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WO2006004824A1 - Oligomeres solubles d'amyloide beta permettant l'arret des fonctions cognitives - Google Patents

Oligomeres solubles d'amyloide beta permettant l'arret des fonctions cognitives Download PDF

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WO2006004824A1
WO2006004824A1 PCT/US2005/023070 US2005023070W WO2006004824A1 WO 2006004824 A1 WO2006004824 A1 WO 2006004824A1 US 2005023070 W US2005023070 W US 2005023070W WO 2006004824 A1 WO2006004824 A1 WO 2006004824A1
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amyloid
protein
oligomers
animal
mammal
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Karen H. Ashe
James P. Cleary
Dominic M. Walsh
Dennis J. Selkoe
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University of Minnesota Twin Cities
University of Minnesota System
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0312—Animal model for Alzheimer's disease

Definitions

  • AD Alzheimer's disease
  • a central unresolved problem in understanding Alzheimer's disease is the nature of the molecular entity causing dementia.
  • the brain in Alzheimer's disease contains soluble and insoluble assemblies of A ⁇ , both of which have been hypothesized to cause dementia (Hardy and Allsop, Trends Pharmacol Sci 1991, 12:383-8; Selkoe, Neuron 1991, 6:487-98; Price et al., Neurobiol Aging 1991, 12:295-312; Lorenzo and Yankner, Proc Natl Acad Sci USA 1994, 91:12243-7; Cummings and Cotman, Lancet 1995, 346:1524-8; McLean et al., Ann Neurol 1999, 46:860-6; and Naslund et al.,
  • the present invention includes a method of disrupting cognitive function in a mammal by administering isolated soluble oligomers of amyloid ⁇ protein intracranially.
  • the cognitive function disrupted is the memory of learned behavior.
  • the oligomers of amyloid ⁇ protein are administered intracerebroventricularly.
  • the oligomers of amyloid ⁇ protein contain no monomers of amyloid ⁇ protein.
  • the oligomers of amyloid ⁇ protein include dimers of amyloid ⁇ protein.
  • the oligomers of amyloid ⁇ protein are dimers of amyloid ⁇ protein.
  • the oligomers of amyloid ⁇ protein include trimers of the amyloid ⁇ protein. In some embodiments, the oligomers of amyloid ⁇ protein are trimers of amyloid ⁇ protein.
  • the mammal serves as an animal model system for Alzheimer's disease. In another aspect, the present invention includes an animal model for a neurological disorder in which soluble oligomers of amyloid ⁇ protein have been administered intracranially to an animal. In some embodiments, the animal model is an animal model for Alzheimer's disease. In some embodiments, the mammal may be a mouse, rat, dog, or non-human primate. In some embodiments, the mammal demonstrates disruption of complex learned behaviors.
  • the mammal demonstrates cognitive deficits of presymptomatic or early Alzheimer's disease.
  • the oligomers of amyloid ⁇ protein contain no monomers of amyloid ⁇ protein.
  • the oligomers of amyloid ⁇ protein include dimers of amyloid ⁇ protein.
  • the oligomers of amyloid ⁇ protein are dimers of amyloid ⁇ protein.
  • the oligomers of amyloid ⁇ protein include trimers of amyloid ⁇ protein.
  • the oligomers of amyloid ⁇ protein are trimers of amyloid ⁇ protein.
  • the present invention includes a method of assaying for the effects of soluble oligomers of the amyloid ⁇ protein on cognitive functioning, the method including administering soluble oligomers of amyloid ⁇ protein intracranially into a mammal and measuring cognitive functioning in the mammal.
  • the cognitive functioning of the mammal is compared to the cognitive functioning of another mammal treated in the same fashion except saline is administered intracranially, rather than soluble oligomers of amyloid ⁇ protein.
  • the present invention includes a method of separating soluble oligomers amyloid ⁇ protein from soluble monomers of amyloid ⁇ protein by subjecting a composition comprising both soluble oligomers of amyloid ⁇ protein and soluble monomers of amyloid ⁇ protein to size exclusion chromatography.
  • a "an,” “the,” and “at least one” are used interchangeably and mean one or more than one.
  • FIG. IA Conditioned medium (CM) from cells secreting human amyloid- ⁇ (A ⁇ ) oligomers and monomers disrupts learned behavior.
  • CM Conditioned medium
  • conditioned medium from 7PA2 cells (lane 1) and untransfected CHO cells (lane 3) were immunoprecipitated with the polyclonal anti-AB antibody R 1282 and immunoblotted with 6E10 (which recognizes an epitope encompassed by residues 6-10 of A ⁇ ).
  • Lane 2 shows the result of a second R1282 immunoprecipitation of the supernatant from lane 1.
  • the faint non-specific band just above the A ⁇ trimer (T) confirms equal protein loading of the 3 samples.
  • the AB concentration of 7PA2 conditioned medium used in lane 1 was measured by A ⁇ l -total ELISA (Johnson-Wood et al., Proc Natl Acad Sci USA 1979, 94:1550-5) and found to be 6412 ⁇ 767 picogram/milliliter (pg/ml).
  • the residual supernatant after the second immunoprecipitation with R 1282 contained no A ⁇ detected by ELISA, and this supernatant was injected as the R1282 immunoprecipitation control. Both mean switching errors (Fig. IB) and mean perseveration errors (Fig.
  • FIG. 2A-2C Oligomers but not monomers of A ⁇ disrupt learned behavior.
  • Fig. 2A AB oligomers and monomers in 7PA2 conditioned medium were fractionated by size exclusion chromatography. Both switching errors (Fig. 2B) and perseveration errors (Fig. 2C) increased two hours after rats received 7PA2 AB oligomers (fraction 7) but not monomers (fraction 9) by ICV injection on day 0. Both types of errors decreased on subsequent days and were at baseline by day 3, indicating that the disruption of learned behavior by AB oligomers is transient. Baseline error rates were means from the previous 7 sessions. Error bars are SEMs and asterisks (*) indicate statistical significance at p ⁇ 0.05.
  • Running response rates at each ratio response requirement value is the rate of responding on either lever, in responses per minute, occurring while a subject is actively pressing levers.
  • Running response rate is a direct function of the size of the response requirement. No significant differences in running response rate were found under ICV injections of 7PA2 conditioned medium (triangles) or A ⁇ oligomers (squares) when tested against baseline running response rates (diamonds) at any ratio value. Overall response rates (total responses/session time) were also not significantly different. Brackets are 1 SEM.
  • FIG. 6A-6B In Fig 6A 7PA2 and CHO cells were metabolically labeled with 35 S-methionine for 16 hours and the conditioned medium collected, cleared of cells and used for R1282 immunoprecipitation and autoradiography. Only p3, A ⁇ monomer, dimers and trimers are specifically detected in 7PA2, and not CHO conditioned medium.
  • Fig. 6B is a full gel view of Fig. IA. 7PA2 and CHO conditioned medium were immunoprecipitated with R 1282, the immunoprecipitation stored and the supernatant immunoprecipitated a second time. The first and second immunoprecipitations were then analyzed by Western blotting using 6E10. As in Fig. 6A, A ⁇ monomer, dimers and trimers are specifically detected in 7PA2, and not CHO conditioned medium.
  • FIG. 7A Incubation of 7PA2 conditioned medium in CSF at 37 0 C for 160 minutes results in no significant decrease in 7PA2-derived monomelic A ⁇ 7PA2 cells were metabolically labeled with 35 S-methionine for 16 hours and the conditioned medium (CM) collected and cleared of cells.
  • This conditioned medium was used as a source of labeled A ⁇ , aliquots Of 35 S 7PA2 conditioned medium (1 milliliter (ml)) were added to human cerebrospinal fluid (CSF) (20 ml) in the presence of proteases inhibitors and incubated at 4 0 C or incubated without protease inhibitors at 37 0 C.
  • CSF human cerebrospinal fluid
  • amyloid ⁇ amyloid ⁇
  • AD Alzheimer's disease
  • other neurodegenerative diseases While ⁇ -amyloidosis has long been hypothesized to affect cognition, the species of the amyloid ⁇ (A ⁇ ) protein associated with Alzheimer's disease (AD) and other neurodegenerative diseases have not been isolated, defined biochemically, or characterized with regard to their effects on cognitive function. With the present invention, discreet amyloid ⁇ moieties that interfere with cognitive functioning have been isolated and characterized. The intracranial delivery of these amyloid ⁇ moieties disrupts cognitive functioning and serves as a model system of Alzheimer's disease and other neurodegenerative disorders.
  • a ⁇ amyloid ⁇
  • Amyloid ⁇ protein also referred to as “amyloid-beta polypeptide,” “amyloid-beta peptide,” “amyloid-beta molecule,” “amyloid-beta protein,” “amyloid- ⁇ polypeptide,” “amyloid- ⁇ peptide,” “amyloid- ⁇ molecule,” “A ⁇ protein,” “A ⁇ polypeptide,” “A ⁇ peptide,” “A ⁇ molecule,” “amyloid-beta,” or “amyloid ⁇ ,” is a major protein component of amyloid plaques in the brains of individuals afflicted with Alzheimer's disease, comprising more than 70% of the total protein in plagues (U. S. Patent No. 6,218,506).
  • Amyloid ⁇ protein is a polypeptide of 39 to 43 amino acid residues, first identified by Glenner and Wong (Glenner et al., (1984) Biochem Biophys. Res Commun 120, 885-890; and Glenner and Wong (1984) Biochem Biophys Res Commun 122, 1131- 1135) and Masters et al. (Masters et al., (1985) Embo 4, 2757-2764; and Masters et al., (1985) Proc Natl Acad Sci USA 82, 4245-4249).
  • amyloid precursor protein (APP) of the amyloid-beta protein has been cloned and sequenced (Kang et al., (1987) Nature 325, 733; Tanzi et al., (1987) Science 235, 880-884; and Selkoe (1994) Annual Review of Neuroscience, 17 489-511).
  • Amyloid ⁇ peptide is generated from the beta-amyloid precursor protein (beta APP) in a two-step process.
  • the first step involves cleavage of the extracellular, amino-terminal domain of beta APP. Protein cleavage is performed by an aspartyl protease termed beta-secretase (BACE).
  • BACE beta-secretase
  • This enzyme is synthesized as a propeptide that must be modified to the mature and active form by the prohormone convertase, furin.
  • Beta APP cleavage by the mature form of BACE results in the cellular secretion of a segment of beta APP and a membrane-bound remnant. This remnant is then processed by another protease termed gamma-secretase.
  • Gamma-secretase cleaves an intra-membrane site in the carboxyl-terminal domain of beta APP, thus generating the amyloid beta peptide.
  • Gamma-secretase is believed to be a multi-subunit complex containing presenilin-1 and 2 as central components.
  • the transmembrane glycoprotein nicastrin is found associated with the presenilins.
  • Nicastrin has been found to bind to the carboxyl-terminus of beta APP and helps to modulate the production of the amyloid beta peptide.
  • Tau is a neuronal microtubule- associated protein found predominantly on axons.
  • tau in its hyperphosphorylated form, is the major component of paired helical filaments (PHF), which is the building block of neurofibrillary lesions in Alzheimer's disease brain (J. Neurosci., 18:1743-1752, 1998 and Neuron, 19:939-945, 1997).
  • PHF paired helical filaments
  • the present invention demonstrates that the intracranial delivery of oligomeric forms of amyloid ⁇ protein disrupts cognitive functioning.
  • This disruption of cognitive functioning may include the disruption of the memory of learned behavior.
  • This disruption of cognitive functioning may be rapid, potent, and/or transient.
  • This disruption of cognitive functioning may be obtained without the induction of permanent neurological deficits.
  • amyloid ⁇ protein to be administered may be any of the many known allelic variants or mutations of the amyloid ⁇ protein.
  • an amyloid ⁇ protein is a monomeric polypeptide, made up of one polypeptide chain and is also referred to herein as a "monomer.”
  • an , oligomer of amyloid ⁇ also referred to herein as an oligomeric form of amyloid ⁇ , is a detergent-stable configuration of more than one amyloid ⁇ protein.
  • An oligomer is not necessarily polymerized.
  • a "dimer” is a detergent-stable configuration of two amyloid ⁇ proteins.
  • a "trimer” is a detergent-stable configuration of three amyloid ⁇ proteins.
  • a “tetramer” is a detergent-stable configuration of four amyloid ⁇ proteins.
  • a "pentamer” is a detergent-stable configuration of five amyloid ⁇ proteins.
  • a "hexamer” is a detergent-stable configuration of six amyloid ⁇ proteins.
  • Oligomers of amyloid ⁇ protein to be administered may remain in solution in a range of any of the various temperatures discussed above.
  • the oligomers of amyloid ⁇ protein to be administered may be non- fibrillar.
  • a "non-fibrillar" protein also referred to herein a "globular” protein, has little alpha helical or beta sheet structure.
  • a fibrillar protein has extensive alpha helix or beta sheet structure.
  • the oligomers of amyloid ⁇ protein to be administered may be preparations from which the fibrillar form of amyloid ⁇ is absent or has been removed.
  • the oligomers of amyloid ⁇ protein to be administered may be multimers of two or more amyloid ⁇ proteins.
  • the oligomers of amyloid ⁇ protein to be administered may be dimers, trimers, tetramers, pentamers, hexamers, and/or other multimers of amyloid ⁇ protein.
  • the oligomers of amyloid ⁇ protein to be administered may be a mixture of monomers, dimers, trimers, tetramers, pentamers, hexamers, and/or other multimers of amyloid ⁇ protein.
  • Oligomers of amyloid ⁇ protein may be obtained from a wide variety of sources. Oligomers of amyloid ⁇ protein may be obtained from natural sources. For example, oligomers of amyloid ⁇ protein may be isolated from natural fluids, cells, or tissues, including, but not limited to, plasma, brain tissue, and cerebrospinal fluid. Oligomers of amyloid ⁇ protein may be isolated from the culture medium of cells expressing endogenous or transfected amyloid ⁇ protein precursor genes. For example, oligomers of amyloid ⁇ protein can be obtained from the culture medium of Chinese hamster ovary (CHO) cells stably transfected to express amyloid ⁇ protein (Podlinsky et al., J Biol. Chem., 1995, 270(16):9564-9570). Oligomers of amyloid ⁇ protein may be synthetically produced. Oligomers of amyloid ⁇ protein may be produced recombinantly.
  • CHO Chinese hamster ovary
  • the oligomers of amyloid ⁇ protein to be administered may be preparations lacking monomers of amyloid ⁇ .
  • Monomers of amyloid ⁇ may be absent or have been removed.
  • Monomers of amyloid ⁇ may be removed by any of a number of means.
  • monomers of amyloid ⁇ may be removed by digestion with insulin-degrading enzyme (Qiu et al., J Biol Chem. 1998 273(49):32730).
  • Monomers of amyloid ⁇ may be removed by various methods of protein size fractionation, for example, by size exclusion chromatography, as described in more detail in the Example 1 (see also, Cleary et al., Nature Neuroscience 2005, 89:79-84).
  • Isolated oligomers of amyloid ⁇ protein are administered.
  • isolated means that a polypeptide or oligomer of polypeptides is either removed from its natural environment or synthetically derived, for instance by recombinant techniques, or chemically or enzymatically synthesized.
  • purified means that a polypeptide or oligomer of polypeptides is essentially free from any other polynucleotides or polypeptides and associated cellular products or other impurities.
  • Intracranial delivery of oligomers of amyloid ⁇ protein may be by any of a wide variety of means.
  • intracranial delivery may be accomplished by oral, subcutaneaous, intraperitoneal, intravenous, and/or intrathecal administration. Delivery may be by local delivery or injection. Delivery may be pump or extended release composition.
  • Intracranial administration may include, for example, intracerebral or intracerebroventricular administration.
  • Oligomers of amyloid ⁇ may be delivered to an animal, for example, a vertebrate animal, including a mammal. Mammals include, for example, a rodent, including, but not limited to, a mouse or a rat, a dog, a non-human primate, or a human.
  • Oligomers of amyloid ⁇ may be administered in a wide range of concentrations. For example, oligomers of amyloid ⁇ may be administered at concentrations that are higher than the concentration of oligomers of amyloid ⁇ found in brain or cerebrospinal fluid (CSF); oligomers of amyloid ⁇ may be administered at concentrations that are the same or similar to the concentration of oligomers of amyloid ⁇ found in brain or CSF; or oligomers of amyloid ⁇ may be administered at concentrations that are less than the concentration of oligomers of amyloid ⁇ found in brain or CSF.
  • CSF cerebrospinal fluid
  • oligomers of amyloid ⁇ may be administered at concentrations that are the same or similar to the concentration of oligomers of amyloid ⁇ found in human brain or human CSF, for example, at concentrations of less than about 4000 picograms per milliliter (less than about 1 nanomolar).
  • the disruption of cognitive function obtained by the administration of oligomers of amyloid ⁇ may be representative of a neurological disorder.
  • the neurological disorder may be a neurodegenerative disorder.
  • a neurological disorder includes, but is not limited to, Alzheimer's disease.
  • Disruptions of cognitive function may be representative of any phase of a neurological disorder, including, but not limited to, a presymptomatic phase, a preclinical phase, or an early phase of a neurological disorder.
  • the disruption of cognitive function obtained by the administration of oligomers of amyloid ⁇ may be representative of age-related memory decline or age-associated memory impairment (see Craik, F. I. in Handbook of the Psychology of Aging (eds. Birren, J. E.
  • the method of the present invention can provide a sensitive assessment of transient cognitive changes, subtle cognitive changes, and cognitive changes over time and treatment conditions.
  • Cognitive disruption may be assayed by any of a variety of methods.
  • One means of assessing cognitive functioning is the Alternating Lever Cyclic Ratio (ALCR) test, which has proven to be sensitive for measuring cognitive function (O ⁇ are et al, Behav Pharmacol 1996, 7:742-753; and Richardson et al., Brain Res 2002, 954:1).
  • ALCR Alternating Lever Cyclic Ratio
  • rats learn a complex sequence of lever-pressing requirements for food reinforcement in a two-lever experimental chamber. Rats must alternate between the two levers, switching to the other lever after pressing the first lever enough to get a food pellet.
  • a delayed non-matching to place test a morris water maze (commonly used to assess working memory in rats and mice), a delayed matching to sample test (an operant procedure for testing working memory), and a fixed-interval operant responding test (a sensitive procedure to assess non-specific cognitive effects, for example, when the type and anatomical location of the cognition being tested is unknown), a delayed conditioning procedure (representing a variety of operant or non-operant tests under which animals are exposed to stimuli paired with a reward or punishment and, after a delay, their ability to respond appropriately to the stimulus-reward combination is assessed), or a repeated acquisition procedure (an operant test, under which subjects are required to repeatedly learn a new stimulus sequence).
  • a morris water maze commonly used to assess working memory in rats and mice
  • a delayed matching to sample test an operant procedure for testing working memory
  • a fixed-interval operant responding test a sensitive procedure to assess non-specific cognitive effects, for example, when the type and anatomical location of the cognition being
  • the administration of oligomers of amyloid ⁇ results in one or more neurological functional deficiencies.
  • Such functional deficiencies may be transient or permanent.
  • Such functional deficiencies may be observed in the absence of neuropathological damage.
  • Such neuropathologies may include, for example, amyloid plaque formation, amyloid deposits, oxidative stress, astrogliosis, microgliosis, cytokine production, dystrophic neurons, formation of neurobifillary tangles, neurodegeneration, gross neuronal atrophy, neuronal loss, synaptic loss, and other manifestations of neuropathology.
  • Also included in the present invention is a method of screening for an agent effective for the treatment of a neurological disorder, including, but not limited to, Alzheimer's disease, by administering an agent to a first animal to which soluble oligomers of the amyloid ⁇ protein has been intracranially administered, measuring cognitive functioning of the first animal, and comparing the cognitive functioning of the first animal to the cognitive functioning of a second animal treated in the same fashion except no agent was administered.
  • An improvement in the cognitive functioning of the first animal compared to the cognitive functioning of the second animal indicates the agent is effective for the treatment of a neurological disorder.
  • An agent may be administered by any of a wide variety of means.
  • an agent may be delivered orally, subcutaneaously, intramuscularly, intravenously, intrathecally, and/or intracranially. Delivery may be by local delivery or injection. Delivery may be by pump or extended release composition.
  • An agent may be delivered prior to, during, and/or after delivery of oligomers of amyloid ⁇ protein.
  • An agent may be delivered prior to and/or during the measurement of cognitive functioning.
  • the present invention includes the agents identified by this screening method and methods of treating a neurological disorder in a subject by administering to the subject one or more agents identified by this screening method. As used herein "treatment" represents the therapeutic and/or prophylactic treatment.
  • Also included in the present invention is a method for assaying the effects of soluble oligomers of amyloid ⁇ protein on cognitive functioning by administering soluble oligomers of amyloid ⁇ protein intracranially into an animal, and measuring cognitive functioning in the animal to determine the long-term disruption of cognitive behavior.
  • the soluble oligomers of amyloid ⁇ protein may be administered intracerebroventricularly.
  • the long-term disruption of cognitive behavior of the animal is compared to the long-term disruption of cognitive behavior of another animal treated in the same fashion except saline rather than soluble oligomers of the amyloid ⁇ protein has been administered intracranially.
  • the methods of the present invention may be used to measure the progression of a neurological disorder.
  • antibodies that bind to soluble oligomers of the amyloid ⁇ protein are also included in the present invention.
  • the antibody binds to an oligomer of the amyloid ⁇ protein and does not bind to monomers of amyloid ⁇ protein.
  • the oligomer of the amyloid ⁇ protein to which the antibody binds is a dimer of the amyloid ⁇ protein.
  • the oligomer of the amyloid ⁇ protein to which the antibody binds is a trimer of the amyloid ⁇ protein.
  • compositions including one or more of the antibodies as discussed herein Such antibodies may be prepared by any of a variety of methods, including, for example, as discussed in U.S. Patent Application 20030068316.
  • Isolated oligomers of amyloid ⁇ protein or fragments thereof may serve as an antigen to immunize an animal to elicit an immune response.
  • the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
  • a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • the present invention also includes a method for detecting the presence of soluble oligomers of amyloid ⁇ protein in a sample taken from a subject by contacting a sample with one of the antibodies as discussed herein and detecting binding of the antibody.
  • Also included in the present invention is a method for counteracting the effects of soluble oligomers of the amyloid ⁇ protein by administering an antibody as discussed herein to a subject in need of such treatment.
  • 7PA2 conditioned medium was injected intracerebroventricularly (ICV) into rats.
  • ICV intracerebroventricularly
  • a persistent obstacle to studying this problem has been the lack of a sufficiently sensitive assessment procedure capable of measuring transient cognitive changes over time and treatment conditions.
  • an operant task was utilized to allow one to assess subtle cognitive effects of very small doses of psychoactive drugs (Weldon et al., Pharmacol Biochem Behav 1996, 54:753-7).
  • Amyloid- ⁇ concentrations in conditioned media were measured by ELISA as previously described (Johnson-Wood et al., Proc Natl Acad Sci USA 1979, 94:1550-5). Size exclusion chromatography. Conditioned medium from either 7PA2 or CHO cells was concentrated ⁇ 10-fold using YM-3 Centriprep filters (Amicon, Millipore, Bedford, MA) and subjected to size exclusion chromatography.
  • Protein was transferred onto 0.2 ⁇ m nitrocellulose and Western blotted with 2G3 (at 1 ⁇ g/ml). Monoclonal antibody 2G3 was raised to amyloid- ⁇ 33- 40) and specifically recognizes amyloid- ⁇ species ending at residue 40 (provided by Elan Pharmaceuticals). Bound 2G3 was detected as described above. Alternating Lever Cyclic Ratio Paradigm (ALCR). Twenty-four male
  • ICV injections ICV injections. Behavioral test sessions were conducted Monday through Friday each week, with injections always given on Tuesday. Only one test injection session was conducted each week. Following the injection, the cannula was capped with a stylet and the rat was placed in a holding box for 2 hours prior to the ALCR session (30-40 minutes for rats receiving scopolamine). On non-injection days, rats were subjected to sham injections, under which the same procedure was followed but no injectate was actually given. In addition, 20 ⁇ l of saline (0.9%) was injected at least once each week in order to help keep the cannulae patent.
  • the first immunoprecipitation substantially reduced the amount of amyloid- ⁇ monomers and oligomers that could be precipitated in a second immunoprecipitation (Fig. IA, lane 2).
  • This A ⁇ -free residual conditioned medium was used as the post-R1282 injectate.
  • Conditioned medium from untransfected CHO cells contained essentially no A ⁇ detectable by either immunoprecipitation or western blot (Fig. IA, lane 3) or ELISA.
  • 7PA2 conditioned medium that was selectively depleted of monomeric A ⁇ by treatment with the protease insulin degrading enzyme blocks the maintenance of hippocampal LTP in vivo, indicating that A ⁇ oligomers, which cannot be digested by this protease and remain in the conditioned medium, may be responsible for the deficit in synaptic plasticity (Walsh et al., Nature 2002, 416:535-9). Therefore whether A ⁇ oligomers were also responsible for the 7PA2 conditioned medium-induced deficits in learned behavior under ALCR was addressed.
  • the approximately 55kD protein present in fraction 7 is not a higher molecular weight aggregate of AB since it is also readily apparent in CHO conditioned medium (Fig. 2A), is not detected by AB immunoprecipitation followed by autoradiography (Fig. 6A), and is not removed by immunoprecipitation under conditions that abolish the ability of 7PA2 conditioned medium to interfere with ALCR performance (Fig. 6B). Therefore, approximately 55kD protein is not involved in disrupting cognitive function in these rats. Injection of fraction 9, containing A ⁇ monomers in much larger amounts than those of the oligomers in fraction 7, did not significantly affect error rates.
  • CSF cerebrospinal fluid
  • a ⁇ oligomers were acting pathophysiologically, in a manner that dissociated their disruptive effects on cognitive function from the neurodegenerative processes characteristic of Alzheimer's disease. If so, then periodic injections of A ⁇ -containing conditioned medium might not result in progressive deterioration of performance across time.
  • 7PA2 conditioned medium or CHO conditioned medium was injected, once a week for four consecutive weeks, into rats trained under the ALCR procedure and compared error rates during the ten days before and after this injection regimen. Rats receiving multiple ICV injections of oligomer-containing 7PA2 conditioned medium did not differ in performance from rats receiving multiple ICV injections of CHO conditioned medium devoid of oligomers (Fig. 3).
  • Rats receiving 7PA2 conditioned medium not only maintained their overall performance, but showed a tendency to improve during the 4-week course of the injection regimen. This finding with natural, low molecular weight oligomers contrasts with studies showing sustained deficits in learned behavior in rats after ICV injection with mixtures of synthetic soluble and fibrillar A ⁇ at high concentrations (Richardson et al., Brain Res 2002, 954:1; and O'Hare et al., Brain Res 1999, 815:1-10).
  • a ⁇ oligomers were remarkably potent at concentrations of A ⁇ monomer in the conditioned medium similar to those found in human CSF.
  • ALCR errors after 20 ⁇ l of 7PA2 conditioned medium, containing approximately 30 fmol (128 pg) A ⁇ were compared to errors after low to moderate ICV doses of the well-known amnestic drug scopolamine (Fig. 4).
  • Typical ICV scopolamine doses used to disrupt cognitive function in rats range from 3 to 50 ⁇ g (Mishima et al., Jpn. J. Pharmacol. 2000, 84(2):163-173; and Pederson et al., Regul Pept.
  • Scopolamine (2 ⁇ g and 5 ⁇ g; approximately 4 and approximately 11 nmol, respectively) was injected into the lateral ventricles of rats responding under ALCR.
  • the disruption in learned behavior due to A ⁇ fell in between the two doses of scopolamine, indicating that the effects of soluble A ⁇ oligomers in 7PA2 conditioned medium were at least four to five orders of magnitude more potent than the typical amnestic dose of ICV scopolamine in the rat.
  • the onset and duration of action of A ⁇ oligomers were also different from those of scopolamine.
  • 7PA2 conditioned medium was found to be more effective when injected 2 hours before testing.
  • scopolamine worked well when injected 30 minutes before the session, but its effects had decayed substantially by 2 hours post-injection.
  • oligomer effects are indicative of a pathophysiological action, similar to that seen with amnestic drugs such as scopolamine.
  • pathophysiological action is usually independent of permanent neuronal injury and, indeed, does not suggest a role for any neurodegenerative processes in the deleterious cognitive effects produced transiently by femtomole quantities of A ⁇ oligomers.
  • a ⁇ -induced cognitive deficits may occur before or in the absence of frank neurodegeneration in APP transgenic mice that model features of Alzheimer's disease (Westerman et al., J Neurosci 2002, 22: 1858-67; Chen et al., Nature 2000, 408:975-9; Koistinaho et al., Proc Natl Acad Sci USA 2001, 98:14675-80; and Palop et al., Proc Natl Acad Sci USA 2003, 100:9572-7).
  • the present data strengthen the notion that memory deficits in these mice may be caused by A ⁇ oligomers.
  • a central message of the present data is that brain dysfunction occurring in presymptomatic stages of Alzheimer's disease, such as those observed with highly sensitive functional imaging techniques (Bookheimer et al., N Engl J Med 2000, 343:450-6; and Small et al., Proc Natl Acad Sci USA 2000, 97:6037-42), might also be related to A ⁇ oligomer effects and might therefore be reversible with appropriate interventions prior to widespread neuronal degeneration.

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Abstract

La présente invention concerne des procédés d'arrêt des fonctions cognitives par administration d'oligomères d'amyloïdes ß.
PCT/US2005/023070 2004-06-30 2005-06-30 Oligomeres solubles d'amyloide beta permettant l'arret des fonctions cognitives Ceased WO2006004824A1 (fr)

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WO2008028939A1 (fr) 2006-09-08 2008-03-13 Vib Vzw moyens et procédés pour la production d'oligomères amyloïdes
US20100081613A1 (en) * 2006-10-11 2010-04-01 The Trustees Of Columbia University In The City Of New York Methods and compositions for enhancing memory
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006047254A1 (fr) * 2004-10-22 2006-05-04 Regents Of The University Of Minnesota Ensembles de proteines beta-amyloides oligomeres et utilisations de ceux-ci
WO2008028939A1 (fr) 2006-09-08 2008-03-13 Vib Vzw moyens et procédés pour la production d'oligomères amyloïdes
US20100081613A1 (en) * 2006-10-11 2010-04-01 The Trustees Of Columbia University In The City Of New York Methods and compositions for enhancing memory
US20130071401A1 (en) * 2010-06-03 2013-03-21 Ramot At Tel-Aviv University Ltd. Methods of treating diabetes and compositions capable of same
US9624285B2 (en) * 2010-06-03 2017-04-18 Ramot a Tel-Aviv University Ltd. Methods of treating diabetes and compositions capable of same
US12352719B2 (en) 2019-09-20 2025-07-08 KYCERA AVX Components Corporation Somatic cell-based electrical biosensor

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