US20080274975A1 - Methods and Compositions for Inhibiting Abad/Abeta Protein Interaction - Google Patents
Methods and Compositions for Inhibiting Abad/Abeta Protein Interaction Download PDFInfo
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- US20080274975A1 US20080274975A1 US11/578,401 US57840105A US2008274975A1 US 20080274975 A1 US20080274975 A1 US 20080274975A1 US 57840105 A US57840105 A US 57840105A US 2008274975 A1 US2008274975 A1 US 2008274975A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- ABAD Human A ⁇ -binding alcohol dehydrogenase
- ERAB Human A ⁇ -binding alcohol dehydrogenase
- HAB Human A ⁇ -binding alcohol dehydrogenase
- 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 ⁇ -induced cellular oxidant stress and cell death (1, 3).
- This invention provides a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to A ⁇ protein and comprises the sequence of amino acid residues 94-114 of ABAD.
- 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 ⁇ 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 ⁇ protein.
- 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 ⁇ protein.
- This invention further provides a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ protein.
- composition of matter comprising (a) a pharmaceutical carrier, and (b) a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ protein.
- This invention further provides a composition of matter comprising a polypeptide consisting of a portion of A ⁇ protein comprising the sequence of amino acid residues 1-20 of A ⁇ protein, wherein the composition binds to ABAD.
- 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 ⁇ protein comprising the sequence of amino acid residues 1-20 of A ⁇ protein, wherein the composition binds to ABAD.
- 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 ⁇ protein and ABAD in the subject's cells.
- 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 ⁇ protein and ABAD.
- 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 ⁇ protein and ABAD.
- 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 ⁇ protein and ABAD.
- 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 ⁇ protein and ABAD.
- 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 ⁇ 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.
- 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 ⁇ 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.
- This invention further provides an article of manufacture comprising a packaging material having therein a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ 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.
- 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 ⁇ protein comprising the sequence of amino acid residues 1-20 of A ⁇ 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.
- This invention further provides a method for determining whether an agent inhibits the binding of A ⁇ protein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to A ⁇ protein and comprises the sequence of amino acid residues 94-114 of ABAD, (ii) A ⁇ protein, and (iii) the agent, under conditions which would permit binding of the polypeptide and A ⁇ protein in the absence of the agent; (b) determining the amount of polypeptide bound to A ⁇ 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 ⁇ protein to ABAD.
- This invention further provides a method for determining whether an agent inhibits the binding of A ⁇ protein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ 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 ⁇ protein to ABAD.
- this invention provides a method for determining whether an agent inhibits the binding of A ⁇ protein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to A ⁇ protein and comprises the sequence of amino acid residues 94-114 of ABAD, (ii) a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ 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
- FIG. 1 ABAD-A ⁇ association in AD patients and transgenic mice.
- A Co-immunoprecipitation of ABAD and A ⁇ 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 ⁇ 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.
- Cox IV cytochrome c oxidase IV
- PDI protein disulfide isomerase
- C Co-localization of ABAD and A ⁇ 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
- Mouse anti-VDAC (20 ⁇ g/ml) guinea pig anti-ABAD (10 ⁇ g/ml) and rabbit anti-A ⁇ (5 ⁇ g/ml) IgGs were used in C-D.
- E Colocalization of ABAD and A ⁇ in mitochondria of the brain of a patient with AD using electron microscopy.
- Double immunogold staining was performed with rabbit anti-A ⁇ IgG and mouse anti-ABAD IgG followed by goat anti-rabbit IgG conjugated to 12 nm gold particles (for A ⁇ 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 ⁇ .
- FIG. 2 Crystal structure of A ⁇ -bound human ABAD.
- A A ribbon diagram with labeled secondary structures and the L D , L E and L F loops.
- B Superposition of A ⁇ -bound human ABAD (magenta) and rat ABAD in complex with NAD (cyan). The L D loop of 3 ⁇ -hydroxysteroid dehydrogenase (3 ⁇ -HSD) (PDB code 1FJH) is shown in yellow. The proposed A ⁇ -binding loop is indicated.
- C Superposition of the active sites of A ⁇ -bound human ABAD (magenta) and rat ABAD (cyan), showing distortion of the NAD binding site and the catalytic triad S155, K162 and Y168.
- FIG. 3 Biochemical and functional effects of ABAD-DP.
- A-B Effect of mutations in the L D loop of ABAD on A ⁇ binding (A) and inhibition of ABAD-A ⁇ 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.
- the mobile (injected) phase was AD (1-40) and for B, the mobile phase was a mixture of A ⁇ (1-42) and either ABAD-DP or ABAD-RP (range of concentrations).
- C Inhibition of A ⁇ -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; t P ⁇ 0.05, versus without ABAD-DP treatment.
- 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.
- 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 ⁇ 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.
- protein extracts prepared from the cerebellum, a region spared from the AD pathology showed no significant differences between AD patient and ND controls.
- FIG. 6 Demonstration of ABAD-A ⁇ complex in brains of Tg mAPP/ABAD mice.
- A Co-immunoprecipitation of ABAD and A ⁇ from mitochondria of transgenic mice. Mitchondrial fractions (500 ⁇ g) from cerebral cortex of nonTg, Tg mAPP and Tg mAPP/ABAD mice were immunoprecipitated with mouse anti-Afi IgG (6E10; 8 ⁇ g/ml), or nonimmune IgG (8 ⁇ g/ml) at 4° 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 ⁇ -actin for crude extracts from mouse brains.
- B Co-localization of ABAD and A ⁇ in the brain of a Tg mAPP/ABAD mouse using confocal microscopy with antibodies to ABAD and A ⁇ (color not shown) (magnification 300-fold).
- C Colocalization of ABAD and A ⁇ in mitochondria of brains from Tg mAPP/ABAD mouse using electron microscopy. Double immunogold staining was performed with rabbit anti-A ⁇ IgG and mouse anti-ABAD IgG followed by goat anti-rabbit IgG conjugated to 12 nm gold particles (for A ⁇ 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 ⁇ .
- FIG. 7 A, SDS-PAGE of washed and dissolved crystals of human ABAD and A ⁇ . Lanes from left to right: ABAD standard, A ⁇ (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 D and L E loops are very similar. The L F loop is ordered in the human structure but disordered in the rat structure.
- FIG. 8 A, Inhibition of ABAD-A ⁇ (1-40) interaction by ABAD-DP.
- ABAD was immobilized on the sensor chip of the Biacore and A ⁇ (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 ⁇ -induced generation of ROS (B), DNA fragmentation (C) and LDH release (D) by ABAD-DP, but not by ABAD-RP.
- ABAD-DP For inhibition by ABAD peptides, cells were pre-incubated with ABAD-DP or ABAD-RP (10 ⁇ M) for 60 min before A ⁇ treatment. *P ⁇ 0.05, versus nonTg cells; t P ⁇ 0.05, versus without ABAD-DP treatment.
- 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.
- 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.
- “Inhibit,” when used in connection with the binding between A ⁇ 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.
- “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.
- “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.
- polypeptide peptide
- protein protein
- Subject shall mean any animal, such as a human, non-human primate, mouse, rat, guinea pig or rabbit.
- Tat HIV Tat protein
- Tat protein 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-lys-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-arg-arg-pro-pro-gln-gly-ser-gln-thr-his-gln-val-ser-leu-ser-lys-lys-gln-pro-thr-thr-his-gln-val-ser-leu-ser-ly
- 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.
- Treating” a disorder shall mean slowing, stopping or reversing the disorder's progression.
- treating a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself.
- ameliorating a disorder and treating a disorder are equivalent.
- 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 ⁇ protein binds to ABAD in the mitochondria.
- this invention provides a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to A ⁇ protein and comprises the sequence of amino acid residues 94-114 of ABAD.
- the ABAD is human ABAD.
- 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).
- 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 ⁇ -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 ⁇ protein.
- the ABAD is human ABAD.
- the composition is further comprised by a pharmaceutical carrier.
- 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 ⁇ protein.
- the ABAD is human ABAD.
- the composition is further comprised by a pharmaceutical carrier.
- This invention further provides for an isolated polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ protein.
- the A ⁇ protein is human A ⁇ protein.
- the full sequence of human A ⁇ is set forth in (1) at page 690, FIG. 1 .
- the sequence of amino acid residues 1-20 of human A ⁇ is as follows: DAEFRHDSGYEVHHQKLVFF (SEQ ID NO. 5).
- composition of matter comprising (a) a pharmaceutical carrier, and (b) a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ protein.
- compositions of matter comprising a polypeptide consisting of a portion of A ⁇ protein comprising the sequence of amino acid residues 1-20 of A ⁇ protein, wherein the composition binds to ABAD.
- the A ⁇ protein is human A ⁇ protein.
- the composition is further comprised by a pharmaceutically acceptable carrier.
- 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 ⁇ protein comprising the sequence of amino acid residues 1-20 of A ⁇ protein, wherein the composition binds to ABAD.
- the A ⁇ protein is human A ⁇ protein.
- the composition is further comprised by a pharmaceutically acceptable carrier.
- 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 ⁇ protein and ABAD in the subject's cells.
- the subject is human.
- the cells are neuronal cells.
- 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.
- the agent is a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to A ⁇ protein and comprises the sequence of amino acid residues 94-114 of ABAD.
- 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 ⁇ protein.
- the agent is a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ protein.
- the agent is a composition of matter comprising a polypeptide consisting of a portion of A ⁇ protein comprising the sequence of amino acid residues 1-20 of A ⁇ protein, wherein the composition binds to ABAD.
- 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 ⁇ protein and ABAD.
- the agent enters the cell's mitochondria once present in the cell's cytosol.
- the cell is a neuronal cell.
- the agent is a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to A ⁇ protein and comprises the sequence of amino acid residues 94-114 of ABAD.
- 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 ⁇ protein.
- the agent is a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ protein.
- the agent is a composition of matter comprising a polypeptide consisting of a portion of A ⁇ protein comprising the sequence of amino acid residues 1-20 of A ⁇ protein, wherein the composition binds to ABAD.
- 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 ⁇ protein and ABAD.
- the cell is a neuronal cell.
- the agent is a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to A ⁇ protein and comprises the sequence of amino acid residues 94-114 of ABAD.
- 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 ⁇ protein.
- the agent is a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ protein.
- the agent is a composition of matter comprising a polypeptide consisting of a portion of A ⁇ protein comprising the sequence of amino acid residues 1-20 of A ⁇ protein, wherein the composition binds to ABAD.
- 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 ⁇ protein and ABAD.
- the cell is a neuronal cell.
- the agent is a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to A ⁇ protein and comprises the sequence of amino acid residues 94-114 of ABAD.
- 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 ⁇ protein.
- the agent is a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ protein.
- the agent is a composition of matter comprising a polypeptide consisting of a portion of A ⁇ protein comprising the sequence of amino acid residues 1-20 of A ⁇ protein, wherein the composition binds to ABAD.
- 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 ⁇ protein and ABAD.
- the cell is a neuronal cell.
- 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.
- 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 ⁇ protein.
- the agent is a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ protein.
- the agent is a composition of matter comprising a polypeptide consisting of a portion of A ⁇ protein comprising the sequence of amino acid residues 1-20 of A ⁇ protein, wherein the composition binds to ABAD.
- 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 ⁇ 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.
- the subject is a human.
- 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 ⁇ 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.
- the subject is a human.
- This invention further provides an article of manufacture comprising a packaging material having therein a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ 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.
- the subject is a human.
- 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 ⁇ protein comprising the sequence of amino acid residues 1-20 of A ⁇ 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.
- the subject is a human.
- This invention further provides a method for determining whether an agent inhibits the binding of A ⁇ protein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to A ⁇ protein and comprises the sequence of amino acid residues 94-114 of ABAD, (ii) A ⁇ protein, and (iii) the agent, under conditions which would permit binding of the polypeptide and A ⁇ protein in the absence of the agent; (b) determining the amount of polypeptide bound to A ⁇ 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 ⁇ protein to ABAD.
- the portion of ABAD and/or of A ⁇ protein is unmodified, i.e., it contains no amino acid residue derivatives and its amino acid residues are bound by peptide bonds.
- the portion of ABAD and/or of A ⁇ 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.
- This invention further provides a method for determining whether an agent inhibits the binding of A ⁇ protein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ 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 ⁇ protein to ABAD.
- This invention further provides a method for determining whether an agent inhibits the binding of A ⁇ protein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to A ⁇ protein and comprises the sequence of amino acid residues 94-114 of ABAD, (ii) a polypeptide consisting of a portion of A ⁇ protein, wherein the portion of A ⁇ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of A ⁇ 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
- 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.
- 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.
- 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° 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.
- Crystallography Expression and purification of human ABAD was performed as described (3,7).
- 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 ⁇ (residues 1-40; Biosource, CA).
- the mixture was crystallized by vapor diffusion at 22° 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 ⁇ -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.
- 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-Thr-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).
- a ⁇ (1-40) or A ⁇ (1-42) alone or with either ABAD-DP or ABAD-RP (range of concentrations) was injected at a flow rate of 30 ⁇ l/min for 2 min at 25° 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 i .
- cytochrome c release 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 ⁇ M) or ABAD-RP (10 ⁇ M) for 60 minutes, followed by incubation with A ⁇ (1-42; 1 ⁇ M) for nonTg and Tg ABAD mice. After 24 hours at 37° 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° C.
- the pellet was resuspended in 400 ⁇ l of lysis buffer containing 50 mM Tris at pH 7.4, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 2 ⁇ g/ml leupeptin, 1 mM PMSF, and 1 ⁇ 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° C. (11). The resulting pellets were resuspended in 50 ⁇ l of lysis buffer and supernatants were concentrated to 20 ⁇ l.
- the protein content of each fraction was determined by the BioRad DC protein assay (BioRad Laboratories, Hercules, Calif.). Samples (40 ⁇ g/lane) were subjected to 12% SDS-PAGE, followed by immunoblotting using anti-cytochrome c antibody (1:500) (Phamergen).
- 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.
- 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 ⁇ 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.
- Immunogold electron microscopy using gold-conjugated antibody systems provided further evidence for the presence of ABAD and A ⁇ within mitochondria (for ABAD, 18 nm gold particles and for A ⁇ , 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 ).
- these microscopic and immunoprecipitation data demonstrate the formation of ABAD-A ⁇ complex within mitochondria in vivo.
- the L F loop is also disordered in the rat ABAD structure in complex with NAD, while the L D loop region is well ordered, suggesting that A ⁇ binding may have influenced the L D loop dynamics and conformation.
- the L 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 ⁇ , it was hypothesized that the L D loop may be a recognition site for A ⁇ .
- ABAD-DP ABAD decoy peptide
- ABAD reversed peptide a peptide of the reverse sequence (residues 120-92, termed ABAD reversed peptide, or ABAD-RP) was completely inactive.
- 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 ⁇ -induced cellular stress and apoptosis.
- HIV human immunodeficiency virus-type 1
- cultured neurons from Tg ABAD mice exposed to A ⁇ display subsequent DNA fragmentation ( FIG. 8C ), and LDH release ( FIG. 8D ).
- mice overexpressing ABAD in an A ⁇ -rich environment i.e., Tg mAPP/ABAD mice
- Electron paramagnetic spin resonance (EPR) spectroscopy was used to measure the level of ROS in intact frozen brain at 77° 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.
Abstract
Description
- 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.
- 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.
- Human Aβ-binding alcohol dehydrogenase (“ABAD,” also known as “ERAB” and “HSD-10”) was the only protein identified from four positive clones that bound Aβ protein (also referred to as “Aβ”) 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 Aβ is highly specific and starts to occur at nanomolar concentrations. At micromolar concentrations, Aβ, 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 Aβ-induced cellular oxidant stress and cell death (1, 3). - This invention provides a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to Aβ protein and comprises the sequence of amino acid residues 94-114 of ABAD.
- 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 Aβ 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 Aβ protein.
- 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 Aβ protein.
- This invention further provides a polypeptide consisting of a portion of Aβ protein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ protein.
- This invention further provides a composition of matter comprising (a) a pharmaceutical carrier, and (b) a polypeptide consisting of a portion of Aβ protein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ protein.
- This invention further provides a composition of matter comprising a polypeptide consisting of a portion of Aβprotein comprising the sequence of amino acid residues 1-20 of Aβ protein, wherein the composition binds to ABAD.
- This invention further provides a composition of matter comprising (a) a Tat protein operatively affixed to (b) a polypeptide consisting of a portion of Aβ protein comprising the sequence of amino acid residues 1-20 of Aβ protein, wherein the composition binds to ABAD.
- 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 Aβ protein and ABAD in the subject's cells.
- 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 Aβ protein and ABAD.
- 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 Aβ protein and ABAD.
- 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 Aβ protein and ABAD.
- 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 Aβ protein and ABAD.
- 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 Aβ 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.
- 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 Aβ 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.
- This invention further provides an article of manufacture comprising a packaging material having therein a polypeptide consisting of a portion of Aβprotein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ 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.
- 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 Aβ protein comprising the sequence of amino acid residues 1-20 of Aβ 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.
- This invention further provides a method for determining whether an agent inhibits the binding of Aβprotein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to Aβ protein and comprises the sequence of amino acid residues 94-114 of ABAD, (ii) Aβ protein, and (iii) the agent, under conditions which would permit binding of the polypeptide and Aβprotein in the absence of the agent; (b) determining the amount of polypeptide bound to Aβ 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 Aβ protein to ABAD.
- This invention further provides a method for determining whether an agent inhibits the binding of Aβprotein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of Aβ protein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ 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 Aβprotein to ABAD.
- Finally, this invention provides a method for determining whether an agent inhibits the binding of Aβprotein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to Aβ protein and comprises the sequence of amino acid residues 94-114 of ABAD, (ii) a polypeptide consisting of a portion of Aβprotein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ 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 Aβ protein to ABAD.
-
FIG. 1 . ABAD-Aβ association in AD patients and transgenic mice. (A) Co-immunoprecipitation of ABAD and Aβ 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 μg 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 Aβ 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 μg/ml), guinea pig anti-ABAD (10 μg/ml) and rabbit anti-Aβ (5 μg/ml) IgGs were used in C-D. (E) Colocalization of ABAD and Aβ in mitochondria of the brain of a patient with AD using electron microscopy. Double immunogold staining was performed with rabbit anti-Aβ IgG and mouse anti-ABAD IgG followed by goat anti-rabbit IgG conjugated to 12 nm gold particles (for Aβ 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 Aβ. -
FIG. 2 . Crystal structure of Aβ-bound human ABAD. (A) A ribbon diagram with labeled secondary structures and the LD, LE and LF loops. (B) Superposition of Aβ-bound human ABAD (magenta) and rat ABAD in complex with NAD (cyan). The LD loop of 3α-hydroxysteroid dehydrogenase (3α-HSD) (PDB code 1FJH) is shown in yellow. The proposed Aβ-binding loop is indicated. (C) Superposition of the active sites of Aβ-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 LD 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. -
FIG. 3 . Biochemical and functional effects of ABAD-DP. (A-B) Effect of mutations in the LD loop of ABAD on Aβ binding (A) and inhibition of ABAD-Aβinteraction 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 Aβ (1-42) and either ABAD-DP or ABAD-RP (range of concentrations). (C) Inhibition of Aβ-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; tP<0.05, versus without ABAD-DP treatment. -
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. -
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 μg 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. -
FIG. 6 . Demonstration of ABAD-Aβ complex in brains of Tg mAPP/ABAD mice. A, Co-immunoprecipitation of ABAD and Aβ from mitochondria of transgenic mice. Mitchondrial fractions (500 μg) from cerebral cortex of nonTg, Tg mAPP and Tg mAPP/ABAD mice were immunoprecipitated with mouse anti-Afi IgG (6E10; 8 μg/ml), or nonimmune IgG (8 μg/ml) at 4° 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 β-actin for crude extracts from mouse brains. B, Co-localization of ABAD and Aβ in the brain of a Tg mAPP/ABAD mouse using confocal microscopy with antibodies to ABAD and Aβ (color not shown) (magnification 300-fold). C, Colocalization of ABAD and Aβ in mitochondria of brains from Tg mAPP/ABAD mouse using electron microscopy. Double immunogold staining was performed with rabbit anti-Aβ IgG and mouse anti-ABAD IgG followed by goat anti-rabbit IgG conjugated to 12 nm gold particles (for Aβ1-42) 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 Aβ. -
FIG. 7 . A, SDS-PAGE of washed and dissolved crystals of human ABAD and Aβ. Lanes from left to right: ABAD standard, Aβ (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 LD and LE loops are very similar. The LF loop is ordered in the human structure but disordered in the rat structure. C, Sequence alignment of the disordered part of the LD 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). -
FIG. 8 . A, Inhibition of ABAD-Aβ (1-40) interaction by ABAD-DP. ABAD was immobilized on the sensor chip of the Biacore and Aβ (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 Aβ-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 μM) for 60 min before Aβtreatment. *P<0.05, versus nonTg cells; tP<0.05, versus without ABAD-DP treatment. - “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.
- “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.
- “Inhibit,” when used in connection with the binding between Aβ 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.
- “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.
- “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.
- The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein, and each means a polymer of amino acid residues.
- “Subject” shall mean any animal, such as a human, non-human primate, mouse, rat, guinea pig or rabbit.
- “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-lys-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-arg-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.
- “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.
- 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 Aβ protein binds to ABAD in the mitochondria.
- Specifically, this invention provides a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to Aβ 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).
- 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 Aβ-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 Aβ 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.
- 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 Aβ 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.
- This invention further provides for an isolated polypeptide consisting of a portion of Aβ protein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ protein. In the preferred embodiment of this polypeptide, the Aβ protein is human Aβ protein. The full sequence of human Aβ is set forth in (1) at page 690,
FIG. 1 . The sequence of amino acid residues 1-20 of human Aβ is as follows: DAEFRHDSGYEVHHQKLVFF (SEQ ID NO. 5). - This invention further provides a composition of matter comprising (a) a pharmaceutical carrier, and (b) a polypeptide consisting of a portion of Aβ protein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ protein.
- This invention further provides a composition of matter comprising a polypeptide consisting of a portion of Aβ protein comprising the sequence of amino acid residues 1-20 of Aβ protein, wherein the composition binds to ABAD. In the preferred embodiment of this composition, the Aβ protein is human Aβ protein. In another embodiment, the composition is further comprised by a pharmaceutically acceptable carrier.
- This invention further provides a composition of matter comprising (a) a Tat protein operatively affixed to (b) a polypeptide consisting of a portion of Aβ protein comprising the sequence of amino acid residues 1-20 of Aβ protein, wherein the composition binds to ABAD. In the preferred embodiment of this composition, the Aβprotein is human Aβ protein. In another embodiment, the composition is further comprised by a pharmaceutically acceptable carrier.
- 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 Aβ protein and ABAD in the subject's cells.
- In the preferred embodiment of this method, the subject is human. In another embodiment, the cells are neuronal cells.
- 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.
- In one embodiment of this method, the agent is a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to Aβ 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 Aβ protein. In yet another embodiment, the agent is a polypeptide consisting of a portion of Aβ protein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ protein. In yet another embodiment, the agent is a composition of matter comprising a polypeptide consisting of a portion of Aβ protein comprising the sequence of amino acid residues 1-20 of Aβ protein, wherein the composition binds to ABAD.
- 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 Aβ 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.
- In one embodiment of this method, the cell is a neuronal cell.
- In one embodiment of this method, the agent is a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to Aβ 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 Aβprotein. In yet another embodiment, the agent is a polypeptide consisting of a portion of Aβ protein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ protein. In yet another embodiment, the agent is a composition of matter comprising a polypeptide consisting of a portion of Aβ protein comprising the sequence of amino acid residues 1-20 of Aβ protein, wherein the composition binds to ABAD.
- 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 Aβ protein and ABAD.
- In one embodiment of this method, the cell is a neuronal cell.
- In one embodiment of this method, the agent is a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to Aβ 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 Aβprotein. In yet another embodiment, the agent is a polypeptide consisting of a portion of Aβ protein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ protein. In yet another embodiment, the agent is a composition of matter comprising a polypeptide consisting of a portion of Aβ protein comprising the sequence of amino acid residues 1-20 of Aβ protein, wherein the composition binds to ABAD.
- 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 Aβ protein and ABAD.
- In one embodiment of this method, the cell is a neuronal cell.
- In one embodiment of this method, the agent is a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to Aβ 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 Aβprotein. In yet another embodiment, the agent is a polypeptide consisting of a portion of Aβ protein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ protein. In yet another embodiment, the agent is a composition of matter comprising a polypeptide consisting of a portion of Aβ protein comprising the sequence of amino acid residues 1-20 of Aβ protein, wherein the composition binds to ABAD.
- 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 Aβ protein and ABAD.
- In one embodiment of this method, the cell is a neuronal cell.
- 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 Aβprotein. In yet another embodiment, the agent is a polypeptide consisting of a portion of Aβ protein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ protein. In yet another embodiment, the agent is a composition of matter comprising a polypeptide consisting of a portion of Aβ protein comprising the sequence of amino acid residues 1-20 of Aβ protein, wherein the composition binds to ABAD.
- 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 Aβ 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.
- 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 Aβ 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.
- This invention further provides an article of manufacture comprising a packaging material having therein a polypeptide consisting of a portion of Aβprotein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ 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.
- 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 Aβ protein comprising the sequence of amino acid residues 1-20 of Aβ 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.
- This invention further provides a method for determining whether an agent inhibits the binding of Aβprotein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to Aβ protein and comprises the sequence of amino acid residues 94-114 of ABAD, (ii) Aβ protein, and (iii) the agent, under conditions which would permit binding of the polypeptide and Aβprotein in the absence of the agent; (b) determining the amount of polypeptide bound to Aβ 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 Aβ protein to ABAD.
- 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 Aβprotein 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 Aβ 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.
- This invention further provides a method for determining whether an agent inhibits the binding of Aβprotein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of Aβ protein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ 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 Aβprotein to ABAD.
- This invention further provides a method for determining whether an agent inhibits the binding of Aβprotein to ABAD, which comprises: (a) admixing (i) a polypeptide consisting of a portion of ABAD, wherein the portion of ABAD binds to Aβ protein and comprises the sequence of amino acid residues 94-114 of ABAD, (ii) a polypeptide consisting of a portion of Aβprotein, wherein the portion of Aβ protein binds to ABAD and comprises the sequence of amino acid residues 1-20 of Aβ 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 Aβ protein to ABAD.
- 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.
- 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 μg/ml PMSF, 1 μg/ml aprotinin), immunoprecipitated with rabbit anti-Aβ IgG (3 μl/500 μg protein) at 4° C. overnight, and Western blotting was done with mouse anti-ABAD IgG (1:1000). Immunoprecipitation was performed on incubation of crude extracts (500 μg) from cerebral cortex with anti-Aβ 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.
- 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.
- 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° 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.
- 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 Aβ (residues 1-40; Biosource, CA). The mixture was crystallized by vapor diffusion at 22° 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 Aβ-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.
- 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-Thr-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, Aβ(1-40) or Aβ(1-42) alone or with either ABAD-DP or ABAD-RP (range of concentrations) was injected at a flow rate of 30 μl/min for 2 min at 25° 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 Ki. - 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 μM) or ABAD-RP (10 μM) for 60 minutes, followed by incubation with Aβ(1-42; 1 μM) for nonTg and Tg ABAD mice. After 24 hours at 37° 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° C. The pellet was resuspended in 400 μl of lysis buffer containing 50 mM Tris at pH 7.4, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 2 μg/ml leupeptin, 1 mM PMSF, and 1 μg/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° C. (11). The resulting pellets were resuspended in 50 μl of lysis buffer and supernatants were concentrated to 20 μl. The protein content of each fraction was determined by the BioRad DC protein assay (BioRad Laboratories, Hercules, Calif.). Samples (40 μg/lane) were subjected to 12% SDS-PAGE, followed by immunoblotting using anti-cytochrome c antibody (1:500) (Phamergen).
- 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 μM 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 Aβ (1-42, 4 μg/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 Aβ-induced and spontaneous DNA fragmentation (Cell Death Detection ELISAPLUS, Roche Diagnostics Co. Indianapolis, Ind.) and LDH release (Sigma).
- 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.
- 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).
- It was speculated that interaction of Aβ with ABAD might induce mitochondrial dysfunction. However, since it had not been established that intracellular Aβ can access mitochondria, it was essential to determine whether ABAD and Aβ interact in pathophysiologically relevant settings. To address this directly, ABAD-Aβ complex was detected in AD brains by immunoprecipitation of cortical protein extracts using anti-Aβ followed by immunoblotting with anti-ABAD IgG (
FIG. 1A ). Age-matched non-demented brain displayed very little ABAD-Aβ 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-Aβ 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. - Confocal microscopy was used to confirm mitochondrial co-localization and interaction of ABAD and Aβ. In the cerebral cortex of AD patients, images of anti-ABAD and anti-Aβ (color not shown), detecting endogenous ABAD and Aβ, extensively co-localize (
FIG. 1C ). Similarly, in the cerebral cortex of Tg mAPP/ABAD mice, there is extensive overlap of immunoreactive ABAD and Aβ (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 Aβ 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 Aβ within mitochondria (for ABAD, 18 nm gold particles and for Aβ, 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-Aβ complex within mitochondria in vivo. - To determine the structural basis of the ABAD-Aβinteraction, human ABAD was crystallized and its structure was determined, in the presence of NAD and a molar excess of Aβ, at 2.3 Å resolution (
FIG. 2A , Table 2). Unexpectedly, NAD is not bound to ABAD in the crystal structure. Under the crystallization condition, Aβ inhibits ABAD activity, though, in the absence of Aβ, ABAD displays normal activity. This suggests that Aβ prevents NAD binding in the crystal structure, which may be the molecular basis for its inhibition of ABAD activity. - SDS-PAGE and amino terminal sequencing of washed and dissolved crystals showed that both ABAD and Aβ are present in the crystals (
FIG. 7A ). However, no electron density is observed for Aβ, suggesting that Aβitself and the region of ABAD that binds to Aβ must be disordered in the crystal. To exclude the possibility that Aβ 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, Aβ(1-42), Aβ(1-40) and Aβ(1-20) demonstrated dose-dependent binding, whereas Aβ(25-35) showed no specific binding (Table 3). - Comparison of the Aβ-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 Aβ-bound ABAD displays significant distortion of the NAD-binding pocket and the catalytic triad (
FIG. 2B , 2C, 7B). The majority of the LD loop, the beginning of the following αD helix, and the latter part of the LF loop of human ABAD are disordered. While the LE loop is ordered, the conformation of the loop and the beginning of the following αE helix is significantly different from the structure without Aβ. - In the absence of substrate, the LF loop is also disordered in the rat ABAD structure in complex with NAD, while the LD loop region is well ordered, suggesting that Aβ binding may have influenced the LD loop dynamics and conformation. In addition, within the NAD-dependent short-chain dehydrogenase/reductase (SDR) superfamily, the LD 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 Aβ, it was hypothesized that the LD loop may be a recognition site for Aβ. - Inspection of crystal packing showed that the ordered ends of the LD loop point into interconnected huge solvent channels with estimated dimensions of 70 Å (
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 Aβ, 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. - To test the idea that the LD loop is important in Aβinteraction, structure-based mutational analyses were performed (Table 1). Binding studies using Aβ and GST-ABAD truncation mutants showed that the amino terminal portion of ABAD (residues 1-158) is responsible for Aβ interaction. Site-directed mutagenesis within and beyond the disordered LD loop region (residues 95-113) specifically located two stretches of ABAD residues important for Aβ binding, residues S98-Y101 and residues T108-T110. An ABAD mutant bearing S98A, K99A and Y101A mutations exhibited no specific interaction to Aβ in a surface plasmon resonance experiment, although wild-type ABAD displayed dose-dependent interaction with Aβ (
FIG. 3A ). - To determine whether the LD loop is sufficient for Aβ 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 Aβ using surface plasmon resonance was tested (
FIG. 3B ). ABAD-DP inhibited binding of Aβ (1-40) (FIG. 8A ) and Aβ (1-42) (FIG. 3B ) to immobilized intact ABAD with inhibitory constants of 4.9 and 1.7 μM, 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 LD region alone could mediate Aβ binding, although it may not be the exclusive site. - To develop a specific inhibitor of the ABAD-Aβinteraction 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 Aβ-induced cellular stress and apoptosis. While cultured wild-type cortical neurons exposed to Aβ(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 Aβ-induced cytochrome c release (
FIG. 3C ). Similarly, ABAD-DP also protected against Aβ-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. - The protective effects of cell permeable ABAD-DP are consistent with the hypothesis that the ABAD-Aβ interaction causes mitochondrial stress and apoptosis. Since mitochondria are the principal sites of generation of reactive oxygen species (ROS) under physiologic conditions, and Aβ is known to trigger oxidative stress, we tested whether the protection from Aβ-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 Aβ, 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 Aβ-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 Aβ-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 Aβ display subsequent DNA fragmentation (FIG. 8C ), and LDH release (FIG. 8D ). In each case, pretreatment with ABAD-DP, not ABAD-RP, attenuated cytotoxicity. - If the above results in cell culture could be extrapolated to in vivo settings, then mice overexpressing ABAD in an Aβ-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° 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. - 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 bytrials 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 ). - The data demonstrate that ABAD and Aβ directly interact in mitochondria in AD, and that this interaction promotes leakage of ROS, mitochondrial dysfunction and cell death, potentially underlying the mechanism of Aβ-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 Aβ may exert an important pathogenic role in the mitochondrial compartment through an interaction with ABAD and that inhibition of ABAD-Aβ interaction may provide a new treatment strategy against AD.
-
TABLE 1 Mutational studies of ABAD. ABAD mutations Aβ Binding* Experiments with ABAD truncations† GST-ABAD(1-186) + GST-ABAD(1-158) + GST-ABAD(159-261) − Experiments with site-directed ABAD mutations† 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. †Binding of 125I-labeled GST-ABAD or ABAD to immobilized Aβ (1-42) as previously described (I). -
TABLE 2 Crystallographic statistics. Crystals of the ABAD/Aβ complex Space group P432 Cell dimensions a = 130.0 Å Diffraction Data Resolution 30-2.3 Å Rsym (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 Å Number of rotations searched 181 Correlation coefficient 32.4% R 44.3% Refinement Resolution 30-2.3 Å 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 Å RMSD bond ansle 1.1° indicates data missing or illegible when filed -
TABLE 3 ABAD interaction. Peptide Kd(nM) Aβ 1-40 38.4 ± 4.6 Aβ 1-42 55.8 ± 10.9 Aβ 1-20 88.9 ± 19.9 Aβ 25-35 >105 PrP 109-141 >106 Amylin >107 *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 (125I-labeled ABAD). -
- 1. S. D. Yan, et al., Nature 389, 689-95 (1997).
- 2. K. Beyreuther, C. L. Masters, Nature 389, 677-8 (1997).
- 3. S. D. Yan, et al., J Biol Chem 274, 2145-56 (1999).
- 4. U. C. Oppermann, S. Salim, L. O. Tjernberg, L. Terenius, H. Jornvall, FEBS Lett 451, 238-42 (1999).
- 5. L. Torroja, D. Ortuno-Sahagun, A. Ferrus, B. Hammerle, J. A. Barbas, J Cell Biol 141, 1009-17 (1998).
- 6. L. Mucke, et al.,
J Neurosci 20, 4050-8 (Jun. 1, 2000). - 7. S. D. Yan, et al., J Biol Chem 275, 27100-9 (2000).
- 8. A. J. Powell, et al., J Mol Biol 303, 311-27 (2000).
- 9. M. A. Abreo, et al., in US Patent and Trademark Office (USA, 2002), vol. 9.
- 10. M. Aarts, et al., Science 298, 846-50 (Oct. 25, 2002).
- 11. M. Becker-Hapak, S. S. McAllister, S. F. Dowdy, Methods 24, 247-56 (July, 2001).
- 12. H. P. Grill, J. L. Zweier, P. Kuppusamy, M. L. Weisfeldt, J. T. Flaherty, J
Am Coll Cardiol 20, 1604-11 (December 1992). - 13. D. Morgan, et al., Nature 408, 982-5 (Dec. 21-28, 2000).
- 14. G. Di Rosa, T. Odrijin, R. A. Nixon, O. Arancio, J Mol Neurosci 19, 135-41 (August-October 2002).
- 15. R. H. Swerdlow, S. J. Kish, Int Rev Neurobiol 53, 341-85 (2002).
- 16. R. Castellani, et al., J Neurosci Res 70, 357-60 (Nov. 1, 2002).
- 17. A. D. Cash, et al., Neuroscientist 8, 489-96 (October 2002).
- 18. J. P. Blass, Int Rev Neurobiol 51, 325-76 (2002).
- 19. A. C. Rego, C. R. Oliveira, Neurochem Res 28, 1563-74 (October 2003).
- 20. G. Aliev, et al.,
Neurol Res 25, 665-74 (September 2003). - 21. J. P. Blass,
Neurol Res 25, 556-66 (September 2003). - 22. M. P. Mattson, Int Rev Neurobiol 53, 387-409 (2002).
- 23. H. K. Anandatheerthavarada, G. Biswas, M. A. Robin, N. G. Avadhani, J Cell Biol 161, 41-54 (Apr. 14, 2003).
- 24. Z. Otwinowski, W. Minor, Methods Enzymol. 276, 307-326 (1997).
- 25. L. Tong, J. Appl. Cryst. 26, 748-751 (1993)
- 26. A. T. Brunger et al., Acta Crystallogr. D54, 905-21 (1998).
- 27. T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, Acta Crystallgr. A47, 110-119 (1991).
- 28. S. V. Evans, J. Mol. Graph. 11, 134-8 (1993).
- 29. T. Valdes-Gonzalez, J. Inagawa, T. Ido, Peptides 22, 1099-106 (July, 2001).
- 30. K. Takuma, et al., J Biol Chem 276, 48093-9 (Dec. 21, 2001).
- 31. J. P. Crow, Nitric Oxide: Biology and
Chemistry 1, 145-157 (1997). - 32. N. W. Kooy, J. A. Royall; H. Ischiropoulos, Free Radic Res 27, 245-54 (September 1997).
- 33. L. F. Lue et al., Experimental Neurology 171, 29-45 (2001).
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