sample and catalase-only control at 412 nm wavelength; L = 0.875 cm,
the equivalent vertical pathlength obtained from the platereader
manufacturer's
specifications; M is the molecular absorbance for NTB (14150 M-' cm-' at 412
nm).
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TCEP is a strong reducing agent, and, hence, will artifactually react with
polypeptides that contain disulfide bonds. This was determined not to be a
source
of artifact for the measurement of HZO, generation from A(3, which does not
possess a disulfide bond.
d) Estimation ojf' OZ
The spectrophotometric absorption peak for O; is 250 nm where its
extinction coefficient is much greater than that of HO, (Bielski et al.,
Philos
Trans R Soc Lond B Biol Sci. 311: 473-482 (1985)). The production of O; was
estimated by measuring the spectrophotometric absorption of polypeptides (10
M, 300 L) after incubation for one hour in PBS, pH 7.4, at 37 C, using a
96-well plate reader. The corresponding blank was the signal from PBS alone.
An absolute baseline for the s;ignal generated by the peptide was not
achievable
in this assay since the absorpi:ion peak for tyrosine (residue 10 of A(3) is
close
(254 nm) to the absorption peak for O. However, attenuation of the absorbance
by co-incubation with superoxide dismutase (100 U/mL) indicated that the
majority of the absorbance signal was due to the presence of O2.
e) Thiobarbituric Acid Reaction Substance (TBARS) Assay - OH-
The Thiobar'bituric Acid-Reactive Substance (TBARS) assay for
incubation mixtures with Fe'-- or Cuz+ was performed in a 96-well microtiter
format modified from established protocols (Gutteridge et al. Biochim.
Biophys.
Acta 759: 38-41 (1983)). Ap peptide species (10 M) or Vitamin C (100 M),
were incubated with ]Fe3+ or C'.u2+ (1 M) and deoxyribose (7.5 mM, Sigma) in
PBS, pH 7.4. Following incubation (37 C,1 hour), glacial (17 M) acetic acid
and
2-thiobarburic acid (l%, w/v in 0.05 M NaOH, Sigma) were added and heated
(100 C, 10 min). The final mixtures were placed on ice for 1-3 minutes
before
absorbances at 532 nm were measured. The net absorbance change for each
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sample were obtained by deducting the absorbance from a control sample
consisting of identical chemical components except for the Vitamin C or Ap
peptides.
Results and Discussion
Oxygen radical involvement in human aging, the predominant risk factor
for Alzheimer's disease (AD), was first proposed by Harman in 1956 (Harman,
D., J. Gerontol. 11:298 (1956)) and increasing evidence has implicated
oxidative
stress in the pathogenesis of AD. Apart from metabolic signs of oxidative
stress
in AD-affected neocortex such as increased glucose-6-phosphate dehydrogenase
activity (Martins, R.N., et al., J. Neurochem. 46:1042-1045 (1986)) and
increased
heme oxygenase-1 levels (Smith, M.A., et al., Am. J. Pathol. 145:42 (1994)),
there are also numerous signs of oxygen radical-mediated chemical attack such
as increased protein and free carbonyls (Smith, C.D., et al., Proc. Natl.
Acad. Sci.
USA 88:10540 (1991); Hensley, K., et al., J. Neurochem. 65:2146 (1995); Smith,
M.A., et al., Nature 382:120 (1996)), lipid peroxidation adducts (Palmer, A.M.
& Burns, M.A., Brain Res. 645:338 (1994); Sayre, L.M. et al., J. Neurochem.
68:2092 (1997)), peroxynitrite-mediated protein nitration (Good, P.F., et al.,
Am.
J. Pathol. 149:21 (1996)); Smith, M.A., et al., Proc. Natl. Acad Sci. USA
94:9866 (1997)), and mitochondrial and nuclear DNA oxidation adducts
(Mecocci, P., et al., Ann. Neurol., 34:609-616 (1993); Mecocci, P., et al.,
Ann.
Neurol., 36:747-751 (1994)). Recently, treatment of individuals with the
antioxidant vitamin E has been reported to delay the progression of clinical
AD
(Sano, M. et al., N.Engl. J. Med. 336:1216 (1997)).
A relationship seems likely to exist between the signs of oxidative stress
and the characteristic A(3 collections (Glenner, G.G. & Wong, C., Biochem.
Biophys. Res. Commun. 120:885 (1984)) found in the cortical interstitium and
cerebrovascular intima media in AD. The brain regional variation of oxidation
biomarkers corresponds with amyloid plaque density (Hensley, K., et al.,
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J. Neurochem. 65:2146 (1995)). Indeed, neurons cultured from subjects with
Down's syndrome, a condition complicated by the invariable premature
deposition of cerebral A(3 (Rumble, B., et al., N. Engl. J. Med. 320:1446
(1989))
and the overexpression of soluble Ap 1 -42 in early life (Teller, J.K., et
al., Nature
Medicine 2:93 (1996)), exhibit lipid peroxidation and apoptotic cell death
caused
by increased generation of hydrogen peroxide (Busciglio, J. & Yankner, B.A.,
Nature 3 78:776 (1995)). Synthetic A(3 peptides have been shown to induce
lipid
peroxidation of synaptosomes (Butterfield, D.A., et al., Biochem. Biophys.
Res.
Commun. 200:710 (1994)), and to exert neurotoxicity (Behl, C., et al., Cell
77:817 (1994); Mattson, M.P., et al., J. Neurochem. 65:1740 (1995)) or
vascular
endothelial toxicity tiilrough a mechanism that involves the generation of
cellular
superoxide/hydrogen peroxide (O2/H2O,) and is abolished by the presence of SOD
(Thomas, T., et al., Nature 380:168 (1996) or catalytic synthetic OFJH,O,
scavengers (Bruce, A.J., et al., Proc. Natl. Acad. Sci. USA 93:2312 (1996)).
Antioxidant vitamin E and the spin-trap compound PBN have been shown to
protect against Ap-niediated neurotoxicity in vitro (Goodman, Y., & Mattson,
M.P., Exp. Neurol. .128:1 (1994); Harris, M.E., el al., Exp. Neurol. 131:193
(1995)).
Ap, a 39-43 amino acid peptide, is produced (Haass, C., et al., Nature
359:322 (1992); Seubert, P., et al., Nature 359:325 (1992); Shoji, M., et al.,
Science 258:126 (1992)) by constitutive cleavage of the amyloid protein
precursor
(APP) (Kang, J., et a!., Nature 325:733 (1987); Tanzi, R.E., et al., Nature
Genet
(1993)) as a mixture of polypeptides manifesting carboxyl-terminal
heterogeneity.
A(3-40 is the major soluble AP species in biological fluids (Vigo-Pelfrey, C.,
et al., J. Neurochem. 61:1965 (1993)) and AP,4, is a minor soluble species,
but
is heavily enriched in interstitial plaque amyloid (Masters, C.L., et al.,
Proc. Natl.
Acad. Sci. USA 82:4245 (1985); Kang, J. et al., Nature 325:733 (1987); Prelli,
F.,
et al., J. Neurochem. 51:648 (1988); Roher et al., J. Cell Biol. 107:2703-2716
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(1988); Roher et al., J. Neurochem. 61:1916-1926 (1993); Miller, D.L., et al.,
Arch. Biochem. Biophys. 301:41 (1993)). The discovery of pathogenic mutations
of APP close to or within the A(3 domain (van Broeckhoven, C., et al., Science
248:1120 (1990); Levy, E., et al., Science 248:1124 (1990); Goate, A., et al.,
Nature 349:704 (1991); Murrell, J., et al., Science 254:94 (1991); Mullan, M.,
et
al., Nature Genet 1:345 (1992)) indicates that the metabolism of Ap is
involved
with the pathophysiology of this predominantly sporadic disease. Familial AD-
linked mutations of APP, presenilin-1 and presenilin-2 correlate with
increased
cortical amyloid burden and appear to induce an increase in the ratio of Ap
1_42 as
part oftheir common pathogenic mechanism (Suzuki, N., et al., Science 264:1336
(1994); Scheuner et al., Nat Med., 2(8):864-870 (1996); Citron, M., et al.,
Nature
Medicine 3:67 (1997)). However, the mechanism by which AP1_42 exerts more
neurotoxicity than AP1_40 and other Ap peptides (Dord, S., et al., Proc. Natl.
Acad.
Sci. USA 94:4772 (1997)) remains unclear.
One of the models proposed for Ap neurotoxicity is based on a series of
observations of Ap-generated oxyradicals generated by a putative AP peptide
fragmentation mechanism which is O,-dependent, metal-independent and
involves the sulfoxation of the methionine at A(3 residue 35 (Butterfield,
D.A.,
et al., Biochem. Biophys. Res. Commun. 200:710 (1994); Hensley, K., et al.,
Proc. Natl. Acad. Sci. USA 91:3270 (1994); Hensley, K., et al., Ann N YAcad
Sci., 786: 120-134 (1996). AP25_35 peptide has been reported to exhibit H,O,-
like
reactivity towards aqueous Fe'-', nitroxide spin probes, and synaptosomal
membrane proteins (Butterfield, D.A., et al., Life Sci. 58:217 (1996)), and
A(31_40
has also been reported to generate the hydroxyl radical by mechanisms that are
unclear (Tomiyama, T., et al., J. Biol. Chem. 271:6839 (1996)). However, there
has been no quantitative appraisal of the ROS-generating capacity of Ap 1-42
versus
that of Ap 1_40 and other A(3 variants, to date.
Ap is a metal binding protein which saturably binds zinc via a histidine-
mediated specific high affinity site (KD = 107 nM) as well as by low affinity
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binding (KD = 5.2 M). The high-affinity zinc binding site was mapped to a
stretch of contiguous residues between positions 6-28 of the Ap sequence
(Bush,
A.I., et al., J. Biol. Chem. 269:12152 (1994)). Concentrations of zinc _ 1 M
rapidly induce aggregation of human Ap 1-40 solutions (Bush, A.I., et al.,
Science
265:1464 (1994)), in:reversible manner which is dependent upon the
dimerization
of peptide in solution, its alpha-helical content, and the concentration of
NaCl
(Huang, X. et al., J. Biol. Chem. 272:26464-26470 (1997)). Rat/mouse A(3,40
("rat Ap", with substitutions o RS-G, Yjf F, and H13-R, as compared to human
A(3) binds zinc less avidly (a single binding site, KA=3.8 M) and, unlike the
human peptide, is not precipitated by zinc at concentrations < 25 M. Since
zinc
is concentrated in the neocortex, we hypothesized that the differential
solubility
of the rat/mouse Ap peptide in the presence of zinc may explain the scarcity
with
which these animals form cei-ebral A(3 deposits (Johnstone, E.M., et al., Mol.
Brain Res. 10:299 (1991); Shivers, B.D., et al., EMBO J. 7:1365 (1988)).
We have also observed interactions of A(3 with Cu'% which stabilizes
dimerization of A(314D on gel chromatography (Bush, A.I., et al., J. Biol.
Chem.
269:12152 (1994)), and which binds to the peptide with an affinity estimated
to
be in the low picomolar rarige. Fe'-+ has been observed to induce partial
aggregation of Ap (Bush, A.I., et al., Science 268:1921 (1995)), and to induce
SDS-resistant polymo.rization of the peptide (Dyrks, T., et al., J. Biol.
Chem.
267:18210-18217 (1992)). We hypothesized that the interactions of A(3 with Fe
and Cu may contribute to the genesis of the oxidation insults that are
observed in
the AD-affected brain.. This is because Fe3+ and Cuz+ are redox-active metal
ions
that are concentrated in brain neurons, and may participate in the generation
of
ROS by transferring electrons in their reduced state (reviewed in Markesbery,
1997).
The levels of Cu and Fe, and their binding proteins, are dysregulated in
AD (Diebel, M.A., et al., J. Neurol. Sci. 143:137 (1996); Good, P.F., et al.,
Ann.
Neurol. 31:286 (1992); Robinson, S.R., et al., Alzheimer's Research 1:191
(1995); Thompson, C.M., et a~, Neurotoxicology 9:1 (1988); Kennard, M.L., et
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al., Nature Medicine 2:1230 (1996); Connor, J.R., et al., Neurosci. Lett.
159:88
(1993)) and may therefore lead to conditions that could promote ROS
production.
While a direct role for Ap in metal-dependent ROS generation has not been
described, the peptide's physiochemical interation with transition metals, the
presence of ferritin (Grudke-Iqbal, I., et al., Acta Neuropathol. 81:105
(1990))
and redox reactive iron (Smith, M.A., et al., Proc. Natl. Acad. Sci. USA
94:9866
(1997)) in amyloid lesions, and the facilitation of AP1_40 neurotoxicity in
cell
culture by nanomolar concentrations of iron (Schubert, D. & Chevion, M.,
Biochem. Biophys. Res. Commun. 216:702 (1995)), collectively support such a
possibility.
We report the simultaneous production of H2O, and reduced metal ions
by Ab, with the consequent generation of the hydroxyl radical. The amounts of
reduced metal and ROS were both greatest when generated by Ap 1_42>A(31_4o rat
Ap 1_40, AP40.1 and Ap_28, a chemical relationship that correlates with the
relative
neurotoxicity of these peptides. These data describe a novel, 02 and biometal-
dependent pathway of ROS generation by Alzheimer Ap peptides which may
explain the occurrence of oxidative stress in AD brain.
a) Metal Ion Reduction by A,13 Peptides
To determine whether A(3 peptides possess metal-reducing properties, the
ability of Ap peptides (Example 1) to reduce Fe3+ and Cu2+, compared to
Vitamin
C and other polypeptides (Example 2) was measured. Vitamin C, serving as a
positive control, reduced Cuz+ efficiently (Figure 13A). However, the
reduction
of Cu'-+ by A(31_42 was as efficient, reducing all of the available Cuz+
during the
incubation period. A(31-40 reduced 60% of the available Cu'+, whereas rat
A(31_40
and A(31-28 reduced no Cu'-+. The reduction of CuZ+ by BSA (25%) and insulin
(10%) was less efficient than that by the human Ap peptides, and was not
unexpected since these polypeptides possess cysteine residues and reduce Cu2+
in the process of forming disulfide bonds.
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Fe3+/FeZ' has lower sl:andard reduction potential (0.11 V) than CuZ+/Cu+
(0.15 V) does under our experimental conditions (Miller, D.M., et al., Free
Radical Biology &A.tedicine 8:95 (1990)), and, in general, Fe3+ was reduced
with
less efficiency by Vitamin C,and the polypeptides that reduced Cu'-+. Vitamin
C
reduced 15% of the available Fe3+, however Ap1_42 was the most efficient (50%)
of the agents tested for Fe'' reduction, reducing more Fe'" in the incubation
period than Vitamin C (15%), AP, _40 (12%) and BSA (8%). Rat AP1_4,, A[3,_28
and
insulin did not signifi.cantly facilitate the reduction of Fe3+. Analysis of
A(3,_42 and
A[31_40 after incubation with Cu'' and Fe3+ under these conditions revealed
that
there was no apparerit mass niodification of the peptides on mass
spectrometry,
and no change in its rnigratior,i pattern on sodium dodecyl sulfate
polyacrylamide
gel electrophoresis (SDS PAGE), nor evidence for increased aggregation of the
peptides by turbidorrietry or sedimentation analysis, suggesting that the
peptides
were not consumed or modified during the reduction reaction. Under these
conditions, the complete kinetics of the peptide-mediated reactions cannot be
appreciated (the presence of A[i, _42 induced the total consumption of the
Cuz+
substrate within the incubation period), but a striking relationship exists
between
the relative efficiencies of the various A[3 peptides to reduce CuZ+/Fe'- in
this
system and their respective pau-ticipation in amyloid neuropathology.
Since the dissolved O, in the buffer vehicle may be expected to react with
the reduced metals being generated [Reaction (1)], the effect of modulating
the
O, tension in the buffer upon the generation of reduced metals by the A[3
peptides (Figure 13B) was examined. Prior to the addition of Vitamin C or
polypeptide, the buffer vehicle was continuously bubbled for 2 hours at 20 C
with 100% O, to create conditions of increased 02 tension, or Argon to create
anaerobic conditions. Increasing the O, tension slightly reduced the levels of
reduced metals being detected., probably due to the diversion of a fraction of
the
Fez+/Cu+ being generated to Reaction (1), and, if H202 is being produced as a
product of Reaction (2), the recruitment of FeZ+/Cu+ into the Fenton reaction
[Reaction (3)]. However, performing the reaction under anaerobic (Argon
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purged) conditions also slightly reduced the levels of reduced metals being
detected. This may be because some of the reduction of Fe3+/Cu'-+ is due to
reaction with
O;:
Ml"'')+ + O; - M , + 02 Reaction (5)
To determine whether the reduction of metal ions in the presence of A(3
was due to the action of the peptide or the generation of O; by the peptide,
the
effects of metal ion chelators on the generation of reduced metal ions (Figure
13B) was studied. It was found that coincubation of AP1_42 with the relatively
Fe3+-specific chelator desferrioxamine (DFO) under ambient oxygenation
conditions nearly halved the production of Fez+. Coincubation of AP,42 with
the
high-affinity Cuz+ chelator TETA abolished 95% of the Cu' generated by the
peptide under ambient oxygenation conditions. These data indicate that the
majority of the Cu+ and a significant amount of the Fe'-+ produced by AP1_42
are
due to the direct action of the peptide and not indirectly due to the
production of
02'
The inhibitory effects of chelation upon A(3-mediated reduction of metal
ions indicates that Ap probably directly coordinates Fe3+ and CuZ+, and also
that
these chelating agents are not potentiating the redox potential of the metals
ions,
suggested to be an artifactual mechanism for the generation of reduced metal
species (Sayre, L.M. et aL, Science 274:1933 (1996)). The reasons for DFO
being less effective than TETA in attenuating metal reduction may relate to
the
respective (unknown) binding affinities for Fe3+ and Cu2+ to the A(3 peptide,
the
stereochemistry of the coordination of the metal ions by the peptide, and the
abilities of the chelating agents to affect electron transfer after
coordinating the
metal ion.
The reduction of metal ions by Ap must leave the peptide, at least
transiently, radicalized, in agreement with the electron paramagnetic
resonance
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(EPR) findings of Hensley et al., Proc. Natl. Acad. Sci. 91:3270 (1994). In
their
report, DFO, EDTA or Chelex 100 could not abolish the EPR signal generated
by AP25-35 in PBS, leading these investigators to conclude that the
radicalization
of A(3 was metal-inclependent. However, the inventors have found that after
treatment with Chelex 100 the concentrations of Fe and Cu in PBS are still as
high as =0.5 M (8), which could be sufficient to induce the radicalization of
the
peptide after metal reduction. Since DFO does not abolish the reduction of
Fe3+
by AR142 (Figure 13B), and since EDTA has been observed to potentiate Fe-
mediated Fenton chernistry (Samuni et al., Eur. J. Biochem. 13 7:119-124(1983
)),
it is suspected that Hensley and colleagues may have inadvertently overlooked
the contribution of metal reduction to A(3-mediated radical formation.
Rat A(31-4. did not reduce metal ions, and has been shown to have
attenuated binding oj- Zn'+ (Bush et al., Science, 265:1464 (1994)). A similar
attenuation of Cu2' and Fe3i binding by rat AD1-4o compared to human AP,_4o is
anticipated. These data also iridicate that the rat Ap substitutions in human
A(3's
zinc binding domain towards the peptide's amino terminus (Bush et al., J.
Biol.
Chem., 269:12152 (1994)) involve residues that mediate the metal-reducing
properties of the peptide. However, the hydrophobic carboxyl-terminal residues
were also critical to the reduc-tion properties of Ap. That AP,-2, did not
reduce
metal ions indicates that an intact Zn'-'-binding site (Bush et al., J. Biol.
Chenz.
269:12152 (1994)) is insufficient to facilitate the metal reduction reaction.
The
mechanism by which the two additional hydrophobic residues (Ile and Ala) on
A(31.4, so substantially enhance the peptide's redox activity compared to AP
1_40 is
still unclear.
It has been observed that sulfoxation of the methionine residue at A(3
position 35 accompanies the EPR changes seen during the incubation of AP25-35
for 3 hours in PBS at 37 C (Hensley, K., et al., Ann N YAcadSci., 786: 120-134
(1996)), however, no evidence was found for a modification of A(31_40 and A(3,-
42
after mass spectrophotometric examination of the peptides incubated under
conditions as described. Therefore, Ap-mediated metal reduction, and the
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subsequent Ap-mediated redox reactions described below, appear to be achieved
by a mechanism that differs from that previously reported.
b) Production of H202 by A,6 Peptides
The reduced metal ions produced by Ap were expected to generate O2 and
H,O2 by Reactions (1) and (2). To study this, a novel assay was developed
(Example 2) which detected the generation of 10 M H,O, by AP,_a, in the
presence of 1 M Fe3+ under ambient O, conditions (Figure 14A). To validate
the assay, coincubation with catalase was observed to abolish the H,O, signal
in
a dose dependent manner. The amount of H,O, produced by the various Ap
peptides was studied, and observed that the order of the production of H,O, by
the
A(3 variants was A(31_4, > A(31_40 >> rat A(31_40 - Ap,_,g (Figure 14B),
paralleling
the amounts of metal reduction by the same peptides (Figure 13A).
H,O, formation is likely to be mediated first by 02-dependent O;
formation [Reaction (1)], followed by dismutation [Reaction (2)]. To appraise
the contribution of Reaction (1) to H,O2 formation, H202formation by AP1_4, in
the presence of chelators was measured (Figure 14C). The amount of H,O1
formed in the presence of 1 M Cu'-+ was 25% greater than the amount formed
in the presence of 1 M Fe3". Coincubation with DFO had no effect on H,O2
formation in the presence of 1 M Fe3+. However, TETA, and the Cu'-specific
indicator BC, both substantially inhibited the formation of H,O, in the
presence
of 1 M Cu21. The reasons why DFO partially inhibited Fe3" reduction, but was
unable to inhibit H2O2 formation are unclear. These data indicate that the
formation of H,OZ by Ap is dependent upon the presence of substoichiometric
amounts of Cu/(II). The possibility that formation of H20, in the presence of
Fe3+ was due to the presence of trace quantities of Cu'-- cannot be excluded.
BC and BP, agents that specifically complex reduced metal ions, were far
more effective than DFO and TETA at inhibiting H202 formation by Ap (Figure
14C) but the reasons for this are not clear. The relatively Fez+-specific
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complexing agent, BP, inhibited H,O, formation in the presence of Cu'-, , and
the
relatively Cu'-speciiic compifexing agent, BC, inhibited H,O, formation in the
present of Fe3+, suggesting that these agents are not totally specific in
their metal
ion affinities. The formation of H,O, by Ap in the absence of BC or BP
confirms
that the reduction of rnetals is :not contingent upon the artifactual
enhancement of
the metal ions' redox potentials (Sayre, L.M., Science 274:1933 (1996)).
To determine whether the formation of OZ/H,O, by A~3 is merely due to
the reduction of inetail ions, or whether Ap also facilitates the recruitment
of the
substrates in Reactio;n (1), the generation of H,02 by AP1_47, AP,_40 and
Vitamin
C under different O, t.ensions in the presence of 1 M Fe3+ (Figure 14D) or 1
M
Cu'-' (Figure 14E) was studied. The presence of Vitamin C was used as a
control
measure to determine the amount H,O, that is generated by the presence of
reduced metals alone. In the presence of either metal ion, there was a
significant
increase in the amount of H,O_, produced under higher Oz tensions. The
presence
of either A(3,_a, and AP,-40 generated more H,O, (Ap 1_4, > A(3,40) than
Vitamin C
under any O, tension studied, and generated H,Oz under conditions where
Vitamin C produced none, even though reduced metal ions must be present due
to the activity of Vitamin C. 'Therefore, under these ambient and argon-purged
conditions, the reduction of metal ions is insufficient to produce H20,. These
data
indicate that Ap indeed facilitates the recruitment of O, into Reaction (1)
more
than would be expected by the interaction of the metals reduced by A(3 with
the
passively dissolved O,. Under relatively anaerobic conditions, the A(3
peptides
were observed to still produce H7O2 in the presence of Cuz+ ( Figure 14E).
This
is probably due to the ability of Ap to recruit O, into Reaction (1) under
conditions of very low O, tension. Since O, is preferentially dissolved in
hydrophobic environments (Halliwell and Gutteridge, Biochem. J., 219:1-14
(1984)), it seems that the hydrophobic carboxyl-terminus of A(3 could attract
O,,
serving as a reservoir for the substrate.
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c) Evidence of the Superoxide Anion Formed by the A/8 -metal Complex
To confirm the production of 02 by A(3, the absorbance of the peptide in
solution at 250 nm, the absorbance peak of Oz (Figure 15A) was measured. The
absorbance generated by A[3,42 in the presence of I M Fe3+ was 60% reduced
when co-incubated with SOD, increased in the presence of high O2 tension and
abolished under anaerobic conditions. These data support the likelihood that
A(3
generates H,O2 by first generating O;.
The absorbance changes at 250 nm for the various A(3 peptides in PBS
(Figure 15B) paralleled the production of H202 from the same peptides (Figure
14B), but the reason for the A250 being much greater for A[3_4, compared to Ap
1_40
is unclear. It is likely that a fraction of the total H202 generated by A[3 is
decomposed by the Fenton reaction [Reaction (3)]. Therefore, the amount of
H,O1 detected may be an attenuated reflection of the amount of O; detected.
d) Detection ofHydroxylRadicals Generatedfrom theA/3-metal Complex
Having demonstrated that human A[3 peptides simultaneously produce
H20, and reduced metals, we proceeded to determine whether the hydroxyl
radical was formed by the Fenton or Haber-Weiss reactions [Reactions (3) and
(4)]. A modified TBARS assay was employed to detect OH= released from
co-incubation mixtures of A(3 peptides and 1 M Fe'+ or Cu'-+. As expected,
A(.i1-42 produced more OH= than A(31-40, and rat Ap did not generate OH=
(Figure
16A). In contrast to the amount of Fe'; and Cu' produced (Figure 13A), Ap
generated more OH= in the presence of Fe3+ than in the presence of Cu''. This
may be because Fez+ is more stable than Cu, which may be more rapidly oxidized
by Reaction (1). Therefore, the Fe2+ generated by A[3 may have a greater
opportunity than the Cu+ generated to react with HzOZ. It is also possible
that the
contribution of the Haber-Weiss reaction to the production of OH= [Reaction
(5)]
is greater in the presence of Fe3+ than in the presence of Cu2+.
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The effects of the OH= scavengers, dimethyl sulfoxide (DMSO) and
mannitol, upon AP,.42-mediated OH= generation were studied. Whereas these
agents suppressed the generation of OH= by Vitamin C in the presence of Fe3+
and DMSO suppressed the generation of OH= by Vitamin C in the presence of
Cuz+, neither were able to quench the generation of OH= by AP 1-42, whether in
the
presence of Fe3+ or Cu2+ (Figure 16B). This suggests that these scavengers
cannot
encounter the OH= generated by Ap before the TBARS reagent does.
e) Similarity Between Bleomycin-Fe and A# -Fe/Cu Complexes
The present Examples provide evidence for a model by which Fe/Cu and
02 are mediators and substrates for the production of OH= by A(3 (Figures 16A
and 16B) in a mannei- that depends upon the presence and length of the
peptide's
carboxyl terminus. T'he brain neocortex is an environment that is rich in both
02
and Fe/Cu, which may expla:in why this organ is predisposed to Ap-mediated
neurotoxicity, if this mechanism is confirmed in vivo. The transport of Fe, Cu
and Zn in the brain is largely energy-dependent. For example, the
copper-transporting gene for 'Wilson's disease is an ATPase (Tanzi, R.E. et
al.,
Nature Genetics 5:344 (1993)), and the re-uptake of zinc following
neurotransmission is highly energy-dependent (Assaf, S.Y. & S.H. Chung,
Nature, 308:734-736 (1984); :Howell et al., Nature, 308:736-738 (1984)).
There is increasing evidence for lesions of brain energy metabolism in
aging and AD (Parker et al., Neurology, 40:1302-1303 (1990); (Mecocci et al.,
Ann. Neurol. 34:609-616 (1993); Beal, M.F. Neurobiol. Aging 15
(Supp12) :S 171-S 174(1994)). 7'herefore, damage to energy-dependent brain
metal
homeostasis may be Em upstream lesion for the genesis of Ap deposition in AD.
Most brain biometals are bound to proteins or other ligands, however,
according
to our findings, only Ap small fraction of the available metals needs to be
derailed to the AP-containing compartment to precipitate the peptide and to
activate its ROS-generating activities. The generation of ROS described herein
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depends upon the sub-stoichiometric amounts of Fe3+/Cu'-+ (1:10, metal:AP),
and
it was estimated that 1% of the zinc that is released during neurotransmission
would be sufficient to precipitate soluble A(3 in the synaptic vicinity
(Huang, X.
et al., J. Biol. Chem. 272:26464-26470 (1997)).
A polypeptide which generates both substrates of the Fenton reaction in
sufficient quantities to form significant amounts of the OH- radical is
unusual.
Therefore, Ap collections in the AD-affected brain are likely to be a major
source
of the oxidation stress seen in the effected tissue. One recent report
describes that
A(3 is released by the treatment of the mammalian lens in culture with H,O2
(Frederikse, P.H., et al., J. Biol. Cheni. 271:10169 (1996)). If a similar
response
mechanism to H,O2 stress exists in neocortex, then the increasing H1O1
concentration generated by the accumulating Ap mass in the AD-affected brain
may induce the production of even more Ap leading to a vicious cycle of A(3
accumulation and ROS stress.
The simultaneous production of Fenton substrates by A(3 is a chemical
property that is brought into therapeutic application in the oxidation
mechanism
of the bleomycin-iron complex. Bleomycin is a glycopeptide antibiotic produced
by Streptomyces verticillus and is a potent antitumor agent. It acts by
complexing
Fe' and then binding to tumor nuclear DNA which is degraded in situ by the
generation of OH- (Sugiura, Y., et al., Biochem. Biophys. Res. Commun.
105:1511 (1997)). Similar to AP-Fe3+/Cu'-+ complexes, incubation of bleomvcin
in aqueous solution also engenders the production of Oz. H2O,1 and OH- in an
Fe3+-dependent manner. DFO could not inhibit HzOZ production from the
AP-Fe3+/Cu'+ complex, and similarly, DFO does not inhibit the OH=-mediated
DNA damage caused by the bleomycin-Fe" complex. Also, low-molecular-mass
OH- scavengers mannitol and DMSO were unable to inhibit the generation of
OH= by A(3-Fe3i/Cu2+, and are similarly unable to inhibit OH- production from
bleomycin-Fe3+
It is proposed herein that inhibition of Ap-mediated OH- provides means
of treatment, e.g. therapy, by compounds that are Fe or Cu chelators. The
clinical
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administration of IJFO was reported as being effective in preventing the
progression of AD (Crapper-McLachlan, D.R. et al., Lancet 337:1304 (1991));
however, since DFO chelates Znz+ as well as Fe3; and Al(III), the effect, if
verifiable, may not tiave been due to the abolition of the redox activity of
AP, but
may have been due to the disaggregation of Zn'-+-mediated A(3 deposits
(Cherny,
R.A. et al., Soc. Neurosci. A'ustr. 23:(abstract)(1997)) which may have
reduced
cortical A(3 burden and, consequently, oxidation stress.
Oxidative Stress and Alzheimer's Disease Pathology
Autopsy tissue from AD subjects has been reported to exhibit higher basal
TBARS formation than control material (Subbarao, K.V. et al., J. Neurochem.
55:342 (1990); Balazs, L. and M. Leon, Neurochem. Res. 19:1131 (1994); Lovell
et al., Neurology 45:1594 (1995)). These observations could be explained, on
the
basis of the present findings, as being due to the reactivity of the AP
content
within the tissue. A13140 recently has been shown to generate TBARS in a dose-
dependent manner when incubated in cell culture, however TBARS reactivity was
reduced by pre-treating the cells with trypsin which also abolished the
binding of
the peptide to the RAGE receptor (Yan et al., Nature 382:685 (1996)). One
possibility for this result is that the RAGE receptor tethers an Ap
microaggregate
sufficiently close to the cell to permit increased penetration of the cell by
H202
which may then cornbine with reduced metals within the cell to generate the
Fenton reaction. Alternatively, A(3 may generate the Fenton chemistry at the
RAGE receptor. The resultitig attack of the cell surface by the highly
reactive
OH = radical, which reacts within nanometers of its generation, may have been
the source of the positive TBARS assay.
APP also reiiuces Cu2+, but not Fe3+, at a site in its amino terminus
(Multhaup, G., et al., Science 271:1406-1409 (1996)), adjacent to a functional
and
specific ZnZ+-binding site that modulates heparin binding and protease
inhibition
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(Bush et al., 1993; Van Nostrand, 1995). Therefore, the amino terminus of APP
reiterates an association with transition metal ions that is found in the AP
domain.
This intriguing theme of tandem Cu/Zn interaction and associated redox
activity
found in two soluble fragments of the parent protein may indicate that the
function and metabolism of APP could be related to biometal homeostasis and
associated redox environments.
The present findings indicate that the manipulation of the brain biometal
environment with specific agents acting directly (e.g. chelators and
antioxidants)
or indirectly (e.g. by improving cerebral energy metabolism) holds promise as
a
means for therapeutic intervention in the prevention and treatment of
Alzheimer's
disease.
Example 5
Reso[ubilization of AO
Considerable evidence now indicates that the accumulation of A(3 in the
brain cortex is very closely related to the cause of Alzheimer's disease. A(3
is a
normal component of biological fluids whose function is unknown. Ap
accumulates in a number of morphologies varying from highly insoluble amyloid
to deposits that can be extracted from post-mortem tissue in aqueous buffer.
The
factors behind the accumulation are unknown, but the inventors have
systematically appraised the solubility of synthetic A(3 peptide in order to
get
some clues as to what kind of pathological environment could induce the
peptide
to precipitate.
It was found that A(3 has three principal vulnerabilities: zinc, copper and
low pH. The precipitation of A(3 by copper is dramatically exaggerated under
mildly acidic conditions (e.g., pH 6.9), suggesting that the cerebral lactic
acidosis
that complicates Alzheimer's disease could contribute to the precipitation of
AP
were this event to be mediated by copper. A consideration of the involvement
of
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zinc and copper in plaque pathology is contemplatable since the regulation of
these metals in the brain has been shown to be abnormal in AD.
Recently direct evidence has been obtained indicating that these metals
are integral components of the Ap deposits in the brain in AD. It was found
that
zinc- and copper-specific chelators dramatically redissolve a significant
proportion (up to 70%) of A(3 extracted from post-mortem AD affected brain
tissue, compared to the amount extracted from the tissue by buffer in the
absence
of chelators.
These data support a strategy of redissolving A(3 deposits in vivo by
chelation. Interestingly, a reported success in attempting to slow down the
progression of A.lzheimer's disease used a chelation strategy with
desferrioxamine. The authors (Crapper-McLachlan, D.R., et al., 337:1304
(1991), thought that they we:re chelating aluminum, but desferrioxamine is
also
a chelator of coppei- and zinc. Treatment with desferrioxamine is impractical
because the therapy requires itwice daily deep intramuscular injections which
are
very painful, and also causes side effects such as anaemia due to iron
chelation.
AP Extraction from Human Brain Post-Mortem Samples
The inventors have recently characterized zinc-mediated AP deposits in
human brain (Cherny, R.A., et al., Soc. Neurosci Abstr. 23:(Abstract) (1997)).
It was recently reported that there is a population of water-extractable A(3
deposit
in the AD-affected brain (Kuo, Y-M., et al., J. Biol. Chem. 271:4077-81
(1996)).
The inventors hypothesized that homogenization of brain tissue in water may
dilute the metal content in the tissue, so lowering the putative zinc
concentration
in Ap collections, and liberating soluble Ap subunits by freeing A(3 complexed
with zinc [Zn2+].
To test this hypothesis, the brain tissue preparation protocol of Kuo and
colleagues was replicated, but phosphate-buffered saline pH 7.4 (PBS) was
substituted as the extraction lbuffer, achieving similar results. Highly
sensitive
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and specific anti-Ap monoclonal antibodies (Ida, N. et al., J. Biol. Chem.
271:22908 1996) were used to assay Ap extraction by western blot. Next, the
extraction of the same material was repeated with PBS in the presence of
chelators of varying specificities (Table 1), and it was determined that the
presence of a chelator increased the amount of A(3 in the soluble extract
several-
fold (Figures 19A-19C, 20A and 20B, 25A; Table 2).
The amount of A(3 detected in the pellet fraction of each sample is
correspondingly lower (data not shown), indicating that the effect of the
chelator
is upon the disassembly of the A(3 aggregate, and not by inhibition of an Ap-
cleaving metalloprotease (such as insulin degrading enzyme cleavage of A(3
reported recently by Dennis Selkoe at the 27'h Annual Meeting for the Society
for
Neuroscience, New Orleans). The extraction of sedimentable Ap into the soluble
phase correlated only with the extraction of zinc from the pellet, and not
with any
other metal assayed (Table 3). Examination of the total amount of protein
released by the treatments revealed that chelation was not merely liberating
more
proteins in a non-specific manner.
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Table 1. Dissociation Constants for Metal Ions of
Various Chelators Used to Extract Human Brain A.
CHELATOR Ca Cu Mg Fe Zn Al Co
EGTA 10.9 17.6 5.3 11.8 12.6 13.9 12.4
EDTA 10.7 18.8 8.9 14.3 16.5 16.5 16.5
Penicillamine 0 i 18.2 0 0 10.2 0 0
TPEN 3.0 20.2 0 14.4 15.4 0 0
Bathophenanthrolin 0 8.8 0 5.6 6.9 0 0
e
Bathocuproine (BC) 0 19.1 0 0 4.1 0 4.2
(Cu')
LogK is illustrated for the chelators, where K= [ML]/[M] [L]. Different
chelators
have greatly differirig affinities for metal ions, as shown. TPEN is
relatively
specific for Zn and,Cu, and has no affinity for Ca and Mg (which are far more
abundant metal ions in tissues). Bathocuproine (BC) has high affinity for zinc
and for cuprous ions. Whereas all the chelators examined have a significant
affinity for zinc, EGTA and EDTA have significant affinities for Ca and Mg.
The ability of chelators to extract Ap from post-mortem brain tissue was
studied in over 40 cases (25 AD, 15 age-matched and young adult controls, all
confrrmed by histopathology). While there is a lot of variation between
samples
as to what is the best concentration of given chelator for the optimum
extraction
of A(3, there are no cases where a chelator does not, at some concentration,
extract
far more Ap than PBS alone.
Figure 19 shows that metal chelators promote the solubilization of Ap
from human brain sarnple horriogenates. Representative curves for three
chelators
(TPEN, EGTA, Bathocuproine) used in extracting the same representative AD
brain sample are shown. 0.5 g of prefrontal cortex was dissected and
homogenized in PBS chelator as indicated. The homogenate was then
centrifuged (100,000 g) and the supernatant removed, and a sample taken for
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western blot assay using anti-A(3 specific antibodies after Tricine PAGE.
Densitometry was performed against synthetic peptide standards. The blots
shown here represent typical results. Similar results were achieved whether or
not protease inhibitors were included in the PBS (extraction was at 4 C).
Furthermore, similar results were achieved when the brain sample was
homogenized in PBS and then pelleted before treated with PBS chelator.
There is also a complex relationship between the dose of the chelator and
the resultant resolubilization of Ap (Figures 19A-C). For the same given
sample,
neither TPEN nor EGTA could increase the extraction of Ap in a does-dependent
manner. Rather, although concentrations of chelators could be very effective
in
the low micromolar range (e.g., TPEN 4 M, Figure 19A), higher concentrations
induced a paradoxical loss of recovery. This kind of response was found in
every
case examined. The extraction of A(3 is abolished by adding exogenous zinc,
but
is enhanced by adding magnesium. Preliminary in vitro data indicate that
whereas Mg has no effect on the precipitation of AD, its presence enhances the
peptide's resolubilization following zinc-induced precipitation. Therefore,
the
"polyphasic" profile of chelator extraction of Ap, with higher concentrations
of
TPEN and EGTA inducing a loss of recovery, may be explained by the chelation
of Mg that is only expected to occur after the chelation of zinc when the
relative
abundance of Mg in the sample, and the relative dissociation constants of TPEN
and EGTA are considered.
In contrast, bathocuproine (BC) exhibits a clear dose-dependent increase
in Ap extraction from human brain, probably due to its relatively high
specificity
for zinc, although an interaction with trace amounts of Cu+ or other metals
not yet
assayed, cannot be excluded.
Western blot analysis of extracts using A(31_4,-specific monoclonals
revealed the presence of abundant Ap 1-42 species. It was observed that z 20%
of
AD cases exhibit clear SDS-resistant Ap dimers in the soluble extract after
treatment with chelators. These dimers are reminiscent of the neurotoxic
A(31_4,
dimers that were extracted by Roher and colleagues from AD-affected brain
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(Roher, A.E., et al., Journal ofBiological Chemistry 271:20631-20635 (1996)).
An estimation of the proportion of total precipitated Ap in the sample was
achieved by extracting the homogenate pellet following centrifugation, into
formic acid, and then performing a western blot on the extract following
neutralization. The, proportion of pelletable Ap that is released by chelation
treatment varies considerably from case to case, from as little as 30% to as
much
as 80%. In the absence of achelator, no more than = 10% of the total
pelletable
Ap is extracted by PBS alone.
One preliminary emerging trend is that samples with a greater proportion
of diffuse or vascular Ap deposit are more likely to have their pelletable AP
resolubilized by chelation treatment. Also, extraction of the tissue
homogenate
overnight with agitation greatly increases the amount of A(3 extracted in the
presence of chelators (compa:red to PBS alone), when compared to briefer
periods
of extraction indicating that the disassembly of Ap deposits by chelation
treatment is a time-dependent reaction and is unlikely to be due to inhibition
of
a protease. A study of braiin cortical tissue from one amyloid-bearing APP
transgenic mouse indicates that, like human brain, homogenization in the
presence of a chelator enhances the extraction of pelletable A(3.
Effects of various chelators on the extraction of A(3 into the supernatant
as a percentage charige from control extractions is summarized below in
Table 2.
Table 2. Effects of Various Chelators Upon Extraction of Ap.
Effect of Chelators (% change from control)
TPEN EGTA BATHOCUP
0.1rnM 2.0mM 0.1mM 2.0mM 0.1mM 2.0mM
Mean (n=6) 182 241 207 46 301 400
+/- SD 79 81 115 48 190 181
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Densitometry of Ap western blots (Figures 19A-19C) was performed for
a series of 6 AD brain samples homogenized in the presence of chelators as
indicated. The mean ( SD) increases in signal, above the signal generated by
PBS extraction alone, are indicated in Table 2. A significantly increased
amount
of chelator-induced Ap resolubilization was achieved by a 16 hour extraction
with agitation in subsequent studies.
Table 3 shows a comparison between pellets of post-centrifugation
homogenates in the presence and absence of a chelator (TPEN).
Table 3. Residual Metals in Pellets of Post-Centrifugation
Homogenatesin the Presence and Absence of Chelator.
METAL Zn Cu Fe Ca Mg Al
PBS 50.7 11.9 227 202 197 44
alone (12.0) (3.5) (69) (69) (94) (111)
mg/kg
(SD)
+TPEN 33.2* 9.8 239 (210) 230 65
mg/kg (9.8) (3.1) (76) (89) (94) (108)
(SD)
Frontal cortex from AD (n=6) and healthy controls (n=4) was homogenized in the
presence and absence of PBS TPEN (0.1 mM). After ultracentrifugation of the
homogenate, the pellets were extracted into concentrated HCI and measured for
metal content by ion coupled plasma - atomic emission spectroscopy (ICP-AES).
Using the same technique, zinc-mediated assembly of A(3 in normal brains
was shown. Figures 20A and 20B show sedimentable Ap deposits in healthy
brain tissue. The effects of chelators in enhancing A(3 extraction from brain
homogenates is also observed in normal tissue. Figure 20A illustrates a
western
blot with anti-A(3 antibody of material extracted from a 27-year-old
individual
with no history of neurological disorder. T= TPEN, E= EGTA, B=
bathocuproine. Bathocuproine is much less effective in extracting AP from
control tissue than from AD tissue. These data are typical of 15 cases.
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As expected, far less total A(3 is present in normal brain samples
compared to AD brain samples, although the content of Ap increases with age.
It is possible that these findings in young adult brains represent the zinc-
mediated
initiation of amyloid formation in deposits that, in youth, are too diffuse to
be
detected by immunohistoche:mistry.
Roher and others have suggested that dimers of A(3 are the toxic
component of amyloid. As shown in Figure 21, dimers appear in response to
chelation in disproportion to the monomeric signal (treatment with PBS alone
does not generate soluble di;mers). This su97ggests that A(3 deposits are
being
dismantled by the chelators iinto SDS-resistant dimeric structural units.
Figure 22 shows that the recovery of total soluble protein is not affected
by the presence of chelators in the homogenization step. The proportionality
of
extracted subfractions, calculated based on total protein as determined by
formic
acid extraction, should not be prone to artifact based on chelator-specific
affects.
Example 6
Differential Effects of Clielatioti of Cerebral A(j Deposits in AD-Affected
Subjects Versus Age-Matched Controls and the Effect of Magnesium
Experiments involviiig extraction of cerebral tissue from AD-affected
subjects and non-A.D, age-matched controls by chelation indicate different
resolubilization responses of'amyloid deposits between the two sample groups
with regard to extraction by specific chelators.
Higher concentrations of chelators with relatively broad specificity (e.g.
EGTA) result in less resolubilization of Ap deposits. Experiments show that
chelation of magnesium negatively affects resolubilzation of A(3 deposits.
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Materia[s and Methods
Cortical tissue was dissected from the frontal poles of frozen AD and age-
matched normal brains for which histopathological and clinical documentation
were provided. AD tissue was selected according to CERAD criteria (Mirra et
al.,
Neurology 41:479-486 (1991)) with particular attention paid to the presence of
neuritic plaques and neurofibrillary tangles. Histological examination of AP
levels
in normal specimens ranged from immunohistochemically undetectable to
substantially present in the form of diffuse plaques.
Suitable quantities of gray matter from each subject were minced to serve
as pools of homogenous tissue. Equal portions (0.5 g unless otherwise
specified)
were homogenized (Ika Ultaturax T-25, Janke and Kunkel, Staufen, Germany) for
3 x 30 second periods at full speed with a 30 second rest between runs in 3 ml
of
ice-cold phosphate-buffered saline (PBS pH 7.4) containing a cocktail of
protease
inhibitors (Biorad, Hercules, CA. - Note: EDTA was not included in the
protease
inhibitor mixture) or in the presence of chelators or metal ions prepared in
PBS.
To obtain the soluble fraction, the homogenates were centrifuged at 100,000 x
g
for 30 min (Beckman J180, Beckman instruments, Fullerton, CA) and the
supernatant collected in 1 ml aliquots and stored on ice or immediately frozen
at
-70 C. In each experiment, all protein was precipitated from 1 ml of
supernatant
from each treatment group using 1:5 ice cold 10% trichloracetic acid and
pelleted
in a bench top microfuge (Heraeus, Osteroder, Germany) at 10,000 x g. The
remaining pellet was frozen at -70 C.
The efficiency of the precipitation was validated by applying the technique
to a sample of whole human serum, diluted 1:10, to which had been added 2 g
of synthetic Ap1_40 or A(3i42 (W. Keck Laboratory, Yale University New Haven,
CT). Protein in the TCA pellet was estimated using the Pierce BCA kit (Pierce,
Rockford, IL). The total A(3 load of unextracted cortex was obtained by
dissolving
0.5 g of grey matter in 2 ml of 90% formic acid, followed by vacuum drying and
neutralization with 30% ammonia.
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Precipitated proteir.i was subjected to SDS polyacrylamide gel
electrophoresis (SI)S-PAGE) on Novex pre-cast 10-20% Tris-Tricine gels
followed by Western transfer onto 0.2 m nitrocellulose membrane (Biorad,
Hercules, CA). Af3 was detected using the W02, G210 or G211 monoclonal
antibodies (Ida, N. et al., J. Biol. Chem. 271:22908 (1996)) in combination
with
HRP-conjugated rabbit anti-mouse IgG (Dako, Denmark), and visualized using
chemiluminescence (ECL, Amersham Life Science, Little Chalfont,
Buckinghamshire, tJK). Each gel included two or more lanes containing known
quantities of synthetic A(3 which served as internal reference standards. Blot
images were captur=ed by a Relisys scanner with transparency adapter (Teco
Information Systems, Taiwan, ROC) and densitometry conducted using the NIII
Image 1.6 program (National Institutes for Health, USA., Modified for PC by
Scion Corporation, :Frederick, MD), calibrated using a step diffusion chart.
For
quantitation of A(3 in brain extracts, the internal reference standards of
synthetic
Ap were utilized to produce standard curves from which values were
interpolated.
In the experiments corresponding to the results shown in Figure 23,
duplicate 0.2 g samples of AD cortical tissue were homogenized and subjected
to
ultracentrifugation as described, but using either I ml or 2 ml of extraction
buffer
(PB S). Protein was precipitated from the entire supernatant and redissolved
in 100
l of sample buffer. Equal volumes of TCA-precipitated protein were subjected
to Tris-Tricine SDS==PAGE and AD was visualized as described above.
In the experiirnents corresponding to the results shown in Figure 24A, 0.2
g specimens of frontal cortex from AD brain were homogenized in the presence
of 2 ml of PBS or varying concentrations of Cu'-+ (Cu(S04),) or Zn2+
(Zn(SO4)2).
Ap in the high speecl supernatant was visualized as described above.
In the experiiments corresponding to the results shown in Figure 24B, 0.2
g specimens of frontal cortex from AD brain were homogenized in the presence
of 2 ml or PBS or 2 mM EGTA. The homogenates were then spun at 100,000 x
g for 30 min and the supernatant discarded. The remaining (metal depleted)
pellets
were rehomogenized in a further 2 ml of either PBS alone, EGTA alone, 2 mM
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Mgz+ (Mg(Cl)2 =6H20) in PBS or 2 mM Ca''+ (CaCI, =2H20) in PBS and the
homogenate subjected to ultracentrifugation. Ap in the soluble fraction was
visualized as described above.
In the experiments corresponding to the results shown in Figures 25A and
25B, frontal cortex from AD (n=6) and age-matched, amyloid-positive (n=5)
subjects were treated with PBS, TPEN, EGTA or BC (0.1 mM and 2 mM) and
soluble Ap assessed as described above.
In the experiments corresponding to the results shown in Figure 26,
representative AD (left panels) and aged-matched control specimens (right
panels)
were prepared as described in PBS or 5mM BC. Identical gels were run and
Western blots were probed with mAbs W02 (raised against residues 5-16,
recognizes Ap1_40 and Ap 1_42), G2 10 (raised against residues 35-40,
recognizes AP ,_
40), or G211 (raised against residues 35-42, recognizes A(31_4,) (Ida, N. et
al., J.
Biol. Chem. 271:22908 (1996).
Results and Discussion
To further explore the involvement of metal ions in the deposition and
architecture of amyloid deposits, the inventors extracted brain tissue from
histologically-confirmed AD-affected subjects and from subjects that were age-
matched to AD-affected subjects but were not clinically demented (age-matched
controls, "AC") in the presence of a variety of chelating agents and metals.
Chelators were selected which displayed high respective affinities for zinc
and/or
copper relative to more abundant metal ions such as calcium and magnesium. See
Table 4 below.
Table 4. Stability constants of metal chelators
Ca Cu Mg Fe Zn Al Co
EGTA 10.86 17.57 5.28 11.8 12.6 13.9 12.35
TPEN 3 20.2 n/a 14.4 15.4 n/a n/a
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BC n/a Cu2+ n/a n/a 4.1 n/a 4.2
6.1
Cu+
19.1
logK10 where K=J=Metal.L:igand]/[Metal][Ligand]. From: NIST database of
critically selected sl:ability constants for metal complexes Version 2.0 1995.
A series of' titratiori curves were prepared to determine the chelator
concentration at which maximal response was obtained. In these experiments,
selected chelators vrere limited to EGTA, TPEN and BC. Figures 19A-C show
interesting dose-dependent patterns of chelator solubilization of A.
It was found that EGTA and TPEN elicited a significant enhancement in
solubilization of A13 in a pattern of response typified by peak values at or
near
0.004 mM and 0.1 mM, anci lower values at concentrations in between. Both
chelators were increasingly imeffective at concentrations over 1 mM, and at 2
mM,
EGTA virtually abolished ttie signal for A. In contrast, BC elicited a typical
concentration-deperident response with no decline in effectiveness in the low
millmolar range even when extended to 20 mM. Total TCA-precipitated protein
in the supernatant was assayed and found to be unaffected by either chelator
kind
or concentration.
Recent findings have demonstrated the presence of neurotoxic dimers in
the soluble (Kuo, Y=-M., el ai:, J. Biol. Chem. 271:4077-81 (1996)) and
insoluble
(Roher, A.E., et al., Journal of Biological Chemistry 271:20631-20635 (1996);
Giulian, D. et al., J. Neurosci., 16:6021-6037 (1996)) fractions of A(3
extracts of
the brains of AD individuals. Figure 21 shows that chelator-promoted
solubilization of A[3 elicits SDS-resistant dimers. Under the preparation
conditions used, SDS-resistant dimers were not generally observed in the
extracts
with PBS alone. Diimers wer-e found to appear, however, in response to
chelator-
promoted solubilization of A.P.
The signal i:or dimeric Ap was frequently disproportionate to that of
monomeric Ap and. the ratio varied with both the type and concentration of
chelator used (Figure 21). In contrast, when synthetic A(31_40 was run under
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identical conditions, the monomer:dimer ratio reflected a predictable and
reproducible concentration-dependent relationship. These data suggest that the
dimers observed in extracts of human brain are predominantly an intermediate
structural unit generated by the dissolution of amyloid, resulting in turn
from the
sequestration of metals by chelating agents.
Figure 24A shows the effect of metals upon the solubility of brain-derived
A. Precipitation of Ap was induced by adding either copper or zinc to
unchelated
extracts. The resulting signal for soluble Ap was attenuated, the threshold
concentration being between 20 and 50 M for copper and between 5 and 20 M
for zinc. At concentrations greater than 100 M solubility was abolished.
Interestingly, at lower concentrations of copper there appears to be a
transitional
stage where Ap is present in the dimeric form prior to complete aggregation,
mirroring the intermediate stage dimers elicited by chelator-mediated
solubilization.
In order to confirm that the chelators were effective at sequestering metals
at the concentrations employed in these experiments, ICP-AES was used to
determine the residual levels of several metals in the post-centrifugation
pellets
retained from the experiment described in Figures 19A-19C. Of the six metals
tested, zinc levels were reduced by TPEN in a dose dependent manner, whereas
EGTA affected calcium and magnesium, particularly at higher concentrations.
See
Table 5 below.
Table 5. Residual Metal Levels in Post-centrifugation (Extracted) Pellets
Mg Al Ca Fe Zn Cu
(mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg)
PBS 202 36 573 411 60 13
0.004 147 22 322 317 28 10
TPEN 0.001 192 34 490 512 42 12
(mM) 0.04 201 22 956 322 22 10
0.1 200 60 708 389 21 12
2.0 200 148 419 376 19 11
5.0 205 16 377 307 17 10
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Mg Al Ca Fe Zn Cu
(mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg)
PBS 223 52 1186 266 45 11
0.004 228 73 795 247 53 11
0.001 237 43 862 281 49 12
EGTA 0.04 247 104 1402 438 71 13
(mM)
0.01 213 61 675 272 54 13
2.0 191 62 519 238 27 13
5.0 168 27 455 230 18 12
0.004 234 33 489 231 47 12
0.001 225 88 1306 275 47 13
BC 0.04 226 38 753 248 56 15
(mM) 0.01 223 73 762 256 49 13
2.0 254 42 1602 271 49 14
5.0 238 38 912 249 53 15
Metal levels were measured in 10 AD specimens treated with 0.1 mM
TPEN. See Table 6 below. The observed increase in extractable Ap correlated
with significant depletion in zinc in every case and to a lesser extent,
copper, when
compared with PBS-treated tissue. No other metal tested was significantly
influenced by treatnient at this concentration.
Table 6. Residual I17etal Levels (Based on 10 AD Specimens)
Zn Cu Fe Ca Mg Al
PBS 50.7 11.9 227 202 197 44
(+/- SEM) (4.9) (1.5) (28.8) (28.3) (39.1) (46.2)
TPEN 33.2 9.8 239 210 230 65
(+/-SEM) 4.1 (1.7) (31.7) (37.0) (39.2) (45.0)
Given the precipitous decline in extractable A(3 observed when employing
high concentrations of TPEN or EGTA (see Figure 19A and 19B), it was
hypothesized that magnesium or calcium might also have a significant role in
the
A(3 solubility equilibrium. Magnesium or calcium added to the homogenization
buffer produced no appreciable alteration in soluble A. However, using an
extract previously depleted of metals by high levels of EGTA, the addition of
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magnesium, and to a lesser extent calcium, led to resolubilization of the
precipitated Ap. Figure 24B shows that Ap solubility in metal-depleted tissue
samples is restored by supplementing with magnesium.
Mindful of the high variability observed between individual subjects, 6 AD
and 5 aged-matched control brains were chosen at random to determine if the
observed phenomena were broadly applicable. These specimens were subjected
to chelation treatment at selected concentrations of 0.1 or 2.0 mM or with PB
S
alone. Figure 25A shows that patterns of chelator-promoted solubilization of
A(3
differ in AD and aged, non-AD tissue. The chelator-promoted solubilization of
A(3 from AD brains represented an increase of up to 7-fold over that seen with
PBS alone; the mean increase for BC being around 4 fold, and that for TPEN
around 2 fold. Treatment with EGTA at 2 mM always produced a diminution in
Ap signal below that observed for the PBS control (See Figure 25B).
The effects observed with non-demented, aged-matched controls were
similar with respect to EGTA and TPEN. However, it is noteworthy that the
effect
of BC was much reduced. In some cases (Figure 25A, lower panel), BC treatment
caused an attenuation in soluble Ap suggesting that the amyloid deposits in AD-
affected brain respond to this chelator in a different fashion than the
deposits
predominating in non-demented elderly brain.
For each subject in the experiments of Figures 25A and 25B, the
extractable Ap was derived and calculated as a proportion of the total pre-
extraction Ap load See Table 7 and 8 below.
Table 7. AD-affected Tissue
AD 1 2 3 4 5 6 X +/-SEM X C/PBS
Total Ap (gg/g) 10.8 77.0 80.3 6.0 14.4 16.8 43.0 14.1
PBS gg/g 0.74 1.39 1.04 0.07 3.0 0.06 1.05 0.44
(% oftotal) (0.1) (1.8) (1.3) (1.1) (2.1) (0.4) (1.2) (0.3)
TPEN 2mM gg/g 0.21 3.40 1.80 5.50 5.00 0.28 2.73 0.85 2.60
(% of total) (0.2) (4.4) (2.25) (9.2) (3.5) (1.75) (4.6) (0.9)
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BC2mM g/g 0.31 5.54 3.62 6.05 6.03 0.54 4.10 0.86 3.90
(% of total) (0.3) (7.2) (4.5) (10.0) (4.2) (3.4) (5.4) (1.2)
Table 8. Age-Matched Control Tissue
AC 1 2 3 4 5 X +/-SEM X C/PBS
Total A(3 ( g/g) 0.7 4.2 2.7 3.2 3.6 2.8 0.60
PBS g/g 0.17 0.13 0.18 0.10 0.66 0.25 0.10
(% of total) (25.0) ;3.1) (6.7) (3.3) (18.3) (11.3) (4.4)
TPEN 2mM g/g 0.22 0.38 0.26 0.09 1.06 0.40 0.17 1.6
(% of total) (32.0) (9.0) (9.7) (3.0) (29.5) (16.7) (5.1)
BC 2mM g/g 0.03 0.24 0.29 0.08 0.98 0.32 0.16 1.28
(% of total) (5) (5.7) (11.0) (2.6) (27.2) (10.3) (4.6)
Total A(3 for AD brains ranged from 6 - 80 g/g wet weight tissue. The
percentage
of Ap extractable (one extr,action/centrifugation sequence) ranged from 0.33 -
10%. The corresponding values for aged-matched control brains were 0.68 -
4.2 g/g total Ap and 2.6 - 29.5% extractable.
In order to further investigate these different responses to chelators,
triplicate blots of AD tissue and control tissue which displayed
cerebrovascular
and diffuse amyloidl deposits were compared using antibodies specific for
AP,_40
and AP1_4,. Figure 26 shows that chelation promotes the solubilization of
API_40
and AP,42 from AD and non-AD tissue. Using 3 different monoclonal antibodies,
attempts to detect whether any particular species of A(3 were selectively
affected
by chelation were performed. Both AP1.40 and AP1_42 were liberated by
chelation,
however the dimeric form of'Ap,_ao in both AD and control tissue predominated.
As reported by Roher et al., Proc. Natl. Acad. Sci., 90:10836-10840 (1993),
the
predominant form af cerebrovascular amyloid is Ap,_42. Somewhat surprisingly,
the dimeric form of this highily aggregating species is absent in the
(control) tissue
in which it is most f'avored.
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It has recently been reported that the zinc-dependent Insulin Degrading
Enzyme (IDE) has significant A(3 cleavage activity (Perez et al., Proc Soc.
for
Neuroscience 20: Abstract 321.13 (1997)). In the experiments presented here,
the
disassembly of amyloid is reflected in the intermediate dimeric species which
result from conversion between soluble and insoluble forms. Thus, simple
inhibition of catalytic enzyme activity cannot account for the observed
increase in
soluble A. However, in the event that a proportion of the chelator-mediated
augmentation of Ap solubilization was due to inhibition of this enzyme,
homogenisations were conducted both in the presence of 1 mM n-ethyl amimide
(NEM), a potent inhibitor of IDE, and at 37 C. No enhancement of Ap signal was
observed above that of PBS alone for NEM , nor was there any diminution of
signal after incubation at 37 C.
Discussion
Metal chelators offer a powerful tool for investigating the role of metals in
the complex environment of the brain, however the strengths of these compounds
may also define their limitations. The broad metal affinities of most
chelators
make them rather a blunt instrument. Attempts were made to sharpen the focus
of the use of chelators by selecting chelators with a range of affinities for
the
metals of interest. These differences may be exploited by appropriate
dilution,
thereby favoring the binding of the relatively high affinity ligand (metal for
which
the chelator has the highest affinity).
The dilution profiles exhibited by EGTA and TPEN (Figure 19A and 19B)
possibly reflect a series of equilibria between different metal ligands and
the
chelators, whereby the influence of low affinity, but abundant, metals is
observed
at high chelator concentrations and that of the high affinity, but more
scarce,
metals predominates at low concentrations of chelator. In the case of Ap
itself, this
explanation is further complicated by the presence of low and high affinity
binding
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sites for zinc (and copper) (Bush, A.I. et al., J. Biol. Chem., 269:12152-
12158
(1994)).
The results shown in Figure 19A and 19B coupled with the hypothesis that
lower affinity metal.s are removed at higher concentrations of chelators
implies a
role for lower affinity metals in meditation of A(3 solubility. Metals such as
Mg
and Ca may be inci-easingly removed at higher chelator concentrations. Figure
24B shows that Mg'', and to a lesser extent, Ca" restore sobulitity to metal
depleted Ap aggregate pellets. This indicates that these metals may function
to
mediate an A(3 solubility equilibrium in vivo.
Bathocuproine with its low affinity for metals other than Cu- is effective
at solubilizing Ap through a dilution range over 3 orders of magnitude, and
interestingly, does riot diminish in effectiveness at the highest levels
tested. The
particular affinity of BC for Cu' has been exploited to demonstrate that in
the
process of binding to APP, Cu'+ is reduced to Cu+ resulting in the liberation
of
potentially destructive free radicals (Multhaup, G., et al., Science 271:1406-
1409
(1996)). It has also been shown that Ap has a similar propensity for reducing
copper with consequent free radical generation (Huang, X., et al., J. Biol.
Chem.
272:26464-26470 (11997)).
Although the predicted reduction in copper in extraction pellets treated
with BC has not been demonstrated, it is possible that the ratio of Cu'-' to
Cu' has
been affected. At this stage, however, the means to evaluate the relative
contributions of divalent anci reduced forms to the total copper content of
such
extraction pellets ari-, not available.
In addition to their primary metal binding characteristics, chelators are a
class of compounds which vary in hydrophobicity and solubility. Their capacity
to infiltrate the highly hydrophobic amyloid deposits may therefore be an
important factor in the disassembly of aggregated Ap. It is also possible that
the
chelators are also acting tc- liberate intracellular stores of A(3 in
vesicular
compartments as metal-bounci aggregates. Preliminary data indicates that this
may
be the case with platelets.
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The variability between subjects is consistent, reflecting the heterogeneity
of the disease in its clinical and histopathological expression. Despite this,
a
consistent pattern of response to the actions of chelators by tissue from both
AD
and non-AD subjects is observed. This universality of the phenomenon of
chelator-mediated solubilization is strongly suggestive that metals are also
involved in the assembly of amyloid deposits in normal individuals, although
the
dissimilar patterns of response suggest that different mechanisms are
operating in
the disease and non-pathological states.
On the basis of the evidence presented here and the in vitro data, it is
proposed that zinc functions in the healthy individual to promote the
reversible
aggregation of Ap, counteracted by magnesium acting to maintain A(3
solubility.
Further, the disease state is characterized by an unregulated interaction with
copper
resulting in the generation of free radicals.
A functional homoeostatic mechanism implies equilibrium between
intracellular copper and zinc (and perhaps other metals) normally present in
trace
amounts, for which Ap has strong affinity, and more abundant metals which bind
less strongly to A(3. Zinc is of particular interest because the anatomical
distribution of zinc correlates with the cortical regions most susceptible to
amyloid
plaque formation (Assaf, S.Y. & Chung, S.H., Nature, 308:734-736 (1984)).
It has recently been demonstrated (Huang, X., et al., J. Biol. Chem.
272:26464-26470 (1997)) that zinc-promoted aggregation of synthetic A(3 is
reversible by the application of EDTA. The tightly-regulated neurocortical
zinc
transport system might provide a physiological parallel for this chelator-
mediated
disaggregation by moving zinc quickly in and out of the intraneuronal spaces.
Copper, while binding less avidly to A(3 than zinc (Bush, A.I., et al., J.
Biol. Chem. 269:12152-12158 (1994)) has greater potential to inflict damage
via
free radical generation, resulting polymers are SDS-resistant (see Example 7,
below). Slight alterations in the transportation and/or metabolism of metals
resulting from age-related deterioration of cellular processes may provide the
environment for a rapid escalation of metal-mediated Ap accretion which
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eventually overwhelms regulatory and clearance mechanisms. In describing a
mechanism for Ap homeostasis this model for amyloid deposition implies a
possible physiological role for A(3 whereby aggregation and disaggregation may
be effected through regulation or cortical metal levels and that the
predominantly
sporadic character of AD reflects individual differences in the brain milieu.
Such
a mechanism by no means rules out other genetic, environmental, inflammatory
or other processes il.nfluencing the progression of the disease. Furthermore,
in
demonstrating the effectiveness of chelators in solubilising amyloid, it is
suggested
herein that suitable zgents of this type are useful for therapeutic or
prophylactic use
in AD.
Example 7
Formation of SDS-Resistant Ajf3 Polymers
The cause fo:rthe permanent deposition ofA(3 in states such as Alzheimer's
Disease (AD) and Down's Syndrome (DS) are unknown, but the extraction of Ap
from the brains of AD and DS patients indicates that there are forms of A(3
that
can be resolubilized in water and run as a monomer on SDS-PAGE (Kuo, Y-M.,
et al., J. Biol. Chern. 271:4077-4081 (1996)), and forms that manifest SDS-,
urea-
and formic acid-resistant polymers on PAGE (Masters, C.L. et al., Proc. Natl.
Acad. Sci. USA 82:4:245-4249 (1985); Dyrks, T., et al., J. Biol. Chem.
267:18210-
18217 (1992); Roher, A.E., et al., Journal of Biological Chemistry 271:20631-
20635 (1996). Thus, the exti-action of SDS-resistant A~ polymers from plaques
implicates polymerization as a pathogenic mechanism that promotes the
formation
of AD amyloid.
The exact mechanism underlying the formation of SDS-resistant polymeric
A(3 species remains unresolved. Recently, Huang, X., et al. have shown that
A(3
reduces both Cu'-+ and Fe3+ (Huang, X., et al., J. Biol. Chem. 272:26464-26470
(1997)), providing a mechanism whereby a highly reactive species could promote
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the modification of proteins via an oxidative mechanism. Here, the inventors
tested the ability of Cu'+ and Fe3+ to promote SDS-resistant Ap
polymerization.
Materials and Methods
Human A(3, _40 peptide was synthesized, purified and characterized as
described above. Rat A(3, _40 was obtained from Quality Control Biochemicals,
Inc.
(Hopkinton, MA). Peptides were analyzed and stock solutions prepared as
described above.
As above, electronic images captured using the Fluoro-S Image Analysis
System (Bio-Rad, Hercules, CA) were analyzed using Multi-Analyst Software
(Bio-Rad, Hercules, CA). This chemiluminescent image analysis system is linear
over 2 orders of magnitude and has comparable sensitivity to film.
Human AD derived SDS-resistant polymers were solublized in formic acid,
and then dialyzed with 5 changes of 100 mM ammonium bicarbonate, pH 7.5. The
solublized peptide was then used for subsequent chelation experiments.
Results and Discussion
The generation of SDS-resistant Ap polymers by metal ions was tested by
incubating Cu'-+ (30,uM) or Zn2+ (30 M) at pH 6.6, 7.4 and 9.0 with AP, _40.
As
shown in Figure 9, Western blot analysis of samples incubated with Cu'+ and
run
under SDS denaturing and P-mercaptoethanol reducing conditions revealed an
increase in dimeric, trimeric and higher oligomeric A(3 species over time. The
dimer and trimer had molecular weights of approximately 8.5 kD and 13.0 kD,
respectively. Image analysis indicated 42% and 9% conversion of the monomer
to dimer and trimer, respectively, in samples incubated at pH 7.4 after 5 d.
The
conversion of monomer to the dimer and trimer was 29% and 2%, respectively, at
pH 6.6 after 5 d.
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In contrast, changes in [H+] alone did not induce SDS-resistant AV40
polymerization. Less than 4% of the peptide was converted to the SDS-resistant
dimer after 5 d in samples ir,icubated at pH 6.6, 7.4 or 9.0, most likely as a
result
of contaminating C'u- in the buffer and A(3 solutions. Cu-+ contamination of
chelex-treated PBS was up to 0.5 4M as determined by ion coupled plasma-atomic
emission spectroscopy (ICP--AES). Although Zn'-+ induces rapid aggregation of
Ap 1_40 (Bush, A.L, etal., J. Biol. Chem. 268:16109 (1993); Bush, A.I., et
al., J.
Biol. Chem. 269:12 a 52 (1994); Bush, A.I., et al., Science 265:1464-1467
(1994);
Bush, A.I., et al., Science .268:1921-1922 (1995); Atwood et al., submitted;
Huang, X. et al., J. l3iol. Chem. 272:26464-26470 (1997)), it did not induce
SDS-
resistant A(3 polymerization (Figure 9) as previously reported (Bush, A.I., et
al.,
Science 268:1921-1922 (1995)).
A(31 _4, is the predominant species found in amyloid plaques (Masters, C.L.
et al., Proc. Natl. Acad. Sci. USA 82: 4245 (1985); Murphy, G.M., et al., Am.
J.
Pathol. 144:1082-1088 (1994); Mak, K., el al., Brain Res. 667:138-142 (1994);
Iwatsubo, T., et al., Ann. Neurol. 37:294-299 (1995); Mann et al., Ann.
Neurol.
40:149-156 (1996)).. Therefore, the ability of AP1_40 and Ap,_4, to form SDS-
resistant polymers mias compared.
In contrast to Cu'+-induced SDS-resistant A(3 1 -40 polymerization over days,
SDS-resisitant Ap ,_,,2 polyme;rization occurred within minutes in the
presence of
Cu2+ (Figure 27A). Unlike A[31_40 where Cu'-+ induces the formation of a SDS-
resistant dimeric species first, A(31_4, initially forms an apparent trimer
species in
the presence of Cu''+. Over time, dimeric and higher polymeric species also
appear
in A(31_4, incubations with Cu:'' at both pH 7.4 and 6.6. The greater Cu2y
induced
A[31_42 polymerization observed at pH 6.6 compared with pH 7.4 in samples
incubated for 30 miri. was reversed after 5 d. At pH 6.6, both A[31_40 and
A[3,_42
exist in an aggregated form within minutes. Therefore, the formation of these
polymeric species occurs within A[3 aggregates and the formation of SDS-
resistant
A(3 polymers is independent of aggregation state (see below). Similar results
were
obtained using the rnonoclonal antibody 4G8.
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Since redox active Fe (Smith, M.A., et al., Proc. Natl. Acad. Sci. USA
94:9866 (1997)) and ferritin (Grudke-Iqbal, I., et al., Acta Neuropathol.
81:105
(1990)) are found in amyloid lesions, experiments were performed to determine
if Fe3+ could induce SDS-resistant polymerization of A(31-40 and AR,-42
(Figure
27A). Fe3+ did not induce A(31-40 polymerization above background levels with
either peptide. The small increase in polymeric A(31 _40 and A[31 _40 in
samples with
no metal ions reflects a small contaminating concentration of Cuz'.
The formation of amyloid plaques is not a feature of aged rats (Johnstone,
E.M., et al., Mol. Brain Res. 10:229 (1991); Shivers et al. (1988)). To test
whether
rat A(3,-ao would form SDS-resistant Ap polymers, rat A(3,_4o was incubated
with
Cu'-' and Fe3+ at pH 7.4 and 6.6 (Figure 27B). Neither metal ion induced SDS-
resistant A[3 polymers ( Huang, X. et al., J. Biol. Chem. 272:26464-26470
(1997)).
The binding and reduction of Cu'' by rat AP 1_40 is markedly decreased
compared
to that of human A[31_40 (Huang, X. et al., J. Biol. Chem. 272:26464-26470
(1997)). This result suggests that the generation of SDS-resistant Ap polymers
is
dependent upon the binding and reduction of Cu2+ by A.
Tests were performed to determine the concentration of Cu'-' required to
induce the formation of SDS-resistant A(31_40 and Ap,_4, polymers. A(3,_40 and
Ap1_4, were incubated with different [Cu2+] (0-30 uM) at pH 7.4 and 6.6 and
the
samples analyzed by Western blot and the signal quantitated using the Fluoro-S
Image Analysis System (Bio-Rad, Hercules, CA) as previously described.
At pH 7.4, the increase in polymerization of A(31_40 was barely detectable
as [Cu2+] was increased from 0.5 to 1 M , but under mildly acidic conditions
(pH
6.6), SDS-resistant polymerization could be detected (over 3-fold increase in
dimerization)(Table 9A).
Table 9A. CuZ+- Induced SDS-Resistant Polymers of A[i,-40
pH 7.4
[Cuz+] Monomer Dimer Trimer Tetramer Pentamer
0 96.8 3.2 <0.1 0 0
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0.5 94.8 4.9 0.3 0 0
1 93.6 5.9 0.6 0 0
84.3 14.2 1.5 0 0
85.2 13.2 1.6 0 0
5 30 76.2 19.1 4.7 0 0
pH 6.6
[Cu'+] Monomer Dimer Trimer Tetramer Pentamer
0 97.9 2.1 <0.1 0 0
0.5 97.5 2.2 0.2 0 0
1 92.6 7.3 0.1 0 0
10 5 90.1 9.8 0.1 0 0
10 79.4 16.1 4.5 0 0
30 74.5 13.2 12.2 0 0
A similar Cu2' concentration and pH dependent increase in SDS-resistant
Ap,_4, polymers also was observed (Table 9B), but SDS-resistant polymerization
occurred at much lower [Cuz+].
Table 9B. CuZ+- Induced SDS-resistant Polymers of A[31_42
pH 7.4
[Cu2+] Monomer Dimer Trimer Tetramer Pentamer
0 76.61 0 16.0 5.5 1.9
0.5 70.7 0 20.5 6.2 2.5
1 64.9 0 23.6 7.4 4.0
5 56.1 0 31.8 8.7 4.1
10 55.1 0 30.3 10.3 4.3
57.1 0 31.1 8.3 4.2
pH 6.6
[Cu2,] Monomer Dimer Trimer Tetramer Pentamer
25 0 61.0 0 27.3 8.6 3.8
0.5 52.1 0 33.8 12.0 3.0
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pH 7.4
[Cu'-+] Monomer Dimer Trimer Tetramer Pentamer
59.6 0 30.0 7.1 3.2
52.3 0 31.7 13.6 2.2
A[31 _40 polymerization was not detected with increasing Fe3 ' concentrations
at any pH. Therefore, of the metal ions known to interact with A(3, only Cu'',
5 whose ability to aggregate and bind Cu'T under mildly acidic conditions is
enhanced, is capable of inducing SDS-resistant A(3 polymerization.
Oxygen radical mediated chemical attack has been correlated with an
increase in protein and free carbonyls (Smith, C.D., et al., Proc. Natl. Acad.
Sci.
USA 88:10540 (1991); Hensley, K., et al., J. Neurochem. 65:2146 (1995); Smith,
10 M.A., et al., Nature 382:120 (1996)) and peroxynitrite-mediated protein
nitration
(Good, P.F., et al., Am. J. Pathol. 149:21 (1996); Smith, M.A., et al., Proc.
Natl.
Acad. Sci. USA 94:9866 (1997)).
A(3 is capable of reducing Cu-2' and H207 is produced in solutions
containing A(3 and Cu'' or Fe3+ (Huang, X. et al., J. Biol. Chem. 272:26464-
26470
(1997)). As shown above, the generation of SDS-resistant Ap polymers in the
order Ap , _4, >> A[3, _40 >> rat Ap , _40 in the presence of Cu'-- correlates
well with
the generation of Cu' and reactive oxygen species (ROS; OH , H,O, and O2:
Huang, X.. et al., J. Biol. Chem. 2 72:26464-26470 (1997)) by each peptide.
The increased generation of SDS-resistant A[3 polymers in the presence of
Cu2+ compared to Fe3+ also was correlated with the generation of the reduced
metal
ions, respectively (Huang, X. et al., J. Biol. Chem. 272:26464-26470 (1997)).
The
increase in SDS-resistant A[3 polymerization seen under mildly acidic
conditions
may be a result of the higher [H+] driving the production of H,Oz dismutated
from
O; with the subsequent generation of OH' via Fenton-like chemistry inducing a
modification of A[3 that results in SDS-resistant A(3 polymers (see Figure 12
showing a schematic of the proposed mechanism of Ap-mediated reduced
metal/ROS production).
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To confirm vvhether ROS were involved in the generation of SDS-resistant
polymers, experiments were performed to determine whether Cu in the presence
or absence of H20, could promote AR polymerization (Figure 28A). A similar
level of AP, _4, polymerization was observed in the presence of Cu'' or Cu+,
indicating that the reduced inetal ion alone was not capable of increasing AP
polymerization. Likewise, polymerization of AP1_42 in the presence of H,O, was
low and equivalent to control levels. However, the addition of Cu2+ or Cu' to
A(3
in the presence of H OZ induced a similar, marked increase in dimers, trimers
and
tetramers within 1 hour. After 1 day, higher molecular weight polymers (> 18
kD)
were generated (from the oligomers), with a subsequent reduction in the levels
of
monomer, dimer, trirner and tetramer only with the coincubation of H,O, and
Cu'+.
Both the reduced ancl oxidized forms of Cu produced similar levels of
polymerization in the presence of H202. In contrast, neither Fe'+ nor Fe''+
induced
as much polymerization as Cu-' in the presence of H20, after 1 day incubation
(Figures 28A and 28B). Since Fe3+ is not reduced as efficiently as Cu2+ by AP
(Huang, X., et al., J. Biol. Chem. 272:26464-26470 (1997)), and Cu+ is rapidly
converted to Cu2' in solution, these results suggest that the reduction
reaction is
required for the polymerization reaction to proceed.
It was confir.med that the reduction of Cu'-+ was required for generating
SDS-resistant Ap polymerization by incubating AP,_q, and Cu2+ with and without
bathocupoinedisulfonic acid i;BC), a Cu' specific chelator (Figure 28C). There
was a marked decrease in polymerization, indicating that Cu+ generation was
crucial for the polymerization of A. It is possible that the decreased
polymerization may be due to chelation of Cu2- by BC, however given the low
binding affinity of 13C for Cu'-' compared with Ap, it seems likely that the
chelation of Cu' by BC prevents it froni inducing SDS-resistant A(3
polymerization. The:refore, Ap may undergo a hydroxyl radical modification
that
promotes its assembly into SI)S-resistant polymers.
If H,Oz is required for the polymerization reaction under physiological
conditions, the removal of H202 and it's precursors O, and OZ (Huang, X., et
al.,
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J. Biol. Chem. 272:26464-26470 (1997)) should decrease SDS-resistant
polymerization. To confirm that H,02 generated in the presence of AP and Cu'-+
was required for the polymerization reaction, Ap, _47 was incubated with or
without
Cuz+ in the presence of TCEP (Figure 29A). TCEP significantly reduced the
level
of polymerization in samples with and without Cu2+ over 3 days. This indicates
that the generation of H,O, is required for the polymerization of AP.
To confirm that the generation of O; was required for SDS-resistant A(3
polymerization, A(3, _4, was incubated with and without Cu'-+ at pH 7.4 and
6.6
under argon in order to decrease the reduction of molecular O, (Figure 29B).
Argon-purging of the solution markedly decreased AP1_42 polymerization under
each condition, indicating that the generation of ROS is required for the
polymerization of A(3.
Taken together, these results indicate that polymerization occurs as a result
of Haber-Weiss chemistry where the continual reduction of Cu2+ by AP provides
a species for the reduction of molecular O, and the subsequent generation of
02,
H,O, and OH-. The binding and reduction of Cu'-+ by Ap is supported by the
finding that the incubation of Fe3+, H2O2 and ascorbic acid with AP, _40
(Figure
29A) and Ap , _47 does not induce SDS-resistant polymerization equivalent to
Cu2+
with H,O, alone. Since ascorbic acid effectively reduces Fe3+, the reduction
of a
metal ion that is not bound to A(3 is insufficient to induce significant SDS-
resistant
polymerization.
The formation of SDS-resistant polymers of A(3 by this metal-catalyzed
oxidative mechanism strongly suggested that a chemical modification to the
peptide backbone allows the formation of the polymer species. To test if the
SDS-
resistant polymers were covalently linked, SDS-resistant polymers generated by
incubating Ap 1_42 with Cu2+ at pH 7 4 and 6.6, or Ap ,_42 with Cu'+ plus H20,
were
subjected to treatment with urea (Figure 30A) and guanidine HCI, chaotrophic
agents known to disrupt H-bonding. Urea and guanidine HCl did not disrupt the
SDS-resistant polymers at 4.5 M, and only slightly at 9M, suggesting that the
SDS-
resistant polymers were held together by high-affinity bonds, but not hydrogen
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bonding alone. HPL C-MS analyses, however, confirmed no covalent modification
of the peptide and no evidence of covalent crosslinking.
Since covalent and/ar hydrogen bonding were not involved in polymer
formation, experiments were performed to detemine whether Cu'' coordination of
the complex by ionic interactions was allowing for the formation of the SDS-
resistant polymer species. To disrupt these ionic interactions, different
chelating
agents were added to a solution containing Cu'+-induced A(3, _40 or A(3, _4,
SDS-
resistant polymers generated at pH 7.4 (Figures 30B and 30C; TETA,
tetraethylenediamine; EDTA, ethylenediaminetetra acetic acid; DTPA,
diethylenetriaminopenta acetic acid; CDTA, trans-1,2-diaminocyclohexanetetra
acetic acid; NTA, nitrilotriacetic acid).
All chelators significantly reduced the amount of Ap, _40 or A(3, _4, SDS-
resistant polymers. EDTA was less effective in destabilizing the polymers,
possibly due to its larger molecular mass, and lower affinity for Cu'-+. EDTA
reduced the amouni. of A(3,._ao polymers, but increased the amount of AP, _40
polymers at pH 7.4. This may be due to the fact that EDTA can enhance the
redox
potential of Cu under certain conditions.
Cu''-induced. SDS-resistant polymers generated at pH 6.6 were also
disrupted with chelation treatment to a similar extent. These results suggest
that
the chelation of Cu'' away iErom AD results in the disruption of the polymer
complex and the release of' monomer species. Thus, there is an absolute
requirement for metal ions in the stabilization of the SDS-resistant polymer
complex.
The SDS-resistant polymers generated with Cu'' are similar to those
extracted from post-mortem AD brains (Rolier, A.E., et al., Journal
ofBiological
Chemistry 2 71:20631-20635 (1996)). To determine if these human oligomeric A(3
species could be disrupted by imetal chelators, TETA and BC were incubated
with
Ap oligomers extracted from fLuman brain. Figure 30E shows that both TETA and
BC significantly increased the amount of monomer A(3 in samples treated with
these chelators. Although the iincrease in the amount of monomer was small,
these
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results suggest that human oligomeric Ap species are partially held together
with
metal ions. Importantly, this result indicates the potential of chelation
therapy as
a means of reducing amyloidosis.
To examine whether conformational changes could disrupt the SDS-
resistant polymers, solutions of SDS-resistant A(3, _,, polymers in the
presence or
absence of Cuz+ were incubated with the a-helical promoting solvent system
DMSO/HFIP, or under acidic conditions (pH 1) (Figure 30D). These conditions
reduced the amount of polymer compared to untreated controls at both pH 7.4
and
6.6, indicating that an alteration in the conformation of Ap,_a, to the a-
helical
conformation could disrupt the strong AP-Cu'' ionic interactions. This
provides
indirect evidence that the polymer structures are likely to be in the more
thermodynamically favorable P-sheet conformation, such as those found in
neuritic
plaques.
SDS-resistant Ap polymers, such as that found in the AD-affected brain,
are likely to be more 'resilient to proteolytic degradation and may explain
the
permanent deposition of Ap in amyloid plaques. Incubation of SDS-resistant Ap
polymers with proteinase K resulted in complete degradation of both monomer
and
oligomeric A(3 species. Since protease treatment is incapable of digesting
hard
core amyloid, covalent crosslinking of the peptide following its deposition
may
occur over time that prevents proteolytic digestion. This may explain the
limited
disruption of human SDS-resistant Ap oligomers compared to the Cu-mediated
SDS-resistant polymers generated in vitro.
Soluble Ap 1 _40 and Ap 1 _42 both exist in phosphate buffered saline as non-
covalent dimers (Huang, X., et al., J. Biol. Chem. 272:26464-26470 (1997); and
unpublished observations). Disruption of ionic and hydrogen bonding of Ap in
the
soluble and aggregated forms (pH or Zn2+) by the ionic detergent SDS results
in
the complete dissociation of Ap into the monomer species as detected on SDS-
PAGE (Figures 9, 32-34). The formation of SDS-resistant polymers of AP over
time in the presence of Cu2+ (Figures 9, 27A-27B, 28A-28C) suggests that
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conformational oi- structural alterations allow for the formation of a
thermodynamically more stable complex.
Although no covalent crosslinking between peptides was detected, it is
possible that a covalent modification(s) takes place within the peptide
backbone
that allows for a high affini7ty association to form between the peptide and
Cu2+.
Thus, a chemical modification to the peptide may increase the affinity of the
polymer for Cuz+ and the formation of a stable complex. Alternatively, the
requirement for molecular oxygen suggests that Cu may be coordinated by oxygen
or ROS in the formation of SDS-resistant polymers.
The formation of SDS-resistant polymers was dependent upon the binding
and reduction of Cuz+. The binding of Cu'-j to A(3 was confirmed by the
detection
of Cu''' in both the monomer and dimer following SDS-PAGE. The [Cu'-] of
PVDF membrane containinl; the immobilized peptide species was measured by
ICP-AES (unpublished observations; Huang, X., et al., J. Biol. Chem. 272:26464-
26470 (1997)) and correlated with the generation of SDS-resistant polymers for
each species.
CuZ+ coordiniation between A[3 molecules was required in order to maintain
the structure since chelatioii treatment disrupted the in vitro generated SDS-
resistant polymer (Figures 30B-30E). Human SDS-resistant Ap polymers also
were disrupted with the Cu+-specific chelator BC indicating Cu coordination in
the
stabilization of these structures (Figure 30E). Together with the fact that Cu-
specific chelators can extract more SDS-resistant A[3 polymers from AD brains
in
aqueous buffer (see Example 6), these results implicate Cu'-+ in the
generation of
SDS-resistant polyniers in vivo.
Fe3 did not induce the formation of SDS-resistant polymers in vitro
(Figures 27A) as previously reported except in the presence of excess H,O, or
ascorbic acid as previously reported (Dyrks, T., et al., J. Biol. Chem.
267:18210-
18217 (1992); and data not shown). Dyrks, T., et al. did, however observe
significant increases in SDS-resistant polymerization with metal-catalyzed
oxidation systems (:Fe-hemin, Fe-hemoglobin or Fe-EDTA) in the presence of
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H20Z. The A(3, _4, used in their experiments was likely to be Cu-bound as it
was
extracted from a wheat germ expression system and already was present as SDS-
resistant oligomers. Thus, it is possible that Cu-bound Ap used in these
experiments contributed to the increased SDS-resistant polymerization observed
in the Fe-catalyzed oxidation systems. Although Fe3+ is reduced by A(3 (Huang,
X., et al., J. Biol. Chem. 272:26464-26470 (1997)), it is unable to
effectively
coordinate the complex like Cu (Figure 28B).
Fe'LL is found in much higher concentrations in the brains of AD patients
compared with age-matched controls (Ehmann, W.D., et al., Neurotoxicol. 7: 197-
206 (1986); Dedman, D.J., et al., Biochem. J. 287:509-514 (1992); Joshi, J.G.,
et
al., Environ. Health Perspect. 102:207-213 (1994)). This is partly
attributable to
the increased ferritin rich microglia and oligodendrocytes that localize to
amyloid
plaques (Grudke-Iqbal, I., et al., Acta Neuropathol. 81:105 (1990); Conner,
J.R.,
et al., J. Neurosci. Res. 31:75-83 (1992); Sadowki, M., et al., Alzheimer's
Res.
1:71-76 (1995)).
Recently, redox active Fe was localized to amyloid lesions (Smith, M.A.,
et al., Proc. Natl. Acad. Sci. USA 94:9866 (1997)). While Fe is normally
sequestered by metalloproteins, this localization of ferritin-rich cells
around
amyloid deposits, and the very high concentrations of iron in amyloid plaques
(Conner, J.R., et al., J. Neurosci. Res. 31:75-83 (1992); Markesbery, W.R. and
Ehmann, W.D., "Brain trace elements in Alzheimer's disease," in Terry, R.D.,
et
al., eds., Alzheimer Disease, Raven Press, New York (1994), pp. 353-368)
suggests that reduced Fe released from ferritin and transferrin under mildly
acidic
conditions could be available for Fenton chemistry and the formation of SDS-
resistant polymers. However, even in the presence of a Fe-ROS generating
system
(ascorbic acid, H202 and Fe) the generation of SDS-resistant Ap polymers in
vitro
was low (Figure 29A) and may be explained by Cu'+ contamination of the
buffers.
Interestingly, diffuse plaques, which may represent the earliest stages of
plaque formation, are relatively free of ferritin-rich cells (Ohgami, T., et
al., Acta
Neuropathol 81:242-247 (1991)). Therefore, the accretion of iron in amyloid
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plaques may be a secondary response to the neurodegeneration caused by the
reduction of Cu"' and the generation of ROS by A(3 and the formation of
neurotoxic SDS-resistant Ap polymers.
Structural differences between AP1_40 and A(3,_42 may allow for the
formation of a thermodynamically stable dimer in the case of Ap , _w and
trimer in
the case of Ap, _42 (Figures 27A , 30B and 30C). Irnespective of this, the
increased
generation of SDS-resistant polymers by A(31_42 compared to AP,_,,o is most
likely
explained by the increased ability of A(i, _42 to reduce Cu and generate ROS.
Since
the addition of exogenous HZO, to A(3, _12 increases the generation ofdimeric
SDS-
resistant polymers of AR_42 (Figures 28A and 28B), this dimeric species may be
an integral intermediate in the formation of the SDS-resistant Ap trimers, and
may
explain why Ap 1 _40 polymerization occurs more slowly.
AD Pathology
The present invention indicates that the manipulation of the brain biometal
environment with specific agents acting directly (e.g., chelators and
antioxidants)
or indirectly (e.g., by improving cerebral energy metabolism) provides a means
for
therapeutic intervention in the prevention and treatment of AlZheimer's
disease.
Those skilled in the art will appreciate that the invention described herein
is susceptible to variations and modifications other than those specifically
described. It is to be understood that the invention includes all such
variations and
modifications. The invention also includes all of the steps, features,
compositions
and compounds referred to or indicated in this specification, individually or
collectively, and any and all combinations of any two or more of said steps or
features.
