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WO1996013513A1 - Peptide d'alzheimer - Google Patents

Peptide d'alzheimer Download PDF

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Publication number
WO1996013513A1
WO1996013513A1 PCT/US1995/013861 US9513861W WO9613513A1 WO 1996013513 A1 WO1996013513 A1 WO 1996013513A1 US 9513861 W US9513861 W US 9513861W WO 9613513 A1 WO9613513 A1 WO 9613513A1
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WIPO (PCT)
Prior art keywords
transgene
peptide
mouse
mammal
transgenic
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Application number
PCT/US1995/013861
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English (en)
Inventor
Gilbert Jay
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American National Red Cross
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Publication date
Application filed by American National Red Cross filed Critical American National Red Cross
Priority to AU40127/95A priority Critical patent/AU4012795A/en
Publication of WO1996013513A1 publication Critical patent/WO1996013513A1/fr

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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
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • 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
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • AD Alzheimer's disease
  • hippocampus cerebral cortex
  • amygdala(2) regions of the brain that play a major role in memory, cognition, and behavior.
  • Amyloid deposits have been the focus of much attention, their role in the pathophysiology of AD remains unclear.
  • Amyloid deposits can occur as diffuse aggregates or as dense deposits that, together with degenerating neuritic structures, are collectively referred to as senile plaques.
  • the principal protein component of these plaques is a 42-amino acid peptide called /-.-amyloid or A0(3,4) that is derived from a larger transme brane protein, the Aj ⁇ precursor protein (APP) , which can exist as several different isoforms(5-7) .
  • APP Aj ⁇ precursor protein
  • a / 3 from APP has been shown to be a normal processing event (8-10) , and A/3 can be detected in the cerebrospinal fluid and plasma of both normal and AD patients (9, 11) .
  • A/3 can be detected in the cerebrospinal fluid and plasma of both normal and AD patients (9, 11) .
  • the process by which A/3 accumulates in the diseased brain is unknown, although mutations in the APP gene linked to familial AD may alter its processing and lead to higher levels of the A/3 peptide.
  • recent in vi tro studies have shown that cells transfected with different mutated forms of APP accumulate at least a six-fold higher level of A ⁇ compared to cells transfected with wild-type APP(12,13) .
  • mice have not yet been reported to develop any significant pathology, perhaps due to technical reasons such as the selection of an inappropriate promoter, low expression levels, the inability of mice to process the human precursor molecule, or inability to distinguish its effect from the genetic background effects of the host mouse strain.
  • the invention relates to a recombinant DNA and a transgene, each comprising a promoter operably linked to a DNA sequence coding for a j ⁇ -amyloid peptide, A ⁇ , and a host cell or whole mammal containing the recombinant DNA or transgene.
  • the A/3 coding sequence can be obtained from the same or different species as the cell or mammal species into which it is added.
  • the host species is a mouse.
  • the A/3 peptide or A/3 is a principal protein component of senile plaques observed in the brain of human patients having Alzheimer's disease.
  • a ⁇ is a 42-amino acid peptide (3,4; Yamada et al. , 1987, Biochem. Biophys . Res . Commun . 149, 665-671) derived from a larger transmembrane protein, the A ⁇ precursor protein (APP) , which exists as several different isoforms (5,6,7). Deposits of the A/3 peptide have been implicated in AD, Down's Syndrome, and HCHWA-D.
  • a nucleic acid sequence consisting essentially of a coding sequence for an A ⁇ peptide means essentially only the nucleic acid sequence that encodes the amino acid sequence of an A ⁇ peptide.
  • a nucleic acid sequence consisting essentially of a coding sequence for a mouse A/3 sequence is shown in Figure 9.
  • the nucleic acid can be, e.g., RNA and/or DNA, and it can comprise degenerate sequences, preferred codons, etc.
  • a nucleic acid sequence coding for an A/3 peptide means a nucleic sequence which, when expressed by a cell, results in the production of an A ⁇ peptide.
  • such a nucleic acid coding sequence can be the complete coding sequence of APP or a fragment thereof, e.g., a 42- amino acid sequence encoding A ⁇ , or the carboxyl terminus of the APP gene. See, e.g., Kammensheidt et al. , Proc. Natl. Acad, Sci . , 89, 10857-10861 (1992) .
  • a recombinant nucleic acid comprising a nucleic acid sequence coding for an A/3 peptide is, therefore, any recombinant nucleic acid which can be used to express A/3, excluding the complete and normal APP gene.
  • a "mouse A ⁇ peptide” denotes a peptide having an amino acid sequence native to mouse tissues. See, e.g., Yamada et al . , Biochem. Biophys. Res. Commun., 149, 665-671 (1987) .
  • a nucleic acid sequence coding for such a peptide includes, degenerate sequences and preferred codons, as well. It is understood that naturally-occurring allelic variations exist in the A ⁇ peptide and occur from individual to individual.
  • variations include amino acid differences, e.g., substitutions, deletions, insertions, or inversions, as well as nucleic acid differences, e.g., substitutions, deletions, insertions, or inversions.
  • amino acid differences e.g., substitutions, deletions, insertions, or inversions
  • nucleic acid differences e.g., substitutions, deletions, insertions, or inversions.
  • sequences are, therefore, mouse A/3 peptides.
  • a mutant mammal A/3 peptide sequence can also be used according to the present invention.
  • Such a mutant sequence can be a non-naturally occurring sequence or a sequence which copies a mutation already found in nature.
  • HSHWA-D hereditary cerebral hemorrhage with amyloidosis Dutch type
  • a ⁇ peptide deposition is found predominantly in cerebral blood vessel walls and, to a lesser extent, in the neuropil.
  • a mutation at position 22 of A ⁇ resulting in the replacement of glutamic acid with glutamine, has been identified that is associated with the disease.
  • the human HCHWA-D sequence encoding the A ⁇ peptide can be used directly, or a derivative sequence can be made in which glutamic acid is replaced with glutamine in the mouse A ⁇ peptide at corresponding position 220. See Figure 9.
  • a mutant sequence can also be identified by mutagenesis, either in whole animals or cell culture, using conventional technology.
  • mutagenized A/3 sequences can be transformed into cells, and the transformed cells can be screened for overexpression or aberrant expression of the A ⁇ peptide, e.g., by immunoassay.
  • the source of the nucleic acid coding sequence can be a natural or mutant A/3 sequence obtained from a mammal in which it is present.
  • the coding sequence can also be synthetic, either wholly or partly, based upon an APP gene or protein sequence.
  • the APP gene which encodes the A/3 peptide has been cloned in a number of different species, including mouse: Yamada et al. , 1987, Biochem. Biophys . Res . Commun . 149, 665-671; rat: Shivers et al. , 1988, EMBO J. 1365-1370; human: Kang et al. , 1987, Nature 325, 773-736; Ponte et al.
  • the source of the A ⁇ peptide can be the same species as the species in which it is to be expressed or it can be different. In a preferred example, the source of the A/3 peptide is mouse when it is to be expressed in a mouse.
  • a heterologous promoter sequence can be operably linked to a D A sequence coding for an A/3 peptide.
  • operably linked it is meant that the heterologous promoter is joined to the A ⁇ peptide coding sequence in a manner to permit the expression of that sequence to be controlled and production of the encoded A/3 peptide.
  • the peptide can accumulate intracellularly or extracellularly.
  • the heterologous promoter sequence is preferably a neuron-specific promoter sequence, i.e., a promoter sequence which is active in neuronal cells, preferably, the promoter sequence is more active in neuronal cells than other cell types.
  • neuron-specific promoter sequences are neuron-enolase, Purkinje-cell protein, dystrophin, neurofilament, preferably, a neurofilament-light (NF-L) gene promoter, more preferably, a mouse NF-L gene promoter (30) .
  • Neuronal specificity can be increased, e.g., by including introns which are part of the gene's coding sequence, such as by including the first intron of the neurofilament gene.
  • the heterologous promoter sequence can also be, e.g., amplifiable, inducible, developmentally-regulated, etc., alone or in combination with a neuron-specific promoter.
  • the heterologous promoter can be a hybrid containing elements of several promoters, including the APP gene promoter itself.
  • a recombinant nucleic acid molecule according to the present invention is a non-naturally occurring nucleic acid molecule.
  • Such a nucleic acid contains a unit of a promoter operably linked to a sequence which codes substantially only for A ⁇ peptides. It can be used in various ways, e.g., as a research tool or probe, e.g., or to effect expression of an A/3 peptide in a cell or whole mammal into which it is introduced.
  • such a recombinant DNA can also comprise a ribosome-binding site, translation initiation sequences, a Kozak consensus sequence, a coding sequence for APP, or a fragment thereof, termination sequences, polyadenylation sequences, i.e., those nucleic acid sequences useful to achieve expression of an A ⁇ peptide.
  • a recombinant nucleic acid can further comprise enhancer sequences which modulate gene expression, intron sequences, and 5' and 3' flanking nucleotide sequences of an APP gene or another desired gene.
  • a nucleic acid sequence according to the present invention can be modified to improve expression of the nucleic acid sequence coding for AjS, e.g., by the addition of enhancer sequences or by altering nucleotide sequence information to eliminate mRNA secondary structure which reduces or interferes with translation. See, e.g., Methods in Enzymology, Volume 185, Academic Press, 1990, especially Chapters 38-44.
  • transgene is meant a recombinant nucleic acid containing information to express an A ⁇ peptide in an animal into which it is introduced, e.g., promoter, a ribosome binding sequence, a Kozak sequence, i.e., CCPuCCAUGG or CCA/TCCA (see Kozak, Nucl. Acid. Res., 12, 857-872, 1984), translation initiation se- quences, enhancer, intron, and/or polyadenylation sequence.
  • Such a transgene can contain more than one coding sequence. These sequences used to effect expression are generally known in the art and discussed above.
  • the present invention also involves a transgenic mammal that contains in some or all of its cells a recombinant DNA, or transgene, comprising a promoter operably linked to a DNA sequence coding substantially for A/3 peptides.
  • a heterologous promoter and A ⁇ peptide are obtained from the same species of mammal into which they are to be introduced by transformation; preferably, this species is a mouse.
  • transgenic mammal can be made by, e.g., directly injecting a transgene into an embryo, using a retrovirus carrying the recombinant nucleic acid, or employing embryonic stem cell methodology. See, e.g., U.S. Patent Nos. 4,736,866; 4,873,191; 4,873,316; 5,082,779; 5,304,489; 5,174,986; 5,175,384; 5,175,385; and 5,221,778.
  • a recombinant DNA comprising the desired A/3 sequence is introduced by microinjection into a pronucleus of ferti ⁇ lized one-cell embryo.
  • a recombinant DNA comprising the desired A/3 sequence is introduced by microinjection into a pronucleus of ferti ⁇ lized one-cell embryo.
  • Palmiter et al. Cell , 41:343-345 (1985) ; Palmiter et al . , Ann . Rev. Genet . , 20:465-499 (1986) .
  • the transgene can be injected into a fertilized mouse egg before fusion between the sperm and egg; thus, if integration into the genome DNA occurs, every cell of the embryo inherits the transgene.
  • transgenic "mammal” is any mammal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus from another organism, other than from its parent.
  • the term is not intended to encompass classical crossbreeding, but rather is meant to comprise mammals in which one or more cells receive a recombinant nucleic acid molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating nucleic acid.
  • transgenic mammal refers to a transgenic mammal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information they, too, are transgenic mammals.
  • a transgenic mammal can also be a chimera in which the genetic information, i.e., the recombinant DNA comprising an A/3 peptide is present in only some cells of the entire organism.
  • the genetic information may be foreign to the species of mammal to which the recipient belongs, foreign only to the particular individual recipient, or genetic information already possessed by the recipient .
  • the introduced gene may be differently expressed than the native gene, e.g., temporally, spatially, or quantitatively.
  • the recombinant nucleic acid or transgene can be introduced into any mammal, including a mouse (Hogan et al . , 1986, in Manipulating the Mouse Embryo: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York) , pig (Hammer et al. , Nature, 315:343-345, 1985), sheep (Hammer et al. , Nature, 315:343-345, 1985) , cattle, rat, or primate. See also, e.g., Church, 1987, Trends in Biotech . 5:13-19; Clark et al., 1987, Trends in Biotech . 5:20-24; and DePamphilis et al., 1988, Biorech- ⁇ igues, 6:662-680. In addition, e.g., custom transgenic rat and mouse production is commercially available.
  • a recombinant D A or transgene comprising a promoter operably linked to a D ⁇ A sequence encoding an A ⁇ peptide can be introduced into a mouse to form a transgenic mouse containing in its cells the recombinant D ⁇ A.
  • the recombinant D ⁇ A can be present in either germ line or somatic cells, or both.
  • the promoter e.g., ⁇ F-L and A/3 coding sequence, is preferably from the mouse.
  • Any known mouse can be used as a host for the transgene or recombinant D ⁇ A.
  • the host can be an in-bred strain, e.g., FVB/ ⁇ , or it can be an out-crossed strain.
  • Another aspect of the invention is also the development of a transgenic mammal which reproduces itself, i.e., in which the germ-line of the transgenic mammal is transformed stably with the recombinant D ⁇ A according to the present invention, permitting its transmission to subsequent generations.
  • a further aspect of the invention is the expression of the A ⁇ peptide in the transgenic mammal.
  • the occurrence of amyloid deposits comprising the A ⁇ peptide is a characteristic feature of Alzheimer's disease (AD) and is a facet of the disease's neuropathology.
  • AD Alzheimer's disease
  • A/3 deposition, or amyloidosis is also observed in other diseases, including, e.g., Down's syndrome and heredity cerebral hemorrhage with amyloidosis Dutch type (HCHWA- D) .
  • HSHWA- D heredity cerebral hemorrhage with amyloidosis Dutch type
  • Expression of the A/3 peptide in a transgenic mammal and its consequent phenotype can therefore be used a model for such diseases and pathologies, e.g., as an AD model.
  • active agents e.g., synthetic, organic, inorganic, or nucleic acids based molecules
  • another aspect of the invention is to provide a method to assist in the advancement of the treatment and/or prevention of the aforementioned symptoms (e.g., neurodegeneration or apoptosis) caused by the APP gene, or a fragment thereof.
  • Such a mammal model can also be used to assay for agents, e.g., zinc, and factors, e.g., environmental, which exacerbate and/or accelerate the diseases. See, e.g., Bush et al . , Science 265, 1464-1467, 1994.
  • a mammal containing a transgene according to the present invention can be used in a method of screening a compound for its effect on a phenotype of an mammal, preferably a mouse, where the phenotype is conferred by the transgene.
  • phenotype is meant, e.g., a collection of morphological, physiological, biochemical, and behavioral traits possessed by a cell or organism that results from the interaction of the genotype and the environment .
  • a phenotype can be behavioral, e.g., occurrence of seizures or cognitive performance, or it can be physiological and/or pathological, e.g., occurrence of neuronal cell degeneration, neuronal cell apoptosis, or accumulation of A ⁇ peptide in the brain of the mammal.
  • a compound can be administered to an mammal containing a transgene and then the existence of an effect on the phenotype of the mammal can be determined. Observation can be accomplished by any means, depending on the specific phenotype which is being examined. For example, the ability of a test compound to suppress a behavioral phenotype can be detected by measuring the latter phenotype before and after administration of the test compound.
  • the invention also relates to a transgenic mammal that contains in its cells, preferably carried in the genome of it, a recombinant DNA comprising a promoter operably linked to a DNA sequence coding for an A/3 peptide and also having a phenotype characterized by the accumulation of the AjS peptide in the brain, e.g., hippocampus, cerebral cortex, and amygdala, of the transgenic mammal, wherein the phenotype is conferred by the expression of the recombinant DNA.
  • a recombinant DNA comprising a promoter operably linked to a DNA sequence coding for an A/3 peptide and also having a phenotype characterized by the accumulation of the AjS peptide in the brain, e.g., hippocampus, cerebral cortex, and amygdala, of the transgenic mammal, wherein the phenotype is conferred by the expression of the recombinant DNA.
  • the level of expression of the AjS peptide can be any amount which can produce a phenotype in the mammal which phenotype can be distinguished from mammals which do not possess the transgene, i.e., a control mammal, e.g., an amount effective to produce neuronal cell degeneration and/or apoptosis and/or an amount effective to cause a behavioral and/or cognitive effect or dysfunction.
  • a mammal containing the AjS transgene can also be characterized by accumulation of the AjS peptide in its brain. The accumulation can be in any quantity which is greater than that observed in mammals not containing the transgene. However, the phenotype conferred by the transgene can occur before or after accumulation can be detected.
  • a ⁇ peptide in the brain of the mammal can be measured conventionally, e.g., by immunoassay or nucleic acid hybridization, either in si tu or from nucleic acid isolated from host tissues.
  • a ⁇ expression was virtually undetectable in controls on antibody-stained cryosections but clearly visible in mammals having the AjS transgene. See, e.g., Figure 4.
  • the identification of agents which prevent and/or treat symptoms associated with expression of the A/3 peptide can be determined routinely.
  • an active agent can be administered to a transgenic mammal expressing a recombinant DNA according to the present invention and then its effect on a behavior or pathology, e.g., A ⁇ deposition in the brain, apoptosis, and/or neurodegeneration, can be determined.
  • the agent can be administered acutely (e.g., once or twice) or chronically by any desired route, e.g., subcutaneously, intravenously, transdermally, or intracathically.
  • the formulation of the agent is conventional, see, e.g., Remington ' s Pharmaceutical Sciences, Eighteenth Edition, Mack Publishing Company, 1990.
  • an agent can be administered in different doses to separate groups of transgenic mammals to establish a dose-response curve to select an effective amount of the active agent.
  • Such effective amount can be extrapolated to other mammals, including humans.
  • the transgenic mammal preferably a mouse
  • the transgenic mammal therefore permits the testing of a wide variety of agents and therapies.
  • AD for example, a number of different agents have been identified which affect the cognitive dysfunction associated with the diseases, e.g., cholinergic agents, such as muscarine agonists, acetylcholinesterase inhibitors, acetylcholine precursors, biogenic amines, nootropics, and angiotensin converting enzyme (ACE) .
  • agents which regulate A/3 expression, AjS deposition, and physiological changes associated with A/3 expression and deposition can also be identified, e.g., calcium homeostasis, inflammation, neurofibrillary tangles.
  • AD effects on AD can be assayed in either behavioral or physiological and/or histological studies.
  • mice spatial learning and memory abilities in mice can be tested in a Morris water maze. See, e.g., Yamaguchi et al. , Neuro- eport, Vol. 2, 781-784 (1991) . Additionally, other behavioral tests can be used, e.g., Swim Test, Morris et al . , Learning and Motivation, 12, 239-260, 1981; Open-field test, Knardahl et al. , Behav. Neurol . Biol . 27, 187-200, 1979; and tests and models used routinely, e.g., in mice, rats, and other rodents.
  • differences in, e.g., levels of expression, cellular localization, and/or onset of expression of AjS can be used to model AD and other diseases associated with AjS expression and the differing stages and progressions of the disease, e.g., cell degeneration, cell death, astrogliosis, and/or amyloidosis.
  • quantities, temporal expression, and spatial localization of a transgenic AjS peptide in a mouse can be achieved. Having a range of A/3 peptide expression phenotypes can be useful to identify different therapies and drug treatments and also diagnostically to identify a disease's progression.
  • the specific treatments can depend on the region of the brain in which A/3 peptide is expressed, how much of it is expressed, and its temporal progression of expression.
  • mammals having different A/3 peptide phenotypes can be used as models for determining therapies which are selective for different stages of the disease and for studying disease progression and intervention.
  • a recombinant D ⁇ A comprising the coding sequence for substantially AjS peptide and cells and/or mammals transformed with it can be used as a source of A/3, i.e., as an A/3 factory.
  • cells e.g., see A ⁇ C Catalogue of Cell Lines and Hybridomas, 7th edition, 1992, can be individually transformed with the recombinant DNA and selected for expression of A/3.
  • methods on how to transform mammalian cells see, e.g., Methods in Enzymology, Volume 185, 1990, especially, Chapters 34-44; Molecular Cloning, Sambrook et al. , 1989, especially, Book 3, Chapter 16; EP 0 451 700.
  • the A ⁇ peptide can be routinely isolated from the cell cultures, e.g., see Masters, C.L. et al. , "Amyloid plaque core protein in Alzheimer disease and Down syndrome," Proc. Natl . Acad. Sci . USA 82:4245, 1985, and used, e.g., as a research tool, such as an antigen to raise antibodies or to determine the physical properties of A/3 for drug therapy design, analogously to how the commercially available amyloid /3-protein fragments listed in the Sigma Chemical Company Catalog, page 1119, 1994, would be used.
  • the AjS peptide can also be isolated from cells or transgenic mammals according to conventional means, e.g., by removing tissues in which A ⁇ is expressed and purifying the peptide according to routine procedures.
  • Cells expressing a recombinant DNA comprising an AjS peptide can also be used as an in vi tro model for studying A ⁇ expression and accumulation.
  • agents can be administered to cell cultures comprising A ⁇ expressing cells, and the effects of such agents can be studied. See, e.g., U.S. Patent No. 5,087,571.
  • such cell cultures can be used as model systems, as well.
  • the present invention also relates to methods of preventing or treating Alzheimer's disease by interfering with the expression of intracellular A ⁇ peptide.
  • a ⁇ intracellular A ⁇
  • secretion of the A ⁇ peptide from the cell and its consequent accretion into extracellular plaques was responsible for the characteristic cell death observed in AD patients
  • intracellular A ⁇ can trigger the physiological cascade leading to neuronal cell death, e.g., apoptosis, and other deleterious events associated with the disease. This indicates an earlier target for AD intervention than had heretofore been identified.
  • an aspect of the present invention is, inhibiting or lowering the expression of intracellular A ⁇ peptide, e.g., by administering to a patient having Alzheimer's Disease, or a related disorder, an amount of a compound effective for such purpose.
  • the amount of A ⁇ expression inhibited or lowered is less than the amount which can lead to the appearance of p53.
  • the A ⁇ peptide initiates the cascade leading to apoptosis and cell death by causing the expression of p53.
  • intracellular A/3 peptide can be reduced to levels at which the p53 gene is not activated, or the p53 gene is not expressed, or expression is insufficient to produce apoptosis.
  • expression it is generally meant any event which leads to the production and/or accumulation of intracellular A ⁇ or p53 peptide, e.g., transcription of the gene, translation of the corresponding mRNA, stability or perdurance of the gene product inside the cell.
  • AD Another aspect of the invention related to this finding is the treatment or prevention of AD by interfering with the expression of a p53 gene or its product. It has now been discovered that expression of intracellular A/3 can induce p53, leading to apoptosis and the extracellular release of AjS peptide. By interfering with, e.g., blocking or inhibiting, the appearance of the p53 gene product inside the cell, AD can be treated or prevented.
  • Intervention with the expression of the APP or p53 gene can be accomplished conventionally, e.g., by interfering with gene transcription, gene translation, or the perdurance of gene product in the cell.
  • anti-sense polynucleotides or ribozyme inhibitors can be employed to inhibit translation of an encoding mRNA into a polypeptide. See, e.g., Yung WK, Curr. Opin . Neurol . , 7(6) : 501-5 (1994) .
  • the methods for selecting a ribozyme or anti-sense nucleic acid compound can be determined based on the known sequence of the APP or p53 mRNA and tested in vi tro for its effect on translation.
  • the nucleic acid compound can be administered conventionally, e.g., by providing it as a pharmaceutical composition administered orally, intravenously, intrathecally, by stereotactic inoculation into the brain, etc., in an amount effective to inhibit gene translation.
  • nucleotide derivatives can be substituted for the naturally-occurring bases.
  • a ribozyme or anti-sense compound can also introduced into the brain by a genetic vector.
  • a retroviral or adenovirus vector can be employed to transfer a gene encoding a ribozyme or anti- sense oligonucleotide into a patient. See, e.g., M.G. Kaplitt et al . , Nat . Genet. 8(2) : 148-54 (1994) ; Horellou et al., Neuroreport (ENGLAND) 6(1) : 49-53 (1994); G. Le Gal La Salle et al. , Science 259: 988-90 (1993) ; S. Chatterjee et al. Science 258: 1485-8 (1992); M. Yamada et al., Jpn . J. Cancer Res . 83: 1244-7 (1992); Y.
  • Herpes simplex virus 1 vectors can also be used to modify neuronal physiology in vivo, e . g . , by the introduction of an anti-sense or ribozyme encoding gene, or other genetic elements which can be used to interfere with the expression of the APP or p53 gene. See, e.g., L. Soroceanu et al. , Proc. Natl . Acad. Sci. 92(5) ; 1411-5 (1995) ; D.S. Latchman, Mol . Biotechnol . 2(2) 179-95 (1994) ; H.J.
  • Compounds can also be administered by liposomes or by implanting cells expressing the desired compound into the brain. See, e.g., E.Y. Snyder et al. , Nature 374 (6520) : 367-70 (1995) .
  • Intervention with the expression of the APP or p53 gene can also be achieved by manipulating the cellular processes responsible for the processing of the gene product, such as cleavage of the precursor protein by regulating the corresponding enzymes or uncoupling the signal of the A/3 peptide to p53 expression.
  • useful agents include, e.g., anti-oxidants, and can be administered routinely.
  • Effective drug dosages can be determined from the animal model according to the present invention.
  • the detection of p53 gene activity e.g., by detecting its mR ⁇ A, protein product, or antibodies to it, can be employed to diagnose or assess disease progression. The latter can be especially useful in the mouse model to assess the efficacy of different drugs in treating or preventing AD and related disorders.
  • the present invention therefore relates to a method of identifying compounds to prevent or treat AD comprising administering a compound to a transgenic animal according to the present invention and measuring the amount of neuronal p53.
  • the p53 can be quantified in vivo or in vitro, e.g., by sacrificing the animal, removing the brain and detecting p53 mRNA or protein on tissue sections or homogenates according to standard procedures, e.g., in si tu hybridization, Northern analysis, PCR.
  • DNA, RNA, and other nucleic acid manipulations can be performed routinely, e.g., as described in Molecular Cloning, Sambrook et al . , 1989. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
  • Figure 1 is a schematic representation of the Hindlll-SacI restriction fragment used to generate transgenic mice.
  • the sequence encoding the A ⁇ peptide was generated by reverse transcriptase-polymerase chain reaction using rat brain RNA because it will yield a peptide identical to the mouse.
  • the forward primer contained an ATG initiation codon flanked by the Kozak consensus sequence(61) (there are no other upstream ATG sites within this transcriptional construct) while the reverse primer contained an in-frame TAA termination codon.
  • the 151-bp PCR fragment was cloned into the BamHI and SacII sites of the pBluescript-KSII vector (Stratagene) , and was subsequently confirmed by sequence analysis.
  • the SV40 polyadenylation-signal seq ⁇ nce was isolated by BairiHI and Bell digestion(62) ; SacII linkers were J igated to this fragment which was then cloned downstream of the AjS region.
  • the mouse NF-L promoter(63) was cloned into the Hindlll and BamHI sites.
  • the 2.2-kb HindllI-Sad fragment was used for the generation of the transgenic mammals.
  • Figure 2 is a characterization of the A/3 mRNA in transgenic mice.
  • Lanes 1 and 2 contain 10 ⁇ g of RNA from the brains of 4-week old non-transgenic and G2 transgenic mice, respectively.
  • the higher molecular weight band in both lanes 1 and 2 correspond to the endogenous APP transcripts.
  • the lower band (indicated by the arrow) is approximately 600 nucleotides (nt) in length and represents the recombinant DNA transcript.
  • the position of the 28S and 18S ribosomal RNA are indicated by the arrowheads.
  • Figure 3 shows detection of the A ⁇ transgenic mRNA in the brain by in si tu hybridization.
  • Brain regions represented include the hippocampus ( a, b) and the cerebral neocortex ( c, d) .
  • Figure 5 shows the incidence of death in the NF_-A/3 transgenic mice. The solid and striped bars reflect the death rates for the control and transgenic mice, respectively. Significance values were calculated using Chi square (Maentel-Haenzel) analysis and are indicted above each time point.
  • Figure 6 shows alterations in neuronal cell morphology in the brains of transgenic mice.
  • High magnification micrographs of hematoxylin-and-eosin stained sagittal sections from the neocortex (a, b) , hippocampus . c, d) , and thalamus ( e, f) are shown.
  • Control mice and transgenic littermates (10 months of age) are on the left and right sides, respectively.
  • c and d the top tract of cells are from the dentate gyrus while the bottom are from the CA3 region.
  • Original magnifications 50X
  • Figure 7 shows biochemical and morphological detection of apoptotic cells in transgenic brains.
  • Cerebral cortex from a control mammal in which no TUNEL- positive cells are evident (a) .
  • Transgenic neocortex from the G2 (Jb) and J3 lines (c) stained with TUNEL showing extensive apoptosis.
  • a control hippocampus from a normal mammal in which no TUNEL-positive cells are evident (f) .
  • the hippocampus (g) and amygdala (h) from mammals of the G2 line with extensive TUNEL staining.
  • Figure 8 shows astrogliosis in the brains of transgenic mice.
  • CA Ammon's horn
  • CX cerebral cortex
  • DG dentate gyrus
  • GL glial limitans.
  • Figure 9 shows the amino acid sequences human (A) , murine (B) , and HCHWA-D (C) AjS peptides.
  • the amino acids are represented by the conventional one-letter symbols.
  • a dash indicates that the amino acid is the same as the human sequence.
  • a murine homolog of the human A ⁇ peptide was selected to interact with species-specific cellular factors to potentiate its toxicity.
  • the murine and human A ⁇ peptides are highly conserved, and, like its human counterpart, the murine peptide possesses the ability to form fibrils in vi tro and is as amyloidogenic as the human sequence(28,29) .
  • To restrict expression of the A ⁇ coding region to neuronal cells 1.8-kb of 5' flanking DNA from the mouse neurofilament-light (NF-L) gene was selected (Figure 1) ; the NF-L gene is transcriptionally active throughout adult life (30) .
  • a methionine initiation codon and a Kozak sequence was placed immediately upstream of the A ⁇ coding sequence.
  • the polyadenylation sequence was derived from a BamHI/BclI digestion of SV40 DNA. There are two polyA signals on this fragment in opposite orientations; the early polyA signal of the T-Ag gene (i.e., 5' BclI-BamHI 3') was used. However, it has been found (Mol . Cell . Biol . 9 , 4248-4258) that the polyA signal may be utilized if inserted in the late orientation, i.e., 5' BamHI-BClI 3' ; thus, this fragment can be used, as well.
  • the Hindlll site is a naturally occurring site at the 5' -end of the promoter, and the BamHI was engineered (J. Cell . Biol . 108, 579-593, 1989) . After processing in the cell to remove the methionine, an authentic 42-amino acid A/3 peptide will be generated.
  • the 2.2-kb NF L -A/3 chimeric recombinant DNA was microinjected into single-cell FVB/N embryos (31-34) . Founder mammals were backcrossed to the parental FVB/N (Taconic, 273 Hover Avenue, Germantown, NY 12526) strain to establish independent lines. The initial transgenic mammals were identified by Southern blot hybridization. Tail DNA was extracted, digested with the restriction enzyme BamHI and subjected to electrophoresis on an agarose gel. The DNA was then transferred to nitrocellulose and probed with a DNA fragments which includes the 3' portion of the neurofilament promoter through the A/3 coding sequence.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • the transgenic transcript is predicted to be approximately 500-600 nucleotides (nt) long, depending on the length of the polyA tract; this includes 111 nt of 5' non-coding sequence, 129 nt from the coding region, and another 175 nt of 3' non-coding sequence.
  • the probe used for the Northern analysis in addition to detecting the transgenic transcript, also detected the endogenous APP transcript, providing an internal reference to which the steady-state level of the recombinant DNA mRNA could be compared. Hybridization to a band of approximately 600 nt, the expected size of the transgenic transcript, is only seen in lane 2, which contains brain RNA from a transgenic mouse of the G2 line.
  • the probe also detects a band of approximately 3.2-kb in length, the reported size of the endogenous APP transcripts(5-7) , which is also present in the lane containing RNA from a nontransgenic brain.
  • a comparison of the levels of the transgenic transcript to the endogenous transcript clearly reveals a substantially higher steady-state level for the former ( Figure 2) . This difference is even more dramatic when one considers that the transgenic mRNA will exclusively encode the AjS peptide while at best only a fraction of the molecules translated from the endogenous APP mRNA will yield AjS.
  • three independent expressing lines (G2, J3, and J4) were selected for further analysis.
  • Example 3 To precisely localize those areas of the brain that expressed the recombinant DNA, in si tu hybridization analyses were performed.
  • Paraffin sections of brain tissue on silane-coated slides were deparaffinized in xylene, rehydrated through an ethanol series, and treated with proteinase K (1 ⁇ g/ml) for 10 min. This was followed by treatment with 2 mg/ml glycine in PBS for 30 sec, a rinse in PBS, a postfixation in 4% paraformaldehyde in PBS for 5 min. Sections were then air dried and hybridized with ⁇ - 4 x 10 6 cpm/50 ⁇ l/slide of tailed probe.
  • Hybridization buffer consisted of 50% formamide, 0.6 M NaCl, 0.05 M Tris, 7.4, 0.004 M EDTA, 0.1% sodium pyrophosphate, 0.2% SDS, 10% dextran sulfate, 0.2% sodium heparin, and 0.1 M DTT. After overnight incubation at 37°C, slides were rinsed in 1 x SSC for 10 min. (twice) , washed once for 10 sec. in water, dipped in 70% ethanol and air dried and then exposed to film.
  • Oligonucleotide probe for in si tu hybridization A/3/SV40 junctional probe, 5'GTGGTATGGCTGATTATGATCCGCGGTTATGCTATGACAACGCCACC3 ' ;
  • cryosections from transgenic and age- matched control mice were immunostained with A/3-specific antibodies.
  • Antibodies used included Boehringer Mannheim Cat. No. 1 381 431; rabbit polyclonal antisera (472,473) prepared conventionally; and antibody 4G8, purchased from H. Wisniewski (SUNY Brooklyn, New York) .
  • Antibody to GFAP was purchased from Dakopatts (Cat. No. Z 334) . No immunostaining was detected in either transgenic or control brain tissues when pre-immune serum was used (data not shown) .
  • Nontransgenic brain sections also failed to immunostain appreciably with any of the A ⁇ antibodies tested, whereas A/3 immunoreactivity was detected in the brains of transgenic mice.
  • Figure 4 compares the regional distribution of immunoreactivity for the A ⁇ peptide in the hippocampal and cortical regions of control and transgenic mice. While significant AjS immunoreactivity was not evident in the control hippocampus ( Figure 4a) , immunostaining was detectable over cells of Ammon's horn (CA cells) as well as over cells of the dentate gyrus of a transgenic mammal (Figure 4Jb) .
  • expression is not widespread in all regions; in some areas, expression appears higher and involves a majority of cells, whereas only focal areas of cells in other regions appear immunoreactive. By and large, there is a good overlap between A ⁇ mRNA and peptide expression in the brains of the transgenic mammals.
  • Example 4 Behavioral phenotype, neuronal cell degeneration results from A3 expression
  • mice 10-30 seconds during which the mice would extend all four limbs, open their mouths, flex their tails so that they became rigid, and shake violently. Following the episodes, the mice appeared to experience a brief period of lethargy. Given the brief nature of these seizures, it is likely that a larger proportion of the transgenic mice may, in fact, manifest this behavior. Furthermore, seizure a: .ivity may ⁇ e triggered by disruption of specific neural pathways. Since not every mouse is affected to the same degree in each brain region, one may not expect all of the mammals to be equally involved. Seizures were never seen in any of the control littermates. Notably, the occurrence of seizures has also been observed in AD patients, where it appears to be a frequent feature in later stages of the disease (37-39) .
  • Transgenic mammals that died prematurely were necropsied to determine the cause of death. Gross anatomic as well as histological examination of visceral organs failed to reveal any pathological changes that would account for their deaths. However, in virtually every mammal, certain regions of the brain contained morphologically altered cells. To confirm this observation, age-matched control and transgenic mammals were anesthetized, transcardially perfused, and immediately processed for histological analysis. In the transgenic mice analyzed, sharply demarcated zones of unstained perinuclear cytoplasm of the neuronal cells were observed in different brain regions, including the cerebral cortex, hippocampus, thalamus, and occasionally the hindbrain.
  • FIG. 6a shows the neocortex from a perfused nontransgenic mouse where the perikarya appear normal .
  • many cells in the transgenic cortex appear to be morphologically altered, as indicated by perinuclear zones of unstained cytoplasm.
  • transgenic mice morphologically altered neuronal cells were observed in 18 of 19 mammals that died unexpectedly, as well as in 7 of 8 mammals that were euthanized by perfusion during the course of the study. Therefore, intracellular accumulation of the AjS peptide may be deleterious to certain neurons or may render them more susceptible to other insults or challenges.
  • Example 5 Neurodegeneration is followed bv apoptotic cell death
  • TUNEL terminal deoxynucleotide transferase-mediated dUTP-biotin nick end labelling
  • the cerebral cortex was one of the most frequently involved regions, and examples from representative mammals of the G2 and J3 lines are shown ( Figures 7Jb, c) . Note that
  • TUNEL-positive cells are scattered throughout the most peripheral layers of the cortex; however, apoptotic cells were occasionally observed in the deeper cortical layers. We also observed that some of the nuclei were stained to different extents by TUNEL; as the nucleus was not condensed in some of these cells, it suggests that they may be at different stages of the apoptotic process ( Figure 7c) . In contrast, TUNEL-positive cells were not observed in the negative littermates ( Figure 7a) . In addition, cells with perinuclear zones of unstained cytoplasm ( Figure 7d) were often found in close proximity to intensely stained TUNEL cells ( Figure 7c) , implying that the degenerating cells will eventually die by apoptosis.
  • the hippocampus was another region that frequently contained striking TUNEL staining in the transgenic mammals. While in some mammals, tracts of cells within the CA1 and CA3 regions contained prominent TUNEL staining (Figure 7g) , in others, cells of the CA2 region and dentate gyrus may also be labelled by TUNEL. No TUNEL-positive cells were observed in the control hippocampus ( Figure If) . Another prominent region that contained apoptotic cells was the amygdala ( Figure Ih) . In the amygdala, as was the case for the cerebral cortex and hippocampus, apoptotic cells were found in close apposition to unstained cells, demonstrating the specificity of TUNEL. Interestingly, these three brain regions that most frequently contained apoptotic cells are among the most compromised regions in AD brains(2) .
  • TUNEL allows for earlier detection of apoptotic cells than morphological criteria alone(41), apoptotic cells were nevertheless recognizable in histological preparations.
  • Example 6 Reactive gliosis accompanies neurodegeneration and apoptosis
  • AjS expression was inducing neurodegeneration and apoptosis in the brains of the transgenic mice, this suggests that there must be functional impairments as well.
  • a prominent response to many types of injuries or insults to the CNS is the activation of astrocytes, a process referred to as gliosis (46); this reactive process is also frequently observed in AD brains (47-49) . Consequently, brain tissues from transgenic and age- matched control mice were immunostained for glial fibrillary acidic protein (GFAP) , an astrocyte-specific intermediate filament (50) .
  • GFAP glial fibrillary acidic protein
  • 50 astrocyte-specific intermediate filament
  • the gliotic areas were less widespread and may be more restricted to either the superficial or deeper cortical layers ( Figure 8e, f, respectively) .
  • the difference in extent of gliosis may be due to the lower level of recombinant DNA expression in the J3 and J4 lines.
  • the astrocytes in the transgenic brains were enlarged, with thicker and increased numbers of processes, all features of reactive astrocytes(46) .
  • Reactive astrocytes are frequently found in areas that contained degenerating cells, either with or without cells progressing to apoptosis (cf. Figures 7c, e) . This was true not only for the cerebral cortex but for other regions including the amygdala and hippocampus (data not shown) . However, regions that appeared hypocellular and spongiotic were often not gliotic, suggesting that once cells have died, astrocytes were no longer present. The development of astrogliosis in the transgenic mice provides further evidence for neurological abnormalities. Astrocytes play an important role in the CNS by responding to injury.
  • Recombinant DNA expression was detected in a variety of tissues including heart, intestine, kidney, liver, muscle, and spleen (data not shown) , an observation that has been reported by others using the same NF-L promoter to drive expression of an heterologous gene in transgenic mammals (53) . Since single cell analysis was not performed, it remains possible that expression was still limited to neurons in all of these tissues. Although recombinant DNA RNA expression was detected in peripheral tissues, histopathological changes were observed only in the brain. Interestingly, A ⁇ deposits can be detected in AD patients outside the CNS in tissues such as skin and intestine, albeit with only subclinical consequences (54) . These observations suggest that the brain may be more vulnerable to the toxic effects of the AjS peptide both in humans and in mice.
  • AD Alzheimer's disease
  • the behavior of each mouse is compared to control mice of the same age using the Morris water maze, and the phenotype is observed. After observing and detecting the behavior, each mouse is sacrificed and assayed for A ⁇ expression as described in the examples above. The experiment is repeated administering different dosages of agent.
  • neocortex from an AjS transgenic mouse were subjected to hematoxylin-and-eosin (H&E) , TUNEL, anti-A/3 and anti-p53 staining.
  • the cortical neurons were morphologically normal-appearing and exhibited an open nuclear morphology. As expected, these cells were TUNEL " because they had no A ⁇ accumulation and no p53 activation.
  • the cortical neurons located at the bottom left revealed a distinctively basophilic cytoplasm and appeared degenerative. They were weakly TUNEL* and showed abundance of both cytoplasmic A ⁇ and nuclear p53 immunoreactivity.
  • cortical neurons at the top right showed highly condensed or fragmented nuclei and were strongly TUNEL*; many of these cells also expressed AjS and p53. Since clusters of neurons invariably undergo apoptosis coordinately, the regional analysis of gene expression may be taken to represent responses in individual cells. This finding demonstrates that intracellular accumulation of AjS correlated perfectly with the activation of p53. Does p53 activa tion precede cell death? Since analysis of the cerebral cortex hinted to the expression of p53 occurring prior to the detection of overt cell death, we analyzed another region of the brain to determine whether even more convincing evidence may be obtained.
  • the top right region lies within the CA3 subregion of the hippocampus and the overlying white matter (Truncus corporis callosi) .
  • the bottom left region is located within the thalamus (possibly Nucleus ventralis thalami, pars basalis) .
  • a high magnification view of the anti-A/3 immunostaining in all three regions shows that the extracellular deposits resemble diffuse plaques.
  • these deposits could also be detected with facility using a silver stain when found not overlying nerve fibers. Not only does this imply significant amounts of the A/3 protein being deposited in the extracellular space, a high magnification view also showed that these deposits assume the appearance of ice crystals and may represent fibrillar aggregates.
  • TUNEL * cells were found to perfectly overlap regions with extracellular AjS immunostaining, confirming the association of cell death with extracellular deposition. Further evidence for cellular injury was obtained from H&E staining of regions around CA1 and CA3, which are hypocellular. The presence of focal regions of basophilia, detected by hematoxylin which stains nuclear chromatin, an overlying A ⁇ immunoreactive areas, suggests that these deposits are released subsequent to cell death. At high magnification, an occasional cell ghost may be detected together with disorganization of the surrounding neuropil in an area marked by distinct basophilia.
  • Kang, J. et al The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature 325, 733-736 (1987) .
  • Amyloid /3-peptide is produced by cultured cells during normal metabolism. Na ture 359, 322-325 (1992) .
  • Citron, M. et al Mutation of the /3-amyloid precursor protein in familial Alzheimer's disease increases j ⁇ -protein production. Nature 360, 672-674 (1992) .
  • GFAP Glial fibrillary acidic protein

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Abstract

L'invention concerne un mammifère qui comprend des cellules contenant un transgène, le transgène comprenant un promoteur hétérologue relié à une séquence d'acide nucléique contenant essentiellement une séquence de codage pour un peptide Aβ. Une méthode de recherche d'un composé ayant un effet sur un phénotype induit par expression d'un peptide Aβ transgénique dans le cerveau d'un mammifère est également décrit. Le procédé consiste à administrer le composé à un mammifère exprimant le peptide Aβ transgénique, et à observer s'il se produit un effet sur un phénotype.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046636A3 (fr) * 1997-04-16 1999-01-28 American Home Prod PROTEINES FIXANT LE PEPTIDE β-AMYLOIDE ET POLYNUCLEOTIDES CODANT CES PROTEINES
US6717031B2 (en) 1995-06-07 2004-04-06 Kate Dora Games Method for selecting a transgenic mouse model of alzheimer's disease
US6787319B2 (en) 1997-04-16 2004-09-07 American Home Products Corp. β-amyloid peptide-binding proteins and polynucleotides encoding the same
US7005295B1 (en) 1997-04-16 2006-02-28 Wyeth β-amyloid peptide-binding proteins and polynucleotides encoding the same
US7097837B2 (en) 2001-02-19 2006-08-29 Pharmexa A/S Synthetic vaccine agents
US7135181B2 (en) 2000-02-21 2006-11-14 Pharmexa A/S Method for down-regulation of amyloid
US8283517B2 (en) 2007-09-12 2012-10-09 Probiodrug Ag Transgenic mouse models of Aβ overexpression
US8871212B2 (en) 2001-08-20 2014-10-28 H. Lundbeck A/S Amyloid-beta polypeptide vaccine

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 149, No. 2, issued 16 December 1987, T. YAMADA et al., "Complementary DNA for the Mouse Homolog of the Human Amyloid Beta Protein Precursor", pages 665-671. *
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 192, No. 2, issued 30 April 1993, M. REEBEN et al., "Tissue-Specific Expression of Rat Light Neuofilament Promoter-Driven Reporter Gene in Transgenic Mice", pages 465-470. *
JOURNAL OF CLINICAL INVESTIGATION, Volume 93, issued May 1994, Y. OGAWA et al., "Molecular Cloning of the Complementary DNA and Gene That Encode Mouse Brain Natriuretic Peptide and Generation of Transgenic Mice That Overexpress the Brain Natriuretic Peptide Gene", pages 1911-1921. *
PROC. NATL. ACAD. SCI. U.S.A., Volume 89, issued November 1992, A. KAMMESHEIDT et al., "Deposition of Beta/A4 Immunoreactivity and Neuronal Pathology in Transgenic Mice Expressing the Carboxyl-Terminal Fragment of the Alzheimer's Amyloid Precursor in the Brain", pages 10857-10861. *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6717031B2 (en) 1995-06-07 2004-04-06 Kate Dora Games Method for selecting a transgenic mouse model of alzheimer's disease
WO1998046636A3 (fr) * 1997-04-16 1999-01-28 American Home Prod PROTEINES FIXANT LE PEPTIDE β-AMYLOIDE ET POLYNUCLEOTIDES CODANT CES PROTEINES
US6787319B2 (en) 1997-04-16 2004-09-07 American Home Products Corp. β-amyloid peptide-binding proteins and polynucleotides encoding the same
US7005295B1 (en) 1997-04-16 2006-02-28 Wyeth β-amyloid peptide-binding proteins and polynucleotides encoding the same
US7101973B2 (en) 1997-04-16 2006-09-05 Wyeth β-amyloid peptide-binding proteins and polynucleotides encoding the same
US7135181B2 (en) 2000-02-21 2006-11-14 Pharmexa A/S Method for down-regulation of amyloid
US7097837B2 (en) 2001-02-19 2006-08-29 Pharmexa A/S Synthetic vaccine agents
US8871212B2 (en) 2001-08-20 2014-10-28 H. Lundbeck A/S Amyloid-beta polypeptide vaccine
US8283517B2 (en) 2007-09-12 2012-10-09 Probiodrug Ag Transgenic mouse models of Aβ overexpression

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