U.S. patent application number 11/578401 was filed with the patent office on 2008-11-06 for methods and compositions for inhibiting abad/abeta protein interaction.
This patent application is currently assigned to THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YOURK. Invention is credited to Joyce W. Lustbader, David M. Stern, Hao Wu, Shi Du Yan.
Application Number | 20080274975 11/578401 |
Document ID | / |
Family ID | 35463248 |
Filed Date | 2008-11-06 |
United States Patent
Application |
20080274975 |
Kind Code |
A1 |
Yan; Shi Du ; et
al. |
November 6, 2008 |
Methods and Compositions for Inhibiting Abad/Abeta Protein
Interaction
Abstract
This invention provides methods, compositions and articles of
manufacture for inhibiting binding between A.beta. protein and ABAD
in cells. Uses of this invention include, for example, treating
Alzheimer's disease; reducing free radical generation, DNA
fragmentation, and cytochrome C release in cells; and preserving
cell viability by preventing LDH release from a cell.
Inventors: |
Yan; Shi Du; (Tenafly,
NJ) ; Stern; David M.; (Augusta, GA) ;
Lustbader; Joyce W.; (Tenafly, NJ) ; Wu; Hao;
(New York, NY) |
Correspondence
Address: |
COOPER & DUNHAM, LLP
1185 AVENUE OF THE AMERICAS
NEW YORK
NY
10036
US
|
Assignee: |
THE TRUSTEES OF COLUMBIA UNIVERSITY
IN THE CITY OF NEW YOURK
New York
NY
CORNELL RESEARCH FOUNDATION, INC.
Ithaca
NY
|
Family ID: |
35463248 |
Appl. No.: |
11/578401 |
Filed: |
April 12, 2005 |
PCT Filed: |
April 12, 2005 |
PCT NO: |
PCT/US2005/012482 |
371 Date: |
January 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60561859 |
Apr 12, 2004 |
|
|
|
Current U.S.
Class: |
514/9.7 ;
435/375; 514/20.1; 530/326 |
Current CPC
Class: |
A61P 25/28 20180101;
A61K 38/00 20130101; C07K 14/4711 20130101 |
Class at
Publication: |
514/13 ; 530/326;
435/375 |
International
Class: |
A61K 38/10 20060101
A61K038/10; C07K 7/08 20060101 C07K007/08; C12N 5/00 20060101
C12N005/00; A61P 25/28 20060101 A61P025/28 |
Claims
1. A polypeptide consisting of a portion of ABAD, wherein the
portion of ABAD binds to A.beta. protein and comprises the sequence
of amino acid residues 94-114 of ABAD.
2. The polypeptide of claim 1, wherein the ABAD is human ABAD.
3. A composition of matter comprising (a) a pharmaceutical carrier,
and (b) the polypeptide of claim 1.
4. The composition of matter of claim 3, wherein the composition
binds to A.beta. protein.
5. The composition of claim 4, wherein the ABAD is human ABAD.
6-9. (canceled)
10. A polypeptide consisting of a portion of A.beta. protein,
wherein the portion of A.beta. protein binds to ABAD and comprises
the sequence of amino acid residues 1-20 of A.beta. protein.
11. The polypeptide of claim 10, wherein the A.beta. protein is
human A.beta. protein.
12. A composition of matter comprising (a) a pharmaceutical
carrier, and (b) the polypeptide of claim 10.
13. The composition of matter of claim 12, wherein the composition
binds to ABAD.
14. The composition of claim 13, wherein the A.beta.protein is
human A.beta. protein.
15-18. (canceled)
19. A method for treating a subject afflicted with Alzheimer's
disease comprising administering to the subject a therapeutically
effective amount of the polypeptide of claim 1.
20. The method of claim 19, wherein the subject is human.
21-25. (canceled)
26. A method for reducing free radical generation in a cell
comprising introducing into the cell an agent that inhibits binding
between A.beta. protein and ABAD.
27. The method of claim 26, wherein the cell is a neuronal
cell.
28. The method of claim 26, wherein the agent is a polypeptide
consisting of a portion of ABAD, wherein the portion of ABAD binds
to A.beta. protein and comprises the sequence of amino acid
residues 94-114 of ABAD.
29. The method of claim 26, wherein the agent is a composition of
matter comprising a polypeptide consisting of a portion of ABAD
comprising the sequence of amino acid residues 94-114 of ABAD,
wherein the composition binds to A.beta. protein.
30. The method of claim 26, wherein the agent is a polypeptide
consisting of a portion of A.beta. protein, wherein the portion of
A.beta. protein binds to ABAD and comprises the sequence of amino
acid residues 1-20 of A.beta. protein.
30. The method of claim 26, wherein the agent is a polypeptide
consisting of a portion of A.beta. protein, wherein the portion of
A.beta. protein binds to ABAD and comprises the sequence of amino
acid residues 1-20 of A.beta. protein.
31. The method of claim 26, wherein the agent is a composition of
matter comprising a polypeptide consisting of a portion of A.beta.
protein comprising the sequence of amino acid residues 1-20 of
A.beta. protein, wherein the composition binds to ABAD.
32-57. (canceled)
Description
[0001] This application claims the benefit of copending U.S.
Provisional Application No. 60/561,859, filed Apr. 12, 2004, the
contents of which are hereby incorporated by reference.
[0002] Throughout this application, various publications are
referenced. Full citations for these publications may be found
immediately preceding the claims. The disclosures of these
publications are hereby incorporated by reference into this
application in order to more fully describe the state of the art as
of the date of the invention described and claimed herein.
BACKGROUND OF THE INVENTION
[0003] Human A.beta.-binding alcohol dehydrogenase ("ABAD," also
known as "ERAB" and "HSD-10") was the only protein identified from
four positive clones that bound A.beta. protein (also referred to
as "A.beta.") in a yeast two-hybrid screen against human brain and
HeLa cDNA libraries (1, 2). Biochemical characterization has
established that the interaction between ABAD and A.beta. is highly
specific and starts to occur at nanomolar concentrations. At
micromolar concentrations, A.beta., likely in its oligomeric form,
inhibits ABAD enzymatic activity (1, 3, 4). ABAD appears to have an
essential physiological role in mitochondria (1, 3), and mutational
inactivation of Drosophilia ABAD (scully) resulted in a lethal
phenotype (5). ABAD is up-regulated in affected neurons in AD (1)
(FIG. 5) and co-expression of ABAD with mutant amyloid precursor
protein (mAPP) exacerbates A.beta.-induced cellular oxidant stress
and cell death (1, 3).
SUMMARY OF THE INVENTION
[0004] This invention provides a polypeptide consisting of a
portion of ABAD, wherein the portion of ABAD binds to A.beta.
protein and comprises the sequence of amino acid residues 94-114 of
ABAD.
[0005] This invention further provides a composition of matter
comprising (a) a pharmaceutical carrier, and (b) a polypeptide
consisting of a portion of ABAD, wherein the portion of ABAD binds
to A.beta. protein and comprises the sequence of amino acid
residues 94-114 of ABAD.
[0006] This invention further provides a composition of matter
comprising a polypeptide consisting of a portion of ABAD comprising
the sequence of amino acid residues 94-114 of ABAD, wherein the
composition binds to A.beta. protein.
[0007] This invention further provides a composition of matter
comprising (a) a Tat protein operatively affixed to (b) a
polypeptide consisting of a portion of ABAD comprising the sequence
of amino acid residues 94-114 of ABAD, wherein the composition
binds to A.beta. protein.
[0008] This invention further provides a polypeptide consisting of
a portion of A.beta. protein, wherein the portion of A.beta.
protein binds to ABAD and comprises the sequence of amino acid
residues 1-20 of A.beta. protein.
[0009] This invention further provides a composition of matter
comprising (a) a pharmaceutical carrier, and (b) a polypeptide
consisting of a portion of A.beta. protein, wherein the portion of
A.beta. protein binds to ABAD and comprises the sequence of amino
acid residues 1-20 of A.beta. protein.
[0010] This invention further provides a composition of matter
comprising a polypeptide consisting of a portion of A.beta.protein
comprising the sequence of amino acid residues 1-20 of A.beta.
protein, wherein the composition binds to ABAD.
[0011] This invention further provides a composition of matter
comprising (a) a Tat protein operatively affixed to (b) a
polypeptide consisting of a portion of A.beta. protein comprising
the sequence of amino acid residues 1-20 of A.beta. protein,
wherein the composition binds to ABAD.
[0012] This invention further provides a method for treating a
subject afflicted with Alzheimer's disease comprising administering
to the subject a therapeutically effective amount of an agent that
inhibits binding between A.beta. protein and ABAD in the subject's
cells.
[0013] This invention further provides a method for reducing free
radical generation in a cell comprising introducing into the cell
an agent that inhibits binding between A.beta. protein and
ABAD.
[0014] This invention further provides a method for reducing DNA
fragmentation in the cytosol of a cell comprising delivering into
the cell an agent that inhibits binding between A.beta. protein and
ABAD.
[0015] This invention further provides a method for reducing
cytochrome c release from mitochondria in a cell comprising
introducing into the cell an agent that inhibits binding between
A.beta. protein and ABAD.
[0016] This invention further provides a method for preserving cell
viability by reducing LDH release from a cell comprising
introducing into the cell an agent that inhibits binding between
A.beta. protein and ABAD.
[0017] This invention further provides an article of manufacture
comprising a packaging material having therein a polypeptide
consisting of a portion of ABAD, wherein the portion of ABAD binds
to A.beta. protein and comprises the sequence of amino acid
residues 94-114 of ABAD, and wherein the packaging material has
affixed thereto a label indicating a use for the polypeptide for
treating a subject afflicted with Alzheimer's disease.
[0018] This invention further provides an article of manufacture
comprising a packaging material having therein a composition of
matter comprising a polypeptide consisting of a portion of ABAD
comprising the sequence of amino acid residues 94-114 of ABAD,
wherein the composition binds to A.beta. protein, and wherein the
packaging material has affixed thereto a label indicating a use for
the composition for treating a subject afflicted with Alzheimer's
disease.
[0019] This invention further provides an article of manufacture
comprising a packaging material having therein a polypeptide
consisting of a portion of A.beta.protein, wherein the portion of
A.beta. protein binds to ABAD and comprises the sequence of amino
acid residues 1-20 of A.beta. protein, and wherein the packaging
material has affixed thereto a label indicating a use for the
polypeptide for treating a subject afflicted with Alzheimer's
disease.
[0020] This invention further provides an article of manufacture
comprising a packaging material having therein a composition of
matter comprising a polypeptide consisting of a portion of A.beta.
protein comprising the sequence of amino acid residues 1-20 of
A.beta. protein, wherein the composition binds to ABAD, and wherein
the packaging material has affixed thereto a label indicating a use
for the composition for treating a subject afflicted with
Alzheimer's disease.
[0021] This invention further provides a method for determining
whether an agent inhibits the binding of A.beta.protein to ABAD,
which comprises: (a) admixing (i) a polypeptide consisting of a
portion of ABAD, wherein the portion of ABAD binds to A.beta.
protein and comprises the sequence of amino acid residues 94-114 of
ABAD, (ii) A.beta. protein, and (iii) the agent, under conditions
which would permit binding of the polypeptide and A.beta.protein in
the absence of the agent; (b) determining the amount of polypeptide
bound to A.beta. protein in step (a); and (c) comparing the amount
of bound polypeptide determined in step (b) with the amount
determined in the absence of the agent, whereby a lower amount of
binding in the presence of the agent indicates that the agent
inhibits the binding of A.beta. protein to ABAD.
[0022] This invention further provides a method for determining
whether an agent inhibits the binding of A.beta.protein to ABAD,
which comprises: (a) admixing (i) a polypeptide consisting of a
portion of A.beta. protein, wherein the portion of A.beta. protein
binds to ABAD and comprises the sequence of amino acid residues
1-20 of A.beta. protein, (ii) ABAD, and (iii) the agent, under
conditions which would permit binding of the polypeptide and ABAD
in the absence of the agent; (b) determining the amount of
polypeptide bound to ABAD in step (a); and (c) comparing the amount
of bound polypeptide determined in step (b) with the amount
determined in the absence of the agent, whereby a lower amount of
binding in the presence of the agent indicates that the agent
inhibits the binding of A.beta.protein to ABAD.
[0023] Finally, this invention provides a method for determining
whether an agent inhibits the binding of A.beta.protein to ABAD,
which comprises: (a) admixing (i) a polypeptide consisting of a
portion of ABAD, wherein the portion of ABAD binds to A.beta.
protein and comprises the sequence of amino acid residues 94-114 of
ABAD, (ii) a polypeptide consisting of a portion of A.beta.protein,
wherein the portion of A.beta. protein binds to ABAD and comprises
the sequence of amino acid residues 1-20 of A.beta. protein, and
(iii) the agent, under conditions which would permit binding of the
portion of ABAD and the portion of A protein in the absence of the
agent; (b) determining the amount of portion of ABAD bound to
portion of A protein in step (a); and (c) comparing the amount of
the bound portion of ABAD determined in step (b) with the amount
determined in the absence of the agent, whereby a lower amount of
binding in the presence or one agent indicates that the agent
inhibits the binding of A.beta. protein to ABAD.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1. ABAD-A.beta. association in AD patients and
transgenic mice. (A) Co-immunoprecipitation of ABAD and A.beta. in
AD patient brains. Results shown are representative of the 3
patients in each group. (B) Subcellular fractionation was used to
prepare fractions of mouse brain enriched for mitochondrial
(fraction I), lysosomal (fraction II) or endoplasmic reticulum
(fraction III) constituents. Each fraction (20 .mu.g total protein
per lane) was immunoblotted with antibodies to Cox IV (cytochrome c
oxidase IV), Cathepsin D and protein disulfide isomerase (PDI).
Protein loading was identical in each case. Lower panel shows the
presence of ABAD in the mitochondrial fraction. (C) Co-localization
of ABAD and A.beta. in cerebral cortex of AD patients
(magnification 200-fold). (D) Mitochondrial localization of ABAD in
cerebral cortex of AD patients (magnification 200-fold). VDAC
(Voltage-Dependent Anion Channel) was used as a mitochondrial
marker. Mouse anti-VDAC (20 .mu.g/ml), guinea pig anti-ABAD (10
.mu.g/ml) and rabbit anti-A.beta. (5 .mu.g/ml) IgGs were used in
C-D. (E) Colocalization of ABAD and A.beta. in mitochondria of the
brain of a patient with AD using electron microscopy. Double
immunogold staining was performed with rabbit anti-A.beta. IgG and
mouse anti-ABAD IgG followed by goat anti-rabbit IgG conjugated to
12 nm gold particles (for A.beta. 1-42) and goat anti-mouse IgG
conjugated to 18 nM gold particles (for ABAD). Arrowheads depict
gold particles localizing ABAD antigen. The smaller gold particles
represent sites of localization of A.beta..
[0025] FIG. 2. Crystal structure of A.beta.-bound human ABAD. (A) A
ribbon diagram with labeled secondary structures and the L.sub.D,
L.sub.E and L.sub.F loops. (B) Superposition of A.beta.-bound human
ABAD (magenta) and rat ABAD in complex with NAD (cyan). The L.sub.D
loop of 3.alpha.-hydroxysteroid dehydrogenase (3.alpha.-HSD) (PDB
code 1FJH) is shown in yellow. The proposed A.beta.-binding loop is
indicated. (C) Superposition of the active sites of A.beta.-bound
human ABAD (magenta) and rat ABAD (cyan), showing distortion of the
NAD binding site and the catalytic triad S155, K162 and Y168. (D) A
section of the crystal packing interactions, showing the large
solvent channels. Each ABAD molecule is shown in a different color.
The ordered ends of the L.sub.D loop, residues 94 and 114, are
marked as red and blue balls, respectively, and the hypothetical
loops are shown in magenta as dotted curvy lines.
[0026] FIG. 3. Biochemical and functional effects of ABAD-DP. (A-B)
Effect of mutations in the L.sub.D loop of ABAD on A.beta. binding
(A) and inhibition of ABAD-A.beta.interaction by ABAD-DP (B),
measured by surface plasmon resonance. ABAD was immobilized on the
sensor chips; for A, wild-type or mutant (S98A, K99A, Y101A) ABAD
was immobilized and for B, wild-type was used. For A, the mobile
(injected) phase was AD (1-40) and for B, the mobile phase was a
mixture of A.beta. (1-42) and either ABAD-DP or ABAD-RP (range of
concentrations). (C) Inhibition of A.beta.-induced and spontaneous
cytochrome c release from mitochondrial or membrane fraction by
ABAD-DP in cultured neurons. (D-E) Inhibition of ROS generation (D)
and DNA fragmentation (E) by ABAD-DP. *P<0.05, versus nonTg
cells; .sup.tP<0.05, versus without ABAD-DP treatment.
[0027] FIG. 4. Generation of free radicals and spatial learning and
memory deficit in Tg mAPP/ABAD mice. (A) Generation of free
radicals in Tg mAPP/ABAD mouse brains, shown by the sharp peak at
3410 Gauss in an EPR spectrum. The amplitude of the spectra for Tg
mAPP, Tg ABAD and nonTg animals has been increased by 10-fold to
display the spectra, which showed only low level changes. (B)
Spatial learning is abnormal in 4.5-5 month old Tg mAPP/ABAD mice
tested in the radial-arm water maze (P<0.05; N=7-8). (C) All
groups (N=7-8) show similar speed and latency to find a visible
platform.
[0028] FIG. 5. Increased expression of ABAD in AD brains (n=19) as
compared with age-matched controls (n=15). Brains from age-matched,
pathologically confirmed 19 patient with AD (means +age 85.5+2.07),
and 15 non-demented (ND) patients (means +age 82.07+1.465) were
harvested according to the rapid autopsy procedure developed at Sun
Health Institute (postmortem time 2.7.+-.0.306 and 2.14.+-.0.162
hour, respectively, for AD and ND patients) (33). Two AD-affected
brain regions were analysed, inferior temporal lobe grey matter and
hippocampus. Protein extracts were prepared by sonication with 5
volumes of extracting buffer (2% SDS, 1 mM EDTA, and protease
inhibitors in PBS) and 8 .mu.g of protein from each brain extract
were loaded onto reducing NuPAGE 4-12% Bis-Tris gel (Invitrogen,
Carlsbad, Calif.). Immunoblotting was performed with specific
antibodies to ABAD (monoclonal antibody to human, generated in our
lab, 1:10,000) or (3-actin (1:10,000, Sigma, St. louis, Mo.). The
immunoreactive bands were detected with Super Signal Pico
Chemiluminescent substrate (Pierce Chemicals). Densitometry was
performed using the Chemilmager 4000 Imaging system (Alpha
Innotech, San Leandro, Calif.) to determine differences in
intensity of the ABAD immunoreactive band, which was normalized
according to intensity of the (3-actin band. Our results
demonstrate a significant increase of ABAD protein in the
AD-pathology-affected regions (approximately 28% increase in
inferior temporal lobe grey matter and 40% in hippocampus) from AD
patients versus ND controls. These data are consistent with our
previous data (1) demonstrating enhanced expression of ABAD in AD
brain by immunoblotting with anti-ABAD antibody. In contrast,
protein extracts prepared from the cerebellum, a region spared from
the AD pathology, showed no significant differences between AD
patient and ND controls.
[0029] FIG. 6. Demonstration of ABAD-A.beta. complex in brains of
Tg mAPP/ABAD mice. A, Co-immunoprecipitation of ABAD and A.beta.
from mitochondria of transgenic mice. Mitchondrial fractions (500
.mu.g) from cerebral cortex of nonTg, Tg mAPP and Tg mAPP/ABAD mice
were immunoprecipitated with mouse anti-Afi IgG (6E10; 8 .mu.g/ml),
or nonimmune IgG (8 .mu.g/ml) at 4.degree. C. overnight followed by
Western blotting with mouse anti-ABAD (1:10,000). The middle panel
shows total input protein reprobed with anti-AR antibody (6E 10).
Lower panel shows immunoblotting of .beta.-actin for crude extracts
from mouse brains. B, Co-localization of ABAD and A.beta. in the
brain of a Tg mAPP/ABAD mouse using confocal microscopy with
antibodies to ABAD and A.beta. (color not shown) (magnification
300-fold). C, Colocalization of ABAD and A.beta. in mitochondria of
brains from Tg mAPP/ABAD mouse using electron microscopy. Double
immunogold staining was performed with rabbit anti-A.beta. IgG and
mouse anti-ABAD IgG followed by goat anti-rabbit IgG conjugated to
12 nm gold particles (for A.beta.1-42) and goat anti-mouse IgG
conjugated to 18 nM gold particles (for ABAD). Arrowheads depict
gold particles localizing ABAD antigen. The smaller gold particles
represent sites of localization of A.beta..
[0030] FIG. 7. A, SDS-PAGE of washed and dissolved crystals of
human ABAD and A.beta.. Lanes from left to right: ABAD standard,
A.beta. (1-40) standard and dissolved crystals. B, Superposition of
rat ABAD in complex with NAD and human ABAD in complex with NAD and
an inhibitor (color not shown). The L.sub.D and L.sub.E loops are
very similar. The L.sub.F loop is ordered in the human structure
but disordered in the rat structure. C, Sequence alignment of the
disordered part of the L.sub.D loop (residues 95-113) among human,
rat, mouse, bovine and Drosophila ABAD and several HSDs, members of
the SDRs, showing the insertion in ABAD relative to other HSDs (SEQ
ID NOS. 6-14, respectively).
[0031] FIG. 8. A, Inhibition of ABAD-A.beta. (1-40) interaction by
ABAD-DP. ABAD was immobilized on the sensor chip of the Biacore and
A.beta. (1-40), in the presence of the indicated concentrations of
ABAD-DP or ABAD-RP, was in the mobile phase. Response data are
plotted in Resonance Units versus ABAD peptide concentrations (nM).
B-D, Inhibition of A.beta.-induced generation of ROS (B), DNA
fragmentation (C) and LDH release (D) by ABAD-DP, but not by
ABAD-RP. For inhibition by ABAD peptides, cells were pre-incubated
with ABAD-DP or ABAD-RP (10 .mu.M) for 60 min before
A.beta.treatment. *P<0.05, versus nonTg cells; .sup.tP<0.05,
versus without ABAD-DP treatment.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0032] "Administering" shall mean delivering in a manner which is
effected or performed using any of the various methods and delivery
systems known to those skilled in the art. Administering can be
performed, for example, intravenously, pericardially, orally, via
implant, trans-mucosally, transdermally, intramuscularly,
sub-cutaneously, intraperitoneally, intrathecally,
intra-lymphatically, intralesionally, or epidurally. Administering
can be performed, for example, once, a plurality of times, and/or
over one or more extended periods.
[0033] "Agent" shall include, without limitation, an organic
compound, a nucleic acid, a polypeptide, a lipid, and a
carbohydrate. Agents include, for example, agents which are known
with respect to structure and/or function, and those which are not
known with respect to structure or function.
[0034] "Inhibit," when used in connection with the binding between
A.beta. and ABAD, shall mean to reduce such binding. In one
embodiment, "inhibit" shall mean to eliminate such binding. In the
preferred embodiment, inhibiting binding between two proteins means
to specifically inhibit such binding, i.e., to reduce or eliminate
binding between those two proteins without reducing or eliminating
binding between other proteins at all or to as great a degree.
[0035] "Operatively affixed," with respect to a first protein
joined to a second protein, means affixed in a manner permitting
each protein to perform at least one function which it would
perform were it not affixed to the other protein.
[0036] "Pharmaceutically acceptable carriers" are well known to
those skilled in the art and include, but are not limited to,
0.01-0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline.
Additionally, such pharmaceutically acceptable carriers can be
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions and suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's and fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers such as
those based on Ringer=s dextrose, and the like. Preservatives and
other additives may also be present, such as, for example,
antimicrobials, antioxidants, chelating agents, inert gases, and
the like.
[0037] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein, and each means a polymer of amino acid
residues.
[0038] "Subject" shall mean any animal, such as a human, non-human
primate, mouse, rat, guinea pig or rabbit.
[0039] "Tat", "HIV Tat protein" and "Tat protein" are equivalent,
and each shall mean any of (a) the HIV protein having the amino
acid sequence
met-glu-pro-val-asp-pro-arg-leu-glu-pro-trp-lys-his-pro-gly-ser-gln-pro-l-
ys-thr-ala-cys-thr-asn-cys-tyr-cys-lys-lys-cys-cys-phe-his-cys-gln-val-cys-
-phe-ile-thr-lys-ala-leu-gly-ile-ser-tyr-gly-arg-lys-lys-arg-arg-gln-arg-a-
rg-arg-pro-pro-gln-gly-ser-gln-thr-his-gln-val-ser-leu-ser-lys-gln-pro-thr-
-ser-gln-ser-arg-gly-asp-pro-thr-gly-pro-lys-glu (SEQ ID NO. 1),
(b) the HIV protein having the amino acid sequence
tyr-gly-arg-lys-lys-arg-arg-gln-arg-arg-arg (SEQ ID NO. 2), and (c)
all naturally occurring variants of proteins (a) and (b). Naturally
occurring variants of HIV protein sequences can be found, inter
alia, in Genbank and the Los Alamos HIV Database, both well know in
the art.
[0040] "Treating" a disorder shall mean slowing, stopping or
reversing the disorder's progression. In the preferred embodiment,
treating a disorder means reversing the disorder's progression,
ideally to the point of eliminating the disorder itself. As used
herein, ameliorating a disorder and treating a disorder are
equivalent.
EMBODIMENTS OF THE INVENTION
[0041] This invention provides methods, compositions and articles
of manufacture for inhibiting neurotoxicity in Alzheimer's disease.
This invention is based on the surprising discovery that A.beta.
protein binds to ABAD in the mitochondria.
[0042] Specifically, this invention provides a polypeptide
consisting of a portion of ABAD, wherein the portion of ABAD binds
to A.beta. protein and comprises the sequence of amino acid
residues 94-114 of ABAD. In the preferred embodiment of this
polypeptide, the ABAD is human ABAD. In the preferred embodiment,
this and other polypeptides of the instant invention are isolated
(e.g. free from other proteins), or enriched (e.g. having only
minimal (<50% total weight) protein impurities). The full
sequence of human ABAD is set forth in (1) at page 690, Figure D.
The sequence of amino acid residues 92-120 of human ABAD is as
follows: AGIAVASKTYNLKKGQTHTLEDFQRVLDV (SEQ ID NO. 3). The sequence
of amino acid residues 94-114 of human ABAD is as follows:
IAVASKTYNLKKGQTHTLEDF (SEQ ID NO. 4).
[0043] This invention further provides a composition of matter
comprising (a) a pharmaceutical carrier, and (b) a polypeptide
consisting of a portion of ABAD, wherein the portion of ABAD binds
to A.beta.-protein and comprises the sequence of amino acid
residues 94-114 of ABAD. This invention further provides a
composition of matter comprising a polypeptide consisting of a
portion of ABAD comprising the sequence of amino acid residues
94-114 of ABAD, wherein the composition binds to A.beta. protein.
In the preferred embodiment of this composition, the ABAD is human
ABAD. In another embodiment, the composition is further comprised
by a pharmaceutical carrier.
[0044] This invention further provides a composition of matter
comprising (a) a Tat protein operatively affixed to (b) a
polypeptide consisting of a portion of ABAD comprising the sequence
of amino acid residues 94-114 of ABAD, wherein the composition
binds to A.beta. protein. In the preferred embodiment of this
composition, the ABAD is human ABAD. In another embodiment, the
composition is further comprised by a pharmaceutical carrier.
[0045] This invention further provides for an isolated polypeptide
consisting of a portion of A.beta. protein, wherein the portion of
A.beta. protein binds to ABAD and comprises the sequence of amino
acid residues 1-20 of A.beta. protein. In the preferred embodiment
of this polypeptide, the A.beta. protein is human A.beta. protein.
The full sequence of human A.beta. is set forth in (1) at page 690,
FIG. 1. The sequence of amino acid residues 1-20 of human A.beta.
is as follows: DAEFRHDSGYEVHHQKLVFF (SEQ ID NO. 5).
[0046] This invention further provides a composition of matter
comprising (a) a pharmaceutical carrier, and (b) a polypeptide
consisting of a portion of A.beta. protein, wherein the portion of
A.beta. protein binds to ABAD and comprises the sequence of amino
acid residues 1-20 of A.beta. protein.
[0047] This invention further provides a composition of matter
comprising a polypeptide consisting of a portion of A.beta. protein
comprising the sequence of amino acid residues 1-20 of A.beta.
protein, wherein the composition binds to ABAD. In the preferred
embodiment of this composition, the A.beta. protein is human
A.beta. protein. In another embodiment, the composition is further
comprised by a pharmaceutically acceptable carrier.
[0048] This invention further provides a composition of matter
comprising (a) a Tat protein operatively affixed to (b) a
polypeptide consisting of a portion of A.beta. protein comprising
the sequence of amino acid residues 1-20 of A.beta. protein,
wherein the composition binds to ABAD. In the preferred embodiment
of this composition, the A.beta.protein is human A.beta. protein.
In another embodiment, the composition is further comprised by a
pharmaceutically acceptable carrier.
[0049] This invention further provides a method for treating a
subject afflicted with Alzheimer's disease comprising administering
to the subject a therapeutically effective amount of an agent that
inhibits binding between A.beta. protein and ABAD in the subject's
cells.
[0050] In the preferred embodiment of this method, the subject is
human. In another embodiment, the cells are neuronal cells.
[0051] Determining a therapeutically effective amount of the
instant polypeptides and compositions can be done based on animal
data using routine computational methods. In one embodiment of the
instant invention, the therapeutically effective amount of
polypeptide or composition is between 0.01 and 1000 mg/kg body
weight/day. In another embodiment, the therapeutically effective
amount is between 0.25 and 50 mg/kg body weight/day. In a preferred
embodiment, the therapeutically effective amount is between 1.0 and
10 mg/kg body weight/day.
[0052] In one embodiment of this method, the agent is a polypeptide
consisting of a portion of ABAD, wherein the portion of ABAD binds
to A.beta. protein and comprises the sequence of amino acid
residues 94-114 of ABAD. In another embodiment, the agent is a
composition of matter comprising a polypeptide consisting of a
portion of ABAD comprising the sequence of amino acid residues
94-114 of ABAD, wherein the composition binds to A.beta. protein.
In yet another embodiment, the agent is a polypeptide consisting of
a portion of A.beta. protein, wherein the portion of A.beta.
protein binds to ABAD and comprises the sequence of amino acid
residues 1-20 of A.beta. protein. In yet another embodiment, the
agent is a composition of matter comprising a polypeptide
consisting of a portion of A.beta. protein comprising the sequence
of amino acid residues 1-20 of A.beta. protein, wherein the
composition binds to ABAD.
[0053] This invention further provides a method for reducing free
radical generation in a cell comprising introducing into the cell
an agent that inhibits binding between A.beta. protein and ABAD. In
the preferred embodiment of this and other instant methods
involving introducing an agent into a cell, the agent enters the
cell's mitochondria once present in the cell's cytosol.
[0054] In one embodiment of this method, the cell is a neuronal
cell.
[0055] In one embodiment of this method, the agent is a polypeptide
consisting of a portion of ABAD, wherein the portion of ABAD binds
to A.beta. protein and comprises the sequence of amino acid
residues 94-114 of ABAD. In another embodiment, the agent is a
composition of matter comprising a polypeptide consisting of a
portion of ABAD comprising the sequence of amino acid residues
94-114 of ABAD, wherein the composition binds to A.beta.protein. In
yet another embodiment, the agent is a polypeptide consisting of a
portion of A.beta. protein, wherein the portion of A.beta. protein
binds to ABAD and comprises the sequence of amino acid residues
1-20 of A.beta. protein. In yet another embodiment, the agent is a
composition of matter comprising a polypeptide consisting of a
portion of A.beta. protein comprising the sequence of amino acid
residues 1-20 of A.beta. protein, wherein the composition binds to
ABAD.
[0056] This invention further provides a method for reducing DNA
fragmentation in the cytosol of a cell comprising delivering into
the cell an agent that inhibits binding between A.beta. protein and
ABAD.
[0057] In one embodiment of this method, the cell is a neuronal
cell.
[0058] In one embodiment of this method, the agent is a polypeptide
consisting of a portion of ABAD, wherein the portion of ABAD binds
to A.beta. protein and comprises the sequence of amino acid
residues 94-114 of ABAD. In another embodiment, the agent is a
composition of matter comprising a polypeptide consisting of a
portion of ABAD comprising the sequence of amino acid residues
94-114 of ABAD, wherein the composition binds to A.beta.protein. In
yet another embodiment, the agent is a polypeptide consisting of a
portion of A.beta. protein, wherein the portion of A.beta. protein
binds to ABAD and comprises the sequence of amino acid residues
1-20 of A.beta. protein. In yet another embodiment, the agent is a
composition of matter comprising a polypeptide consisting of a
portion of A.beta. protein comprising the sequence of amino acid
residues 1-20 of A.beta. protein, wherein the composition binds to
ABAD.
[0059] This invention further provides a method for reducing
cytochrome c release from mitochondria in a cell comprising
introducing into the cell an agent that inhibits binding between
A.beta. protein and ABAD.
[0060] In one embodiment of this method, the cell is a neuronal
cell.
[0061] In one embodiment of this method, the agent is a polypeptide
consisting of a portion of ABAD, wherein the portion of ABAD binds
to A.beta. protein and comprises the sequence of amino acid
residues 94-114 of ABAD. In another embodiment, the agent is a
composition of matter comprising a polypeptide consisting of a
portion of ABAD comprising the sequence of amino acid residues
94-114 of ABAD, wherein the composition binds to A.beta.protein. In
yet another embodiment, the agent is a polypeptide consisting of a
portion of A.beta. protein, wherein the portion of A.beta. protein
binds to ABAD and comprises the sequence of amino acid residues
1-20 of A.beta. protein. In yet another embodiment, the agent is a
composition of matter comprising a polypeptide consisting of a
portion of A.beta. protein comprising the sequence of amino acid
residues 1-20 of A.beta. protein, wherein the composition binds to
ABAD.
[0062] This invention further provides a method for preserving cell
viability by reducing LDH release from a cell comprising
introducing into the cell an agent that inhibits binding between
A.beta. protein and ABAD.
[0063] In one embodiment of this method, the cell is a neuronal
cell.
[0064] In one embodiment of this method, the agent is a polypeptide
consisting of a portion of ABAD, wherein the portion of ABAD binds
to AA protein and comprises the sequence of amino acid residues
94-114 of ABAD. In another embodiment, the agent is a composition
of matter comprising a polypeptide consisting of a portion of ABAD
comprising the sequence of amino acid residues 94-114 of ABAD,
wherein the composition binds to A.beta.protein. In yet another
embodiment, the agent is a polypeptide consisting of a portion of
A.beta. protein, wherein the portion of A.beta. protein binds to
ABAD and comprises the sequence of amino acid residues 1-20 of
A.beta. protein. In yet another embodiment, the agent is a
composition of matter comprising a polypeptide consisting of a
portion of A.beta. protein comprising the sequence of amino acid
residues 1-20 of A.beta. protein, wherein the composition binds to
ABAD.
[0065] This invention further provides an article of manufacture
comprising a packaging material having therein a polypeptide
consisting of a portion of ABAD, wherein the portion of ABAD binds
to A.beta. protein and comprises the sequence of amino acid
residues 94-114 of ABAD, and wherein the packaging material has
affixed thereto a label indicating a use for the polypeptide for
treating a subject afflicted with Alzheimer's disease. In the
preferred embodiment of this article, the subject is a human.
[0066] This invention further provides an article of manufacture
comprising a packaging material having therein a composition of
matter comprising a polypeptide consisting of a portion of ABAD
comprising the sequence of amino acid residues 94-114 of ABAD,
wherein the composition binds to A.beta. protein, and wherein the
packaging material has affixed thereto a label indicating a use for
the composition for treating a subject afflicted with Alzheimer's
disease. In the preferred embodiment of this article, the subject
is a human.
[0067] This invention further provides an article of manufacture
comprising a packaging material having therein a polypeptide
consisting of a portion of A.beta.protein, wherein the portion of
A.beta. protein binds to ABAD and comprises the sequence of amino
acid residues 1-20 of A.beta. protein, and wherein the packaging
material has affixed thereto a label indicating a use for the
polypeptide for treating a subject afflicted with Alzheimer's
disease. In the preferred embodiment of this article, the subject
is a human.
[0068] This invention further provides an article of manufacture
comprising a packaging material having therein a composition of
matter comprising a polypeptide consisting of a portion of A.beta.
protein comprising the sequence of amino acid residues 1-20 of
A.beta. protein, wherein the composition binds to ABAD, and wherein
the packaging material has affixed thereto a label indicating a use
for the composition for treating a subject afflicted with
Alzheimer's disease. In the preferred embodiment of this article,
the subject is a human.
[0069] This invention further provides a method for determining
whether an agent inhibits the binding of A.beta.protein to ABAD,
which comprises: (a) admixing (i) a polypeptide consisting of a
portion of ABAD, wherein the portion of ABAD binds to A.beta.
protein and comprises the sequence of amino acid residues 94-114 of
ABAD, (ii) A.beta. protein, and (iii) the agent, under conditions
which would permit binding of the polypeptide and A.beta.protein in
the absence of the agent; (b) determining the amount of polypeptide
bound to A.beta. protein in step (a); and (c) comparing the amount
of bound polypeptide determined in step (b) with the amount
determined in the absence of the agent, whereby a lower amount of
binding in the presence of the agent indicates that the agent
inhibits the binding of A.beta. protein to ABAD.
[0070] In one embodiment of the instant assays, as well as the
instant polypeptides, compositions, therapeutic methods and other
methods, the portion of ABAD and/or of A.beta.protein is
unmodified, i.e., it contains no amino acid residue derivatives and
its amino acid residues are bound by peptide bonds. In another
embodiment, the portion of ABAD and/or of A.beta. protein contains
at least one amino acid residue derivative (e.g., a residue having
a non-naturally occurring side chain) and/or contains at least one
non-peptide bond joining two amino acid residues. These portions
can also include modifications such as glycosylation, lipid
attachment, sulfation, hydroxylation, and ADP-ribosylation.
[0071] This invention further provides a method for determining
whether an agent inhibits the binding of A.beta.protein to ABAD,
which comprises: (a) admixing (i) a polypeptide consisting of a
portion of A.beta. protein, wherein the portion of A.beta. protein
binds to ABAD and comprises the sequence of amino acid residues
1-20 of A.beta. protein, (ii) ABAD, and (iii) the agent, under
conditions which would permit binding of the polypeptide and ABAD
in the absence of the agent; (b) determining the amount of
polypeptide bound to ABAD in step (a); and (c) comparing the amount
of bound polypeptide determined in step (b) with the amount
determined in the absence of the agent, whereby a lower amount of
binding in the presence of the agent indicates that the agent
inhibits the binding of A.beta.protein to ABAD.
[0072] This invention further provides a method for determining
whether an agent inhibits the binding of A.beta.protein to ABAD,
which comprises: (a) admixing (i) a polypeptide consisting of a
portion of ABAD, wherein the portion of ABAD binds to A.beta.
protein and comprises the sequence of amino acid residues 94-114 of
ABAD, (ii) a polypeptide consisting of a portion of A.beta.protein,
wherein the portion of A.beta. protein binds to ABAD and comprises
the sequence of amino acid residues 1-20 of A.beta. protein, and
(iii) the agent, under conditions which would permit binding of the
portion of ABAD and the portion of A protein in the absence of the
agent; (b) determining the amount of portion of ABAD bound to
portion of A protein in step (a); and (c) comparing the amount of
the bound portion of ABAD determined in step (b) with the amount
determined in the absence of the agent, whereby a lower amount of
binding in the presence of the agent indicates that the agent
inhibits the binding of A.beta. protein to ABAD.
[0073] This invention is illustrated in the Experimental Details
section which follows. This section is set forth to aid in an
understanding of the invention but is not intended to, and should
not be construed to, limit in any way the invention set forth in
the claims which follow.
Experimental Details
Methods
[0074] Immunoprecipitation/immunoblotting. Human brain tissues from
AD patients and age-matched non-demented controls (N=3 in each
case) were homogenized in Tris buffer (10 mM Tris, 0.1 M NaCl, 1 mM
EDTA, 100 .mu.g/ml PMSF, 1 .mu.g/ml aprotinin), immunoprecipitated
with rabbit anti-A.beta. IgG (3 .mu.l/500 .mu.g protein) at
4.degree. C. overnight, and Western blotting was done with mouse
anti-ABAD IgG (1:1000). Immunoprecipitation was performed on
incubation of crude extracts (500 .mu.g) from cerebral cortex with
anti-A.beta. antibody followed by immunoblotting with anti-ABAD
IgG. Peroxidase-conjugated goat anti-mouse IgG (specific for heavy
chain, Jackson Lab) was used as a secondary antibody.
Electrophoresis was performed with 12% Tris-Glycine SDS-PAGE.
Results shown are representative of the 3 patients in each group.
The same methodology was employed for
immunoprecipitation/immunoblotting studies of mitochondria derived
from brains of Tg mAPP/ABAD mice.
[0075] Transgenic (Tg) mice overexpressing a mutant human form of
amyloid precursor protein (mAPP), the latter a minigene encoding
hAPP695, 751 and hAPP770 bearing V717/F, K670M, N671L; J-20 line)
in the C57BL6 background were provided by Dr. Lennart Mucke (6).
The latter are termed Tg mAPP mice (>N10 in the C57BL/6 strain).
Tg ABAD mice (N8 in the C57BL/6 strain), which overexpress ABAD
under control of the PDGF B-chain promoter have described
previously (7). Tg mAPP and Tg ABAD animals were crossed to
generated Tg mAPP/ABAD, Tg mAPP, Tg ABAD and nontransgenic
littermates. Offspring were identified by PCR using primers for
specific for each transgene.
[0076] Isolation of mitochondria. Brain tissue in isolation buffer
(20 mM Hepes at pH 7.2 and 1 mM EDTA) was subjected to 10 strokes
of a glass teflon potter homogenizer. The homogenate was
centrifuged at 1,700 rpm for 6 min at 4.degree. C. The resulting
supernatant was then centrifuged at 5000 g for 10 min. The pellet
was resuspended in isolation buffer, and layered on a Ficoll
gradient generated from 5 ml of 11% Ficoll and 3 ml of 7.5% Ficoll
and centrifuged in an AH-628 rotor at 79,000 g for 30 minutes. The
pellet was resuspended again in isolation buffer, incubated with
fresh digitonin (1.25 mg/100 mg brain tissues) for 15 min on ice,
and then centrifuged at 6,500 rpm for 10 min. The resulting new
pellet, containing highly purified mitochondria, was washed with
0.2% BSA and resuspended in isolation buffer.
[0077] Crystallography. Expression and purification of human ABAD
was performed as described (3,7). For crystallization, ABAD was
concentrated to 10 mg/ml in 25 mM MES at pH 6.0, 0.1 M NaCl and 5
mM DTT and mixed with 5 mM NAD and 3- to 4-fold molar excess of
A.beta. (residues 1-40; Biosource, CA). The mixture was
crystallized by vapor diffusion at 22.degree. C. using a
precipitant solution containing 0.1 M MES at pH 6.0, 2.5 M NaCl, 5
mM benzamidine and 5 mM NAD. Diffraction data were collected at the
COM-CAT beamline of Advanced Photon Source and processed with the
HKL package (23). The structure was determined by molecular
replacement calculations in the program Replace (24, 25) using the
rat ABAD structure as a search model. Limited six-dimensional
search and a high large term cutoff (2.0) were used in the
structure determination. Retrospectively, these strategies were
crucial in overcoming the hurdle provided by the high symmetry of
the space group and the large conformational differences between
rat ABAD and human A.beta.-bound ABAD structures. Refinement was
carried out by the simulated annealing protocol in CNS (26). Model
building was performed in program (27, 28). The final atomic model
contains residues 6-94, 114-207 and 229-253.
[0078] Studies with ABAD-derived peptides. ABAD decoy peptide
(ABAD-DP, residues 92-120, with a sequence of
Ala-Gly-Ile-Ala-Val-Ala-Ser-Lys-Thr-Tyr-Asn-Leu-Lys-Lys-Gly-Gln-Thr-His-T-
hr-Leu-Glu-Asp-Phe-Gln-Arg-Val-Leu-Asp-Val; SEQ ID NO. 3) and ABAD
reverse peptide (ABAD-RP, residues 120-92) were synthesized by
Biotechnology Lab at Yale University. For binding studies, ABAD was
immobilized on CM5 sensor chip (29). As indicated, A.beta.(1-40) or
A.beta.(1-42) alone or with either ABAD-DP or ABAD-RP (range of
concentrations) was injected at a flow rate of 30 .mu.l/min for 2
min at 25.degree. C. using Biacore x (Pharmacia). The
reaction/binding buffer contained 50 mM Hepes at pH 7.4, 0.15 M
NaCl, 1 mM EDTA, and 0.005% Tween 20. Data analysis was performed
using Biacore X biosensor system (Uppsala, Sweden) and BIA
evaluation 3.0 software (Biacore, Sweden). Response data are
plotted in Resonance Units versus ABAD peptide concentrations (nM),
and were fit to a one-site model for competitive inhibition to
obtain the K.sub.i.
[0079] The effect of ABAD-DP on cytochrome c release. Primary
cortical neurons (4 day) from mice of each of the genotypes were
cultured. The effect of ABAD-DP on cytochrome c release was studied
by pre-treating cultures with ABAD-DP (10 .mu.M) or ABAD-RP (10
.mu.M) for 60 minutes, followed by incubation with A.beta.(1-42; 1
.mu.M) for nonTg and Tg ABAD mice. After 24 hours at 37.degree. C.,
cytosolic cytochrome c was determined by the following procedure.
Briefly, cells plated on 100 mm dishes were washed with cold PBS,
scraped using a rubber policeman, and collected by centrifugation
at 300 g for 5 min at 4.degree. C. The pellet was resuspended in
400 .mu.l of lysis buffer containing 50 mM Tris at pH 7.4, 1 mM
EDTA, 1 mM EGTA, 250 mM sucrose, 2 .mu.g/ml leupeptin, 1 mM PMSF,
and 1 .mu.g/ml pepstatin A and disrupted with 10 strokes of a
Dounce homogenizes Cytosol and membrane fractions were separated by
centrifugation at 105,000 g for 1 hour at 4.degree. C. (11). The
resulting pellets were resuspended in 50 .mu.l of lysis buffer and
supernatants were concentrated to 20 .mu.l. The protein content of
each fraction was determined by the BioRad DC protein assay (BioRad
Laboratories, Hercules, Calif.). Samples (40 .mu.g/lane) were
subjected to 12% SDS-PAGE, followed by immunoblotting using
anti-cytochrome c antibody (1:500) (Phamergen).
[0080] The effect of ABAD-DP on intracellular generation of ROS.
Studies were also performed to determine the effect of ABAD-DP on
intracellular generation of ROS by neurons from Tg mice using DCFDA
fluorescent probe (31, 32). Cells plated on 100 mm dishes were
washed with Hanks balanced saline solution, incubated with 0.05%
trypsin-EDTA containing 1 .mu.M DCFDA for 25 minutes and collected
by centrifugation at 300 g for 5 min at room temperature. The
pellet was resuspended in 1.5 ml Earle's balanced saline solution
and exposed to A.beta. (1-42, 4 .mu.g/ml) for 5 minutes.
Fluorescence was measured with excitation at 490 nm and emission at
530 nm using a FluoroMax-2 spectrofluorometer (Jobin Yvon Inc.,
Edison, N.J.). Other studies investigated the effect of ABAD-DP on
A.beta.-induced and spontaneous DNA fragmentation (Cell Death
Detection ELISA.sup.PLUS, Roche Diagnostics Co. Indianapolis, Ind.)
and LDH release (Sigma).
[0081] EPR spectra were recorded on frozen brain tissues in liquid
nitrogen using a quartz dewar at X-band. Brain tissue was extruded
in the form of a cylinder with 5 mm diameter at room temperature
and quickly frozen in liquid nitrogen. The measurements were
performed on frozen intact tissue. No processing (grinding) was
done on the frozen sample to avoid artifacts. The spectrometer was
set at a frequency of 9.561 GHz, a modulation amplitude of 2.5 G
and a microwave power of 1 mW. Spectral acquisitions were performed
for 10 min on each sample.
[0082] The radial-arm water maze task (P<0.05; N=7-8) was
performed as previously described. Investigators were blinded to
mouse genotypes (14). Mice had to find a platform hidden beneath
the water surface at the end of one of the six arms. The four
groups of animals under study in our behavioral experiments were
littermates. This breeding strategy and the C57BL/6 background were
employed to enhance the reproducibility and reliability of our
results in the radial arm water maze, based on work of other
investigators. Time to reach the platform and speed of swimming
were recorded and analyzed with a video-tracking system (HVS-2020,
HVS Image, UK).
Results
[0083] It was speculated that interaction of A.beta. with ABAD
might induce mitochondrial dysfunction. However, since it had not
been established that intracellular A.beta. can access
mitochondria, it was essential to determine whether ABAD and
A.beta. interact in pathophysiologically relevant settings. To
address this directly, ABAD-A.beta. complex was detected in AD
brains by immunoprecipitation of cortical protein extracts using
anti-A.beta. followed by immunoblotting with anti-ABAD IgG (FIG.
1A). Age-matched non-demented brain displayed very little
ABAD-A.beta. complex. Substitution of nonimmune IgG for specific
antibodies prevented appearance of the band (not shown). Since
cellular and mitochondrial integrity may start to deteriorate soon
after death, allowing non-physiological interactions to occur,
mitochondria were isolated from the cerebral cortex of 12 month-old
mice expressing mAPP (6), ABAD (7) or both, driven by the PDGF
B-chain promoter. The purity of mitochondrial preparations was
confirmed by enrichment of Cox IV (cytochrome c oxidase IV), and
relative absence of lysosomal (cathepsin D) and endoplasmic
reticulum (PDI, protein disulfide isomerase) markers (FIG. 1B).
ABAD-A.beta. complex was evident in the mitochondria of both
transgenic (Tg) mAPP and Tg mAPP/ABAD mice (FIG. 6A) Similar bands
were only present at very low levels in samples from age- and
strain-matched nonTg littermates or when nonimmune IgG replaced
specific antibodies.
[0084] Confocal microscopy was used to confirm mitochondrial
co-localization and interaction of ABAD and A.beta.. In the
cerebral cortex of AD patients, images of anti-ABAD and
anti-A.beta. (color not shown), detecting endogenous ABAD and
A.beta., extensively co-localize (FIG. 1C). Similarly, in the
cerebral cortex of Tg mAPP/ABAD mice, there is extensive overlap of
immunoreactive ABAD and A.beta. (FIG. 6B). Because ABAD is mainly
localized to mitochondria, as shown by the overlap of anti-ABAD and
anti-VDAC (voltage-dependent anion channel) images (color not
shown) (FIG. 1D), these data demonstrate that A.beta. is also
present in the mitochondria of AD patients. Immunogold electron
microscopy using gold-conjugated antibody systems provided further
evidence for the presence of ABAD and A.beta. within mitochondria
(for ABAD, 18 nm gold particles and for A.beta., 12 nm gold
particles). The two different sizes of gold particles are
concentrated in the mitochondria of brains from a patient with AD
(FIG. 1E) and from Tg mAPP/ABAD mice (FIG. 6C). Taken together,
these microscopic and immunoprecipitation data demonstrate the
formation of ABAD-A.beta. complex within mitochondria in vivo.
[0085] To determine the structural basis of the
ABAD-A.beta.interaction, human ABAD was crystallized and its
structure was determined, in the presence of NAD and a molar excess
of A.beta., at 2.3 .ANG. resolution (FIG. 2A, Table 2).
Unexpectedly, NAD is not bound to ABAD in the crystal structure.
Under the crystallization condition, A.beta. inhibits ABAD
activity, though, in the absence of A.beta., ABAD displays normal
activity. This suggests that A.beta. prevents NAD binding in the
crystal structure, which may be the molecular basis for its
inhibition of ABAD activity.
[0086] SDS-PAGE and amino terminal sequencing of washed and
dissolved crystals showed that both ABAD and A.beta. are present in
the crystals (FIG. 7A). However, no electron density is observed
for A.beta., suggesting that A.beta.itself and the region of ABAD
that binds to A.beta. must be disordered in the crystal. To exclude
the possibility that A.beta. interacts with ABAD non-specifically,
additional experiments were performed, which showed that other
amyloid species such as a prion-derived peptide and amylin did not
bind to ABAD. In addition, A.beta.(1-42), A.beta.(1-40) and
A.beta.(1-20) demonstrated dose-dependent binding, whereas
A.beta.(25-35) showed no specific binding (Table 3).
[0087] Comparison of the A.beta.-bound ABAD structure with the rat
ABAD structure in complex with NAD (8) and the human ABAD structure
in complex with NAD and a small molecule inhibitor (9) shows that
the A.beta.-bound ABAD displays significant distortion of the
NAD-binding pocket and the catalytic triad (FIG. 2B, 2C, 7B). The
majority of the L.sub.D loop, the beginning of the following
.alpha..sub.D helix, and the latter part of the L.sub.F loop of
human ABAD are disordered. While the L.sub.E loop is ordered, the
conformation of the loop and the beginning of the following
.alpha..sub.E helix is significantly different from the structure
without A.beta..
[0088] In the absence of substrate, the L.sub.F loop is also
disordered in the rat ABAD structure in complex with NAD, while the
L.sub.D loop region is well ordered, suggesting that A.beta.
binding may have influenced the L.sub.D loop dynamics and
conformation. In addition, within the NAD-dependent short-chain
dehydrogenase/reductase (SDR) superfamily, the L.sub.D loop of ABAD
from different species contains a unique insertion that is absent
in all other SDRs (FIG. 7C). Because ABAD is the only SDR that has
been observed to bind A.beta., it was hypothesized that the L.sub.D
loop may be a recognition site for A.beta..
[0089] Inspection of crystal packing showed that the ordered ends
of the L.sub.D loop point into interconnected huge solvent channels
with estimated dimensions of 70 .ANG. (FIG. 2D). It is estimated
that the ordered part of the crystal only occupies about 30% of the
total crystal volume. Sufficient space is therefore available for
the disordered loops and the bound A.beta., which could drift
freely in the large solvent channels in the crystal to cause
disorder or nonspecifically bind and clog the active site region to
inactivate the enzyme.
[0090] To test the idea that the L.sub.D loop is important in
A.beta.interaction, structure-based mutational analyses were
performed (Table 1). Binding studies using A.beta. and GST-ABAD
truncation mutants showed that the amino terminal portion of ABAD
(residues 1-158) is responsible for A.beta. interaction.
Site-directed mutagenesis within and beyond the disordered L.sub.D
loop region (residues 95-113) specifically located two stretches of
ABAD residues important for A.beta. binding, residues S98-Y101 and
residues T108-T110. An ABAD mutant bearing S98A, K99A and Y101A
mutations exhibited no specific interaction to A.beta. in a surface
plasmon resonance experiment, although wild-type ABAD displayed
dose-dependent interaction with A.beta. (FIG. 3A).
[0091] To determine whether the L.sub.D loop is sufficient for
A.beta. interaction, a peptide was synthesized encompassing this
region (residues 92-120) of human ABAD (termed ABAD decoy peptide,
or ABAD-DP) and its ability to inhibit the interaction of intact
ABAD with A.beta. using surface plasmon resonance was tested (FIG.
3B). ABAD-DP inhibited binding of A.beta. (1-40) (FIG. 8A) and
A.beta. (1-42) (FIG. 3B) to immobilized intact ABAD with inhibitory
constants of 4.9 and 1.7 .mu.M, respectively. On the other hand, a
peptide of the reverse sequence (residues 120-92, termed ABAD
reversed peptide, or ABAD-RP) was completely inactive. These
competitive binding studies confirmed that the L.sub.D region alone
could mediate A.beta. binding, although it may not be the exclusive
site.
[0092] To develop a specific inhibitor of the
ABAD-A.beta.interaction in cultured neurons, the cell-membrane
transduction domain of the human immunodeficiency virus-type 1
(HIV) Tat protein (10,11) was added to ABAD-DP and ABAD-RP (thereby
enabling the peptides to cross cell membranes). Cytochrome c
release from mitochondria was used as a marker of A.beta.-induced
cellular stress and apoptosis. While cultured wild-type cortical
neurons exposed to A.beta.(1-42) suffered loss of cytochrome c from
the mitochondrial or membrane fraction to the cytosol fraction,
pre-incubation of the cells with ABAD-DP, but not with ABAD-RP,
largely prevented A.beta.-induced cytochrome c release (FIG. 3C).
Similarly, ABAD-DP also protected against A.beta.-induced
mitochondrial cytochrome c release in neurons from Tg ABAD mice,
although these neurons displayed enhanced cytochrome c release
compared with nonTg mice. Furthermore, pre-treatment with ABAD-DP,
but not with ABAD-RP, dramatically reduced spontaneous loss of
cytochrome c from the mitochondrial/membrane fraction of cultured
neurons derived from Tg mAPP/ABAD mice.
[0093] The protective effects of cell permeable ABAD-DP are
consistent with the hypothesis that the ABAD-A.beta. interaction
causes mitochondrial stress and apoptosis. Since mitochondria are
the principal sites of generation of reactive oxygen species (ROS)
under physiologic conditions, and A.beta. is known to trigger
oxidative stress, we tested whether the protection from
A.beta.-induced cytotoxicity by ABAD-DP is accompanied by
attenuated generation of ROS. Cultured neurons loaded with a probe
for ROS, dichlorofluorescin diacetate (DCFDA), demonstrated
fluorescence on exposure to A.beta., a phenomenon accentuated in
neurons from Tg ABAD mice compared with nonTg littermates (FIG.
8B). Pre-treatment with ABAD-DP virtually completely suppressed
fluorescence in DCFDA-loaded and A.beta.-exposed neurons, from both
nonTg and Tg ABAD animals (FIG. 8B). In contrast, there was no
effect exerted by ABAD-RP. Furthermore, spontaneous generation of
ROS by cultured neurons from Tg mAPP/ABAD mice was suppressed by
pre-treatment with ABAD-DP, not ABAD-RP (FIG. 3D). Linkage between
A.beta.-ABAD-induced generation of ROS, and subsequent DNA
fragmentation (FIG. 3E) and LDH release (FIG. 8D) was shown in
cultured neurons from Tg mAPP/ABAD mice. Similarly, cultured
neurons from Tg ABAD mice exposed to A.beta. display subsequent DNA
fragmentation (FIG. 8C), and LDH release (FIG. 8D). In each case,
pretreatment with ABAD-DP, not ABAD-RP, attenuated
cytotoxicity.
[0094] If the above results in cell culture could be extrapolated
to in vivo settings, then mice overexpressing ABAD in an
A.beta.-rich environment, i.e., Tg mAPP/ABAD mice, might exhibit
exaggerated oxidative stress and elevated generation of ROS.
Electron paramagnetic spin resonance (EPR) spectroscopy was used to
measure the level of ROS in intact frozen brain at 77.degree. K
(FIG. 4A). Dramatically higher amounts of radicals, as shown by the
sharp peak at 3410 Gauss, were observed in the Tg mAPP/ABAD mouse
brains, in comparison with brains from nonTg, Tg ABAD or Tg mAPP
mice. It is evident that this species of free radicals, which might
be from ascorbyl or the one-electron reduced ubiquinone radical, is
generated as a result of higher levels of oxidative stress in Tg
mAPP/ABAD (12), compared to the other genotypes.
[0095] Excessive generation of ROS could result in neuronal
dysfunction, or, alternatively, could be buffered by protective
anti-oxidant mechanisms without changes in neuronal function. The
radial-arm water maze test was used to detect hippocampal-dependent
learning/memory deficits in Tg mAPP/ABAD mice (13, 14). Young mice
(4.5-5 months of age) of nonTg, Tg mAPP or Tg ABAD transgenic
littermates, all showed strong learning and memory capacity. As
they sought out the new platform location, they averaged less than
1-2 errors by trials 4 or 5 (FIG. 4B). In contrast, Tg mAPP/ABAD
mice failed to learn efficiently and still averaged about 4 errors
by trials 4 or 5 (FIG. 4B), indicative of severe impairment in
spatial and temporal memory. Double transgenic expression of ABAD
and mAPP did not cause impairment in vision, motor coordination or
motivation. The visible platform test showed that the four
genotypes exhibited no difference in their speed of swimming and in
their latencies to find the platform (FIG. 4C).
[0096] The data demonstrate that ABAD and A.beta. directly interact
in mitochondria in AD, and that this interaction promotes leakage
of ROS, mitochondrial dysfunction and cell death, potentially
underlying the mechanism of A.beta.-induced mitochondrial toxicity
(15-22). Such events are likely to induce changes in behavior,
characterized by exaggerated impairment of hippocampal function in
Tg mAPP/ABAD mice. Taken together, these studies establish that
A.beta. may exert an important pathogenic role in the mitochondrial
compartment through an interaction with ABAD and that inhibition of
ABAD-A.beta. interaction may provide a new treatment strategy
against AD.
TABLE-US-00001 TABLE 1 Mutational studies of ABAD. ABAD mutations
A.beta. Binding* Experiments with ABAD truncations.sup..dagger.
GST-ABAD(1-186) + GST-ABAD(1-158) + GST-ABAD(159-261) - Experiments
with site-directed ABAD mutations.sup..dagger. G93A + S98A, K99A +
S98A, K99A, T100A, Y101A - S98A, K99A, Y101A - N102A + N102A, L103A
+ T108A, H109A, T110A - V156A + Q162A + *+: Specific binding
comparable to that observed with wild type ABAD; -: No observed
specific binding. .sup..dagger.Binding of .sup.125I-labeled
GST-ABAD or ABAD to immobilized A.beta. (1-42) as previously
described (I).
TABLE-US-00002 TABLE 2 Crystallographic statistics. Crystals of the
ABAD/A.beta. complex Space group P432 Cell dimensions a = 130.0
.ANG. Diffraction Data Resolution 30-2.3 .ANG. R.sub.sym (last
shell) 5.1% (15.2%) Completeness (last shell) 99.8% (99.8%) l/Sigl
(last shell) 57.7 (13.0) Molecular Replacement Resolution 10-4.0
.ANG. Number of rotations searched 181 Correlation coefficient
32.4% R 44.3% Refinement Resolution 30-2.3 .ANG. Sigma cutoff 0.0
Number of protein residues 208 Number of protein atoms 1480 Number
of solvent and ion atoms 121 Number of reflections used 17049 R (R
) 23.1% (26.1%) RMSD bond length 0.006 .ANG. RMSD bond ansle
1.1.degree. indicates data missing or illegible when filed
TABLE-US-00003 TABLE 3 ABAD interaction. Peptide K.sub.d(nM)
A.beta. 1-40 38.4 .+-. 4.6 A.beta. 1-42 55.8 .+-. 10.9 A.beta. 1-20
88.9 .+-. 19.9 A.beta. 25-35 >10.sup.5 PrP 109-141 >10.sup.6
Amylin >10.sup.7 *These studies were performed by immobilizing
the indicated amyloid-related peptide on microtiter wells followed
by blocking excess sites in the well, and then doing a binding
assay by addition of fluorescein-labeled ABAD. Similar results were
obtained using a radioligand binding assay (.sup.125I-labeled
ABAD).
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Sequence CWU 1
1
14186PRTHuman immunodeficiency virus 1Met Glu Pro Val Asp Pro Arg
Leu Glu Pro Trp Lys His Pro Gly Ser1 5 10 15Gln Pro Lys Thr Ala Cys
Thr Asn Cys Tyr Cys Lys Lys Cys Cys Phe 20 25 30His Cys Gln Val Cys
Phe Ile Thr Lys Ala Leu Gly Ile Ser Tyr Gly 35 40 45Arg Lys Lys Arg
Arg Gln Arg Arg Arg Pro Pro Gln Gly Ser Gln Thr 50 55 60His Gln Val
Ser Leu Ser Lys Gln Pro Thr Ser Gln Ser Arg Gly Asp65 70 75 80Pro
Thr Gly Pro Lys Glu 85211PRTHuman immunodeficiency virus 2Tyr Gly
Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5 10329PRTHuman 3Ala Gly Ile
Ala Val Ala Ser Lys Thr Tyr Asn Leu Lys Lys Gly Gln1 5 10 15Thr His
Thr Leu Glu Asp Phe Gln Arg Val Leu Asp Val 20 25421PRTHuman 4Ile
Ala Val Ala Ser Lys Thr Tyr Asn Leu Lys Lys Gly Gln Thr His1 5 10
15Thr Leu Glu Asp Phe 20520PRTHuman 5Asp Ala Glu Phe Arg His Asp
Ser Gly Tyr Glu Val His His Gln Lys1 5 10 15Leu Val Phe Phe
20619PRTHuman 6Ala Val Ala Ser Lys Thr Tyr Asn Leu Lys Lys Gly Gln
Thr His Thr1 5 10 15Leu Glu Asp719PRTRat 7Ala Val Ala Ile Lys Thr
Tyr His Glu Lys Lys Asn Gln Val His Thr1 5 10 15Leu Glu
Asp819PRTMouse 8Ala Val Ala Ile Lys Thr Tyr His Gln Lys Lys Asn Lys
Ile His Thr1 5 10 15Leu Glu Asp919PRTBovine 9Ala Val Ala Ser Lys
Thr Tyr Asn Leu Lys Lys Ser Gln Ala His Thr1 5 10 15Leu Glu
Asp1019PRTDrosophila 10Ala Thr Ala Val Lys Thr Phe Asn Phe Asn Lys
Asn Val Ala His Arg1 5 10 15Leu Glu Asp116PRTHuman 11Gly Pro Gln
Thr Lys Val1 51212PRTHuman 12Gly Gly Pro Lys Pro Phe Asp Met Pro
Met Ala Asp1 5 101313PRTHuman 13Gly Leu Leu Gly Pro Leu Glu Ala Leu
Gly Glu Asp Ala1 5 101413PRTStreptomyces exfoliatus 14Ser Thr Gly
Met Phe Leu Glu Thr Glu Ser Val Glu Arg1 5 10
* * * * *