EP0833901A1 - Methode zur identifizierung von arzneimitteln gegen die alzheimer krankheit mittels transgener tiermodelle - Google Patents

Methode zur identifizierung von arzneimitteln gegen die alzheimer krankheit mittels transgener tiermodelle

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Publication number
EP0833901A1
EP0833901A1 EP96919314A EP96919314A EP0833901A1 EP 0833901 A1 EP0833901 A1 EP 0833901A1 EP 96919314 A EP96919314 A EP 96919314A EP 96919314 A EP96919314 A EP 96919314A EP 0833901 A1 EP0833901 A1 EP 0833901A1
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European Patent Office
Prior art keywords
app
glu
protein
val
ala
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EP96919314A
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English (en)
French (fr)
Inventor
Kate D. Games
Dale B. Schenk
Lisa Claire Mcconlogue
Peter A. Seubert
Russell E. Rydel
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Elan Pharmaceuticals LLC
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Athena Neurosciences Inc
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Publication of EP0833901A1 publication Critical patent/EP0833901A1/de
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    • 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
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • 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
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • Transgenic animal models of Alzheimer's disease are described along with a method of using the transgenic animal models to screen for therapeutics useful for the treatment of Alzheimer's disease.
  • AD Alzheimer's disease
  • AD patients have increased problems with memory loss and intellectual functions which progress to the point where they cannot function as normal individuals. With the loss of intellectual skills the patients exhibit personality changes, socially inappropriate actions and schizophrenia (A Guide to the Understanding of Alzheimer's Disease and Related Disorders, edited by Jorm (New York University Press, New York 1987). AD is devastating for both victims and their families, for there is no effective palliative or preventive treatment for the inevitable neurodegeneration.
  • AD Alzheimer's disease
  • AD patients At a macroscopic level, the brains of AD patients are usually smaller, sometimes weighing less than 1,000 grams.
  • the histopathological hallmarks of AD include neurofibrillary tangles (NFT), neuritic plaques, and degeneration of neurons.
  • AD patients exhibit degeneration of nerve cells in the frontal and temporal cortex of the cerebral cortex, pyramidal neurons of hippocampus, neurons in the medial, medial central, and cortical nuclei of the amygdala, noradrenergic neurons in the locus coeruleus, and the neurons in the basal forebrain cholinergic system.
  • AD Alzheimer's Disease and Related Disorders, Research and Development edited by Kelly (Charles C. Thomas, Springfield, IL. 1984)). In fact, AD is defmed by the neuropathology of the brain.
  • AD is associated with neuritic plaques measuring up to 200 ⁇ m in diameter in the cortex, hippocampus, subiculum, hippocampal gyrus, and amygdala.
  • One of the principal constituents of neuritic plaques is amyloid, which is stained by Congo Red (Fisher (1983); Kelly (1984)).
  • Amyloid plaques stained by Congo Red are extracellular, pink or rust-colored in bright field, and birefringent in polarized light.
  • the plaques are composed of polypeptide fibrils and are often present around blood vessels, reducing blood supply to various neurons in the brain.
  • AD neuropathy Various factors such as genetic predisposition, infectious agents, toxins, metals, and head trauma have all been suggested as possible mechanisms of AD neuropathy. However, available evidence strongly indicates that there are distinct types of genetic predispositions for AD.
  • AD-stricken families have provided evidence for mutations in the amyloid precursor protein (APP) gene in certain AD-stricken families (Goate et al. Nature 349:704-706 (1991); Murrell et al. Science 254:97-99 (1991); Chartier-Harlin et al. Nature 353:844-846 (1991); Mullan et al, Nature Genet. 1:345-347 (1992)). Additional genes for dominant forms of early onset AD reside on chromosome 14 and chromosome 1 (Rogaev et al.
  • AD apolipoprotein E
  • Amyloid plaques are abundantly present in AD patients and in Down's Syndrome individuals surviving to the age of 40.
  • the overexpression of APP in Down's Syndrome is recognized as a possible cause of the development of AD in Down's patients over thirty years of age (Rumble et al. , New England J. Med. 320: 1446-1452 (1989); Mann et al, Neurobiol. Aging 10:397-399 (1989)).
  • the plaques are also present in the normal aging brain, although at a lower number. These plaques are made up primarily of the amyloid ⁇ peptide (A ⁇ ; sometimes also referred to in the literature as ⁇ -amyloid peptide or ⁇ peptide) (Glenner and Wong, Biochem. Biophys. Res.
  • amyloid is a filamentous material that is arranged in beta-pleated sheets.
  • A/3 is a hydrophobic peptide comprising up to 43 amino acids. The determination of its amino acid sequence led to the cloning of the APP cDNA (Kang et al, Nature 325:733-735 (1987); Goldgaber et al, Science 235:877-880 (1987); Robakis et al , Proc. Natl Acad. Sci.
  • a ⁇ consists of up to 28 amino acids just outside the hydrophobic transmembrane domain and up to 15 residues of this transmembrane domain.
  • a ⁇ is a cleavage product derived from APP which is normally found in brain and other tissues such as heart, kidney and spleen.
  • a ⁇ deposits are usually found in abundance only in the brain.
  • APP751, APP770 The larger alternate forms of APP (APP751, APP770) consist of APP695 plus one or two additional domains.
  • APP751 consists of all 695 amino acids of APP695 plus an additional 56 amino acids which has homology to the Kunitz family of serine protease inhibitors (KPI) (Tanzi et al. (1988); Weidemann et al , Cell 57: 115-126 (1989); Kitaguchi et al, Nature 331:530-532 (1988); Tanzi et al, Nature 329:156 (1987)).
  • KPI Kunitz family of serine protease inhibitors
  • APP770 contains all 751 amino acids of APP751 and an additional 19 amino acid domain homologous to the neuron cell surface antigen OX-2 (Weidemann et al (1989); Kitaguchi et al (1988)). Unless otherwise noted, the amino acid positions referred to herein are the positions as they appear in APP770. The amino acid number of equivalent positions in APP695 and APP751 differ in some cases due to the absence of the OX-2 and KPI domains. By convention, the amino acid positions of all forms of APP are referenced by the equivalent positions in the APP770 form. Unless otherwise noted, this convention is followed herein.
  • APP is post-translationally modified by the removal of the leader sequence and by the addition of sulfate and sugar groups.
  • FAD familial Alzheimer's disease
  • FAD familial Alzheimer's disease
  • These mutations co-segregate with the disease within the families and are absent in families with late-onset AD.
  • This mutation at amino acid 717 increases the production of the Aj3 M2 form of A ⁇ from APP (Suzuki et al , Science 264:1336-1340 (1994)).
  • Another mutant form contains a change in amino acids at positions 670 and 671 of the full length protein (Mullan et al.
  • AD-like symptoms may be induced by electrolysis, transplantation of AD brain samples, aluminum chloride, kainic acid or choline analogs (Kisner et al, Neurobiol Aging 7:287-292 (1986); Mistry et al, J Med Chem 29:337-343 (1986)).
  • transgenic rodent lines have been produced that express either the human APP gene or human APP complementary DNA regulated by a variety of promoters.
  • Transgenic mice with the human APP promoter linked to E. coli /3-galactosidase (Wirak et al, The EMBO J 10:289-296 (1991)) as well as transgenic mice expressing the human APP751 cDNA (Quon et al. Nature 352:239-241 (1991)) or subfragments of the cDNA including the A ⁇ (Wirak et al, Science 253:323-325 (1991); Sandhu et al, J. Biol. Chem.
  • Kawabata et al (1991) was later retracted by Kawabata et al, Nature 356:23 (1992) and Kawabata et al, Nature 356:265 (1992).
  • transgenic mice expressing the APP751 cDNA from the neuron-specific enolase promoter of Quon et al. (1991) rare, small extracellular deposits of material reactive with antibody prepared against synthetic A ⁇ were observed.
  • a review of the papers describing these early transgenic mice indicate that do not produce characteristic Alzheimer pathologies (see Marx, Science 255:1200-1202 (1992)).
  • mice expressing APP751 from a neuron-specific enolase (NSE) promoter were recently described by McConlogue et al , Neurobiol Aging 15:S12 (1994), Higgins et al, Ann Neurol. 35:598-607 (1995), Mucke et al, Brain Res. 666:151-167 (1994), Higgins et al, Proc. Natl. Acad. Sci. USA 92:4402-4406 (1995), and U.S. Patent 5,387,742 to Cordell.
  • Higgins et al, Ann Neurol 35:598-607 (1995) describe results with the same mice as described by Quon et al. (1991).
  • mice have only sparse A ⁇ deposits which are more typical of very early AD and young Down's syndrome cases.
  • the deposits seen in this transgenic mouse were also seen, although at a lower abundance, in non-transgenic control animals. Mature lesions such as frequent compacted plaques, neuritic dystrophy and extensive gliosis are not seen in these mice (Higgins et al, Ann Neurol. 35:598-607 (1995)). McConlogue et al. (1994) reported finding no A ⁇ deposits in these mice.
  • Transgenic mice in which APP is expressed from the neuronal specific synaptophysin promoter express APP at low levels equivalent to that in brain tissue from the NSE APP mice described above. These mice were also reported not to display any brain lesions (Higgins et al).
  • mice containing yeast artificial chromosome (YAC) APP constructs have also been made (Pearson and Choi, Proc. Natl. Acad. Sci. USA 90:10578-10582 (1993); Lamb et al, Nature Genetics 5:22-30 (1993); Buxbaum et al, Biochem. Biophys. Res. Comm. 197:639-645 (1993)). These mice contain the entire human APP genomic gene and express human APP protein at levels similar to endogenous APP; higher levels of expression than that obtained in mice using the NSE promoter. None of these mice, however, show evidence of pathology similar to AD.
  • YAC yeast artificial chromosome
  • Alzheimer's disease animal models including transgenic models, have been recently reviewed by Lannfelt et al, Behavioural Brain Res. 57:207- 213 (1993), and Fukuchi et al, Ann. N Y. Acad. Sci. 695:217-223 (1993).
  • Lannfelt et al. points out that none of the prior transgenic animals that show apparent plaques demonstrate neuropathological changes characteristic of AD.
  • Lannfelt et al. also discusses possible reasons for the "failure" of previous transgenic animal models.
  • Fukuchi et al. discusses the failure of prior transgenic animal models to display most of the characteristics known to be associated with AD.
  • the transgenic mouse reported by Quon et al. is reported to produce A ⁇ immunoreactive deposits that stain only infrequently with thioflavin S and not at all with Congo Red, in contrast to the staining pattern of AD A ⁇ deposits.
  • Alzheimer's disease is characterized by numerous changes in the expression levels of various proteins, the biochemical activity and histopathology of brain tissue, as well as cognitive changes in affected individuals. Such characteristic changes associated with AD have been well documented. The most prominent change, as noted above, is the deposition of A ⁇ into amyloid plaques (Haass and Selkoe, Cell 75:1039-1042 (1993)). A variety of other molecules are also present in plaques, such as apolipoprotein E, laminin, amyloid P component, and collagen type IV
  • Alzheimer's disease is also known to stimulate an immunoinflammatory response, increasing such inflammatory markers as glial fibrillary acidic protein (GFAP), ⁇ 2-macroglobulin, and interleukins 1 and 6 (IL-1 and IL-6) (Frederickson and Brunden, Alzheimer Disease and
  • AD Alzheimer's disease
  • transgenic animal models for testing potential treatments for Alzheimer's disease is described.
  • the models are characterized by a greater similarity to the conditions existing in naturally occurring Alzheimer's disease, based on the ability to control expression of one or more of the three major forms of the /3-amyloid precursor protein (APP), APP695, APP751, and APP770, or subfragments thereof, as well as various point mutations based on naturally occurring mutations, such as the FAD mutations at amino acid 717, and predicted mutations in the APP gene.
  • APP /3-amyloid precursor protein
  • the APP gene constructs are prepared using the naturally occurring APP promoter of human, mouse, or rat origin, efficient promoters such as human platelet derived growth factor ⁇ chain (PDGF-B) gene promoter, as well as inducible promoters such as the mouse metallothionine promoter, which can be regulated by addition of heavy metals such as zinc to the animal's water or diet.
  • PDGF-B platelet derived growth factor ⁇ chain
  • inducible promoters such as the mouse metallothionine promoter
  • Neuron-specific expression of constructs can be achieved by using the rat neuron specific enolase promoter.
  • constructs are introduced into animal embryos using standard techniques such as microinjection or embryonic stem cells.
  • Cell culture based models can also be prepared by two methods. Cells can be isolated from the transgenic animals or prepared from established cell cultures using the same constructs with standard cell transfection techniques.
  • constructs disclosed herein generally encode all or a contiguous portion of one of the three forms of APP: APP695, APP751, or APP770, preferably an A/3-containing protein, as described herein.
  • a ⁇ - containing proteins are proteins that include all or a contiguous portion of APP770, APP770 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, APP751, APP751 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, APP695, and APP695 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, where each of these A ⁇ - containing proteins includes amino acids 672 to 714 of human APP.
  • Some specific constructs that are described employ the following protein coding sequences: the APP770 cDNA; the APP770 cDNA bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; the APP751 cDNA containing the KPI protease inhibitor domain without the OX-2 domain in the construct; the APP751 cDNA bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; the APP695 cDNA; the APP695 cDNA bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; APP695, APP751, or APP770 cDNA truncated at amino acid 671 or 685, the sites of ⁇ -secretase or ⁇ -secretase cleavage, respectfully; APP cDNA truncated to encode
  • leader sequences specifying the transport and secretion of the encoded A ⁇ related protein.
  • a preferred leader sequence is the APP leader sequence.
  • These combined protein coding sequences are in turn operably linked to a promoter that causes high expression of A ⁇ in transgenic animal brain tissue.
  • a preferred promoter is the human platelet derived growth factor ⁇ chain (PDGF-B) gene promoter.
  • Additional constructs include a human yeast artificial chromosome construct controlled by the PDGF-B promoter; a human yeast artificial chromosome construct controlled by the PDGF-B promoter with the addition of a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; the endogenous mouse or rat APP gene modified through the process of homologous recombination between the APP gene in a mouse or rat embryonic stem (ES) cell and a vector carrying the human APP cDNA bearing a mutation at amino acid position 669, 670, 671, 690, 692, 717, or a combination of these mutations, such that sequences in the resident rodent chromosomal APP gene beyond the recombination point (the preferred site for recombination is within APP exon 9) are replaced by the analogous human sequences bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of
  • transgenic animals can be introduced into the transgenic animals and then combined by mating of animals expressing the different constructs.
  • the transgenic animals, or animal cells are used to screen for compounds altering the pathological course of Alzheimer's disease as measured by their effect on the amount and/or histopathology of Alzheimer's disease markers in the animals, as well as by behavioral alterations.
  • markers include APP and APP cleavage products; A ⁇ ; other plaque related molecules such as apolipoprotein E, laminin, and collagen type IV; cytoskeletal markers, such as spectrin, tau, neurofilaments, and MAP-2; inflammatory markers, such as GFAP, ⁇ 2-macroglobulin, IL-1, and IL-6; and neuronal and synaptic neurotransmitter related markers, such as GAP43 and synaptophysin, and those associated with the cholinergic, muscarinic, serotinergic, adrenergic, and adensosine receptor systems.
  • the boxed portions of the drawings indicate the amino acid coding portions of the constructs. Filled portions indicate the various domains of the protein as indicated in the Figure Legend. Lines indicate sequences in the clones that are 5' or 3' untranslated sequences, flanking genomic sequences, or introns. The break in the line to the left of the constructs in Figures 7 and
  • Figure la is a schematic of the APP770 cDNA coding sequence.
  • Figure lb is a schematic of the APP770 cDNA coding sequence bearing a mutation at position 717.
  • Figure 2a is a schematic of the APP751 cDNA coding sequence.
  • Figure 2b is a schematic of the APP751 cDNA coding sequence bearing a mutation at position 717.
  • Figure 3a is a schematic of the APP695 coding sequence.
  • Figure 3b is a schematic of the APP695 cDNA coding sequence bearing a mutation at position 717.
  • Figure 4a is a schematic of a coding sequence for the carboxy terminal portion of APP.
  • Figure 4b is a schematic of a coding sequence for the carboxy terminal portion of APP bearing a mutation at position 717.
  • Figure 5 is a schematic of a coding sequence for the A ⁇ portion of APP.
  • Figure 6a is a schematic of a combination cDNA/genomic coding sequence allowing alternative splicing of the KPI and OX-2 exons.
  • Figure 6b is a schematic of a combination cDNA/genomic coding sequence bearing a mutation at position 717 and allowing alternative splicing of the KPI and OX-2 exons.
  • Figure 7a is a schematic of a human APP YAC coding sequence.
  • Figure 7b is a schematic of a human APP YAC coding sequence bearing a mutation at position 717.
  • Figures 8a and 8b are schematics of genetic alteration of the mouse APP gene by homologous recombination between the mouse APP gene in a mouse ES cell and a vector carrying the human APP cDNA (either of the wild-type ( Figure 8a) or FAD mutant form ( Figure 8b)) directed to the exon
  • Figure 9 is a schematic map of the PDAPP vector, a combination cDNA/genomic APP construct.
  • Figure 10 is a diagram of the genomic region of APP present in the
  • FIG. 11 is a diagram of the intermediate constructs used to construct the APP splicing cassette and the PDAPP vector.
  • Figure 12 is a diagram of the PDAPP-wt vector and the plasmids used to make the PDAPP-wt vector.
  • Figure 13 is a diagram of the PDAPP-Sw/Ha vector and the plasmids and intermediate constructs used to make the PDAPP-Sw/Ha vector.
  • Figure 14 is a diagram of the PDAPP695 V-F vector and the plasmids and intermediate constructs used to make the PDAPP695 V . F vector.
  • Figure 15 is a diagram of the PDAPP751 V _ F vector and the plasmids and intermediate constructs used to make the PDAPP751 V . F vector. Detailed Description of the Invention
  • the constructs and transgenic animals and animal cells are prepared using the methods and materials described below. Sources of materials.
  • Restriction endonucleases are obtained from conventional commercial sources such as New England Biolabs (Beverly, MA.), Promega Biological Research Products (Madison, WI.), and Stratagene (La Jolla CA.). Radioactive materials are obtained from conventional commercial sources such as Dupont/NEN or Amersham. Custom-designed oligonucleotides for site-directed mutagenesis are available from any of several commercial providers of such materials such as Bio-Synthesis Inc., Lewisville, TX. Kits for carrying out site-directed mutagenesis are available from commercial suppliers such as Promega Biological Research Products and Stratagene. Clones of cDNA including the APP695, APP751, and APP770 forms of APP mRNA were obtained directly from Dr.
  • Libraries of DNA are available from commercial providers such as Stratagene, La Jolla, CA., or Clontech, Palo Alto, CA.
  • PC12 and 3T3 cells were obtained from ATCC (#CRL1721 and #CCL92, respectively).
  • An additional PC12 cell line was obtained from Dr. Charles Marotta of Harvard Medical School, Massachusetts General Hospital, and McLean Hospital.
  • Standard cell culture media appropriate to the cell line are obtained from conventional commercial sources such as Gibco/BRL.
  • Murine stem cells, strain D3, were obtained from Dr. Rolf Kemler (Doetschman et al, J. Embryol. Exp. Morphol. 87:27 (1985)).
  • Lipofectin for DNA transfection and the drug G418 for selection of stable transformants are available from Gibco/BRL. Definition of APP cDNA clones.
  • the cDNA clone APP695 is of the form of cDNA described by Kang et al, Nature 325:733-735 (1987), and represents the most predominant form of APP in the brain.
  • the cDNA clone APP751 is of the form described by Ponte et al, Nature 331:525-527 (1988). This form contains an insert of 168 nucleotides relative to the APP695 cDNA. The 168 nucleotide insert encodes the KPI domain.
  • the cDNA clone APP770 is of the form described by Kitaguchi et al Nature 331:530-532 (1988). This form contains an insert of 225 nucleotides relative to the APP695 cDNA.
  • This insert includes the 168 nucleotides present in the insert of the APP751 cDNA, as well as an addition 57 nucleotide region that does not appear in APP751 cDNA.
  • the 225 nucleotide insert encodes for the KPI domain as well as the OX-2 domain. All three forms arise from the same precursor RNA transcript by alternative splicing.
  • the 168 nucleotide insert is present in both APP751 cDNA and APP770 cDNA.
  • SEQ ID NO: l The sequence encoding APP695 is shown in SEQ ID NO: l . This sequence begins with the first base of the initiation codon AUG and encodes a 695 amino acid protein. The region from nucleotide 1789 to 1917 of SEQ ID NO: l encodes the A ⁇ . The amino acid sequence of APP695 is shown in SEQ ID NO:2. Amino acids 597 to 639 of SEQ ID NO:2 form the A ⁇ .
  • the amino-acid composition of the APP695 is A57, C12, D47, E85, F17, G31, H25, 123, K38, L52, M21, N28, P31, Q33, R33, S30, T45, V62, W8, Y17 resulting in a calculated molecular weight of 78,644.45.
  • These sequences are derived from Kang et al. (1988).
  • the sequence encoding APP751 is shown in SEQ ID NO: 3. This sequence begins with the first base of the initiation codon AUG and encodes a 751 amino acid protein. Nucleotides 866 to 1033 of SEQ ID NO:3 do not appear in APP695 cDNA.
  • the region from nucleotide 1957 to 2085 of SEQ ID NO: 3 encodes the A ⁇ .
  • the amino acid sequence of APP751 is shown in SEQ ID NO:4.
  • Amino acids 289 to 345 of SEQ ID NO:4 do not appear in APP695.
  • This 57 amino acid region includes the KPI domain.
  • Amino acids 653 to 695 of SEQ ID NO: 4 form the A ⁇ .
  • APP770 The sequence encoding APP770 is shown in SEQ ID NO:5. This sequence begins with the first base of the initiation codon AUG and encodes a 770 amino acid protein. Nucleotides 866 to 1090 of SEQ ID NO:5 do not appear in APP695 cDNA. Nucleotides 1034 to 1090 of SEQ ID NO: 5 do not appear in APP751 cDNA. The region from nucleotide 2014 to 2142 encodes the A ⁇ . The amino acid sequence of APP770 is shown in SEQ ID NO:6. Amino acids 289 to 364 of SEQ ID NO: 6 do not appear in APP695. This 76 amino acid region includes the KPI and OX-2 domains.
  • Amino acids 345 to 364 of SEQ ID NO: 6 do not appear in APP751. This 20 amino acid region includes the OX-2 domain. Amino acids 672 to 714 form the A ⁇ . A probable membrane-spanning region of the APP occurs from amino acid 700 to 723. Unless otherwise stated, all references herein to nucleotide positions refer to the numbering of SEQ ID NO:5. This is the numbering derived from the APP770 cDNA. Unless otherwise stated, all references herein to amino acid positions refer to the numbering of SEQ ID NO:6. This is the numbering derived from APP770.
  • amino acid position 717 refers to amino acid 717 of APP770, amino acid 698 of APP751, and amino acid 642 of APP695.
  • the above sequences are derived from Kang et al. (1988) and Kitaguchi et al. (1988).
  • all forms of APP and fragments of APP, including all forms of A ⁇ , referred to herein are based on the human APP amino acid sequence.
  • a ⁇ refers to the human A ⁇
  • APP refers to human APP
  • APP770 refers to human APP770.
  • cDNA refers not only to DNA molecules actually prepared by reverse transcription of mRNA, but also any DNA molecule encoding a protein where the coding region is not interrupted, that is, a DNA molecule having a continuous open reading frame encoding a protein.
  • cDNA as used herein provides a convenient means of referring to a protein encoding DNA molecule where the protein encoding region is not interrupted by intron sequences (or any other sequences not encoding protein). Definition of the APP genomic locus.
  • Table 2 indicates where the 17 introns interrupt the APP coding sequence.
  • the numbering refers to the nucleotide positions of APP770 cDNA as shown in SEQ ID NO:5.
  • the starting nucleotide of exon 1 represents the first transcribed nucleotide. It is negative because the + 1 nucleotide is the first nucleotide of the AUG initiator codon by convention (Kang et al. (1988)).
  • the ending nucleotide of exon 18 represents the last nucleotide present in the mRNA prior to the poly(A) tail (Yoshikai et al. (1990)). It has been discovered that Yoshikai et al.
  • Yoshikai et al (1991) contain an error in the location of exon 8.
  • Figure 1 of Yoshikai et al (1991) includes an EcoRI fragment between EcoRI fragments containing exon 7 and exon 8. In fact, this intervening EcoRI fragment is actually located immediately after exon 8, so that the EcoRI fragment containing exon 7 and the EcoRI fragment containing exon 8 are adjacent to each other.
  • Table 2 Location of Introns in APP Gene Sequence.
  • Certain families are genetically predisposed to Alzheimer's disease, a condition referred to as familial Alzheimer's disease (FAD), through mutations resulting in an amino acid replacement at position 717 of the full length protein (Goate et al. (1991); Murrell et al. (1991); Chartier-Harlin et al. (1991)). These mutations co-segregate with the disease within the families. For example, Murrell et al. (1991) described a specific mutation found in exon 17 (which Murrell et al. refers to as exon 15) where the valine of position 717 is replaced by phenylalanine.
  • Another FAD mutant form contains a change in amino acids at positions 670 and 671 of the full length protein (Mullan et al. (1992)).
  • the lysine at position 670 is replaced by asparagine and the methionine at position 671 is replaced by leucine.
  • the effect of this mutation is to increase the production of A ⁇ in cultured cells approximately 7-fold (Citron et al., Nature 360: 672-674 (1992); Lai et al. , Science 259:514-516 (1993)).
  • Replacement of the methionine at position 671 with leucine by itself has also been shown to increase production of A ⁇ .
  • Additional mutations in APP at amino acids 669, 670, and 671 have been shown to reduce the amount of A ⁇ processed from APP (Citron et al. , Neuron 14:661-670 (1995)).
  • the APP construct with Val at amino acid 690 produces an increased amount of a truncated form of A/3.
  • APP expression clones can be constructed that bear a mutation at amino acid 669, 670, 671, 690, 692, or 717 of the full length protein.
  • the mutations from Lys to Asn and from Met to Leu at amino acids 670 and 671, respectively, are sometimes referred to as the Swedish mutation. Additional mutations can also be introduced at amino acids 669, 670, or 671 which either increase or reduce the amount of A/3 processed from APP.
  • Mutations at these amino acids in any APP clone or transgene can be created by site-directed mutagenesis (Vincent et al, Genes & Devel. 3:334-347 (1989)), or, once made, can be incorporated into other constructs using standard genetic engineering techniques. Some mutations at amino acid 717 are sometimes referred to as the Hardy mutation. Such mutations can include conversion of the wild-type Val717 codon to a codon for Ile, Phe, Gly, Tyr, Leu, Ala, Pro, Trp, Met, Ser, Thr, Asn, or Gin. A preferred substitution for Val717 is Phe. These mutations predispose individuals expressing the mutant proteins to develop Alzheimer's disease.
  • Mutations at amino acid 669 can include conversion of the wild-type Val669 codon to a codon for Trp, or deletion of the codon.
  • Mutations at amino acid 670 can include conversion of the wild-type Lys670 codon to a codon for Asn or Glu, or deletion of the codon.
  • Mutations at amino acid 671 can include conversion of the wild-type Met671 codon to a codon for Leu, Val, Lys, Tyr, Glu, or Ile, or deletion of the codon.
  • a preferred substitution for Lys670 is Asn
  • a preferred substitution for Met671 is Leu.
  • Truncated forms of APP can also be expressed from transgene constructs.
  • APP cDNA truncated to encode amino acids 646 to 770 of APP The APP cDNA construct truncated to encode amino acids 646 to 770 of APP, and operatively linked to the PDGF-B promoter, is referred to as PDAPPcl25.
  • Constructs for use in transgenic animals include a promoter for expression of the construct in a mammalian cell and a region encoding a protein that includes all or a contiguous portion of one of the three forms of APP: APP695, APP751, or APP770, with or without specific amino acid mutations as described herein. It is preferred that protein encoded is an A ⁇ - containing protein.
  • an A/3-containing protein is a protein that includes all or a contiguous portion of one of the three forms of APP:
  • A/3-containing proteins include amino acids 672 to 714 of human APP.
  • Preferred forms of such A/3- containing proteins include all or a contiguous portion of APP770, APP770 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, APP751, APP751 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/ or 717, APP695, and APP695 bearing a mutation in amino acid 669,
  • each of these A/3-containing proteins includes amino acids 672 to 714 of human APP.
  • Preferred forms of the above A/3-containing proteins are APP770; APP770 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; APP751; APP751 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670,
  • APP695 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; a protein consisting of amino acids 646 to 770 of APP; a protein consisting of amino acids 670 to 770 of APP; a protein consisting of amino acids 672 to 770 of APP; and a protein consisting of amino acids 672 to 714 of APP.
  • the DNA encoding the A/3- containing protein can be cDNA or a cDNA/genomic DNA hybrid, wherein the cDNA/genomic DNA hybrid includes at least one APP intron sequence wherein the intron sequence is sufficient for splicing.
  • Preferred constructs contain DNA encoding APP770; DNA encoding APP770 bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of DNA encoding APP770 which encodes an amino acid sequence comprising amino acids 672 to 714 of APP770; DNA encoding APP751; DNA encoding APP751 bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of DNA encoding APP751 which encodes an amino acid sequence comprising amino acids 672 to 714 of APP770; DNA encoding APP695; DNA encoding APP695 bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of DNA encoding APP695 which encodes
  • Preferred forms of such constructs are APP770 cDNA; APP770 cDNA bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of APP770 cDNA encoding an APP amino acid sequence, the amino acid sequence comprising amino acids 672 to 714 of APP770; APP751 cDNA; APP751 cDNA bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of APP751 cDNA encoding an APP amino acid sequence, the amino acid sequence comprising amino acids 672 to 714 of APP770; APP695 cDNA; APP695 cDNA bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of APP
  • APP transgenes can be accomplished using any suitable genetic engineering technique, such as those described in Sambrook et al. , Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y., 1989). Regions of APP clones that have been engineered or mutated can be interchanged by using convenient restriction enzyme sites present in APP cDNA clones.
  • a Nrul site starts at position -5 (relative to the first nucleotide of the AUG initiator codon).
  • a Kpnl and an Aspl 18 site both start at position 57 (these are isoschizomers leaving different sticky ends).
  • a Xcml site starts at position 836 and cuts at position 843.
  • a Seal site starts at position 1004.
  • a Xhol site starts at position 1135.
  • BamHI site starts at position 1554.
  • a Bglll site starts at position 1994.
  • An EcoRI site starts at position 2020.
  • a Spel site starts at position 2583.
  • Another EcoRI site starts at position 3076.
  • the clones bearing various portions of the human APP gene sequence shown in Figures 1 to 5 can be constructed in a common manner using standard genetic engineering techniques. For example, these clones can be constructed by first cloning the poly A addition signal from SV40 virus, as a 253 base pair Bell to BamHI fragment (Reddy et al, Science 200:494-502 (1978), into a modified vector from the pUC series. Next, the cDNA coding sequences (APP770, APP751, or APP695) can be inserted. Correct orientation and content of the fragments inserted can be determined through restriction endonuclease mapping and limited sequencing.
  • the clones bearing various carboxy terminal portions of the human APP gene sequence shown in Figures 4 and 5 can be constructed through several steps in addition to those indicated above.
  • an APP770 cDNA clone (SEQ ID NO:5) can be digested with Aspl 8 which cleaves after nucleotide position 57.
  • the resulting 5' extension is filled in using the Klenow enzyme (Sambrook et al. (1989)) and ligated to a hexanucleotide of the following sequence: AGATCT, the recognition site for Bglll. After cleavage with Bg ⁇ ll, which also cuts after position 1994, and re-ligation, the translational reading frame of the protein is preserved.
  • the truncated protein thus encoded contains the leader sequence, followed by approximately 6 amino acids that precede the A/3, followed by the A ⁇ , and the 56 terminal amino acids of APP.
  • the clone in Figure 5 is created by converting the nucleotide at position 2138 to a T by site directed mutagenesis in the clone of Figure 4a, thus creating a termination codon directly following the last amino acid codon of the A ⁇ .
  • APP cDNA clones naturally contain an Nrul site that cuts 2 nucleotides upstream from the initiator methionine codon. This site can be used for attachment of the different promoters used to complete each construct.
  • APP transgenes can also be constructed using PCR cloning techniques. Such techniques allow precise coupling of DNA fragments in the transgenes.
  • Endogenous APP expression results from transcription of precursor mRNA followed by alternative splicing to produce three main forms of APP. It is believed that this alternative splicing may be important in producing the pattern of APP expression involved in Alzheimer's disease. It is also believed that the presence of introns in expression constructs can influence the level and nature of expression by, for example, targeting precursor mRNA to mRNA processing and transport pathways (Huang et al. , Nucleic Acids Res. 18:937-947 (1990)). Accordingly, transgenes combining cDNA and genomic DNA, which include intron sequences, are a preferred type of construct. The RNA splicing mechanism requires only a few specific and well known consensus sequences.
  • transgenes can be constructed using one or more complete and intact intron sequences. However, it is preferred that the transgenes are constructed using truncated intron sequences that contain an effective amount of intron sequence to allow splicing. In general, truncated intron sequences that retain the splicing donor site, the splicing acceptor site, and the splicing branchpoint sequence will constitute an effective amount of an intron. The sufficiency of any truncated intron sequence can be determined by testing for the presence of correctly spliced mRNA in transgenic cells using methods described below.
  • intron sequences and splicing signals which are not derived from APP gene sequences may also be used in the transgene constructs. Such intron sequences will enhance expression of the transgene construct.
  • a preferred heterologous intron is a hybrid between the adenovirus major late region first exon and intron junction and an IgG variable region splice acceptor.
  • This hybrid intron can be constructed, for example, by joining the 162 bp Pvull to Hindlll fragment of the adenovirus major late region, containing 8 bp of the first exon and 145 bp of the first intron, and the 99 bp Hindlll to Pstl fragment of the IgG variable region splice acceptor clone-6, as described by Bothwell et al. , Cell 24:625-637 (1981). A similar splice signal has been shown to enhance expression of a construct to which it was attached, as described by Manley et al , Nucleic Acids Res. 18:937-947 (1990).
  • a preferred APP combination cDNA/genomic expression clone includes an effective amount of introns 6, 7 and 8, as shown in Figure 6.
  • Such a transgene can be constructed as follows. A preferred method of construction is described in Example 5.
  • a plasmid containing the cDNA portion of the clone can be constructed by first converting the Taql site at position 860 in an APP770 cDNA clone to an Xhol site by site-directed mutagenesis.
  • Cleavage of the resulting plasmid with Xhol cuts at the new Xhol site and a pre-existing Xhol site at position 1135, and releases the KPI and OX-2 coding sequence.
  • the plasmid thus generated serves as the acceptor for the KPI and OX-2 alternative splicing cassette.
  • the alternative splicing cassette can be created through a series of cloning steps involving genomic DNA.
  • the Taql site at position 860 in a genomic clone containing exon 6 and the adjacent downstream intron can be converted to an Xhol site by site-directed mutagenesis.
  • Cleavage of the resulting plasmid with Xhol cuts at the new Xhol site and an Xhol site within either intron 6 or 7.
  • This fragment, containing a part of exon 6 and at least a part of adjacent intron 6, can then be cloned into the Xhol site in a plasmid vector.
  • a genomic clone containing exon 9 and the adjacent upstream genomic sequences is cleaved with Xhol, cleaving the clone at the Xhol site at position 1135 (position 910 using the numbering system of Kang et al (1987)) and an Xhol site in either intron 7 or 8.
  • This fragment, containing a part of exon 9 and at least a part of adjacent intron 8, can then be cloned into the Xhol site of another plasmid vector.
  • exon intron junction fragments can then be released from their respective plasmid vectors by cleavage with Xhol and either BamHI or BgHl, and cloned together into the Xhol site of another plasmid vector. It is preferred that the exon/intron junction fragments be excised with BamHI. It is most preferable that BamHI sites are engineered in the intron portion of the exon/intron junction fragments prior to their excision. This allows the elimination of lengthy extraneous intron sequences from the cDNA/genomic clone.
  • the Xhol fragment resulting from cloning the two exon/intron junction fragments together can be cleaved with either BamHI or BgHl, depending on which enzyme was used for excision step above, and the genomic 6.8 kb
  • BamHI segment containing the KPI and OX-2 coding region along with their flanking intron sequences, can be inserted.
  • This fragment was identified by Kitaguchi et al. (1988) using Southern blot analysis of -S ⁇ HI-digested lymphocyte DNA from one normal individual and eight Alzheimer's disease patients using a 212 bp Taql-Aval fragment, nucleotides 862 to 1,073, of APP770 cDNA as the hybridization probe.
  • Genomic DNA clones containing the region of the 225 bp insert can be isolated, for example, from a human leukocyte DNA library using the 212 bp Taql-Aval fragment as a probe.
  • the 225 bp sequence is located in a 168 bp exon (exon 7) and a 57 bp exon (exon 8), separated by an intron of approximately 2.6 kb (intron 7), with both exons flanked by intron-exon consensus sequences.
  • the exon 7 corresponds to nucleotides 866 to 1,033 of APP770, and the exon 8 to nucleotides 1,034 to 1,090.
  • Exon 7 encodes the highly conserved region of the Kunitz-type protease inhibitor family domain.
  • this alternative splicing cassette containing both exon and intron sequences, can then be excised by cleavage with Xhol and inserted into the Xhol site of the modified APP770 cDNA plasmid (the acceptor plasmid) constructed above.
  • These cloning steps generate a combination cDNA/genomic expression clone that allows cells in a transgenic animal to regulate the inclusion of the KPI and OX-2 domains by a natural alternative splicing mechamsm.
  • An analogous gene bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations, can be constructed either directly by in vitro mutagenesis.
  • a mutation to amino acid 717 can also be made by using the mutated form of APP770 cDNA described above to construct an acceptor plasmid.
  • Promoters Different promoter sequences can be used to control expression of nucleotide sequences encoding A/3-containing proteins.
  • the ability to regulate expression of the gene encoding an A/3-containing protein in transgenic animals is believed to be useful in evaluating the roles of the different APP gene products in AD.
  • the ability to regulate expression of the gene encoding an A/3-containing protein in cultured cells is believed to be useful in evaluating expression and processing of the different A/3-containing gene products and may provide the basis for cell cultured drug screens.
  • a preferred promoter is the human platelet derived growth factor ⁇ (PDGF-B) chain gene promoter (Sasahara et al, Cell 64:217-227 (1991)).
  • Preferred promoters for the disclosed APP constructs are those that, when operatively linked to the protein coding sequences, mediate expression of one or more of the following expression products to at least a specific level in brain tissue of a two to four month old animal transgenic for one of the disclosed APP constructs.
  • the products and their expression levels are A/3 tot to a level of at least 30 ng/g (6.8 pmoles/g) brain tissue and preferably at least 40 ng/g (9.12 pmoles/g) brain tissue, A ⁇ l 2 to a level of at least 8.5 ng/g (1.82 pmoles/g) brain tissue and preferably at least 11.5 ng/g (2.5 pmoles/g) brain tissue, full length APP (FLAPP) and APP ⁇ combined (FLAPP+APP ⁇ ) to a level of at least 150 pmoles/g brain tissue, APP/3 to a level of at least 42 pmoles/g brain tissue, and mRNA encoding human A/3- containing protein to a level at least twice that of mRNA encoding the endogenous APP of the transgenic animal.
  • a ⁇ loi is the total of all forms of A ⁇ .
  • A/3 1-42 is a form of A/3 having amino acids 1 to 42 of A ⁇ (corresponding to amino acids 672 to 714 of APP).
  • FLAPP+APP ⁇ refers to APP forms containing the first 12 amino acids of the A/3 region (corresponding to amino acids 672 to 684 of APP).
  • FLAPP+APP ⁇ represents a mixmre of full length forms of APP and APP cleaved at the ⁇ -secretase site (Esch et al. ,
  • APP/3 is APP cleaved at the /3-secretase site (Seubert et al , Nature 361:260-263 (1993)).
  • the levels of expression described above refer to amounts of expression product present and are not limited to the specific units of measure used above.
  • an expression level can be measured, for example, in moles per gram of tissue, grams per grams of tissue, moles per volume of tissue, and in grams per volume of tissue. The equivalence of these units of measure to the measures listed above can be determined using known conversion methods.
  • the levels of expression described above need not occur in all brain tissues. Thus, a promoter is considered preferred if at least one of the levels of expression described above occurs in at least one type of brain tissue.
  • the promoter can mediate expression of the above expression products to the levels described above either coristitutively or by induction. Induction can be accomplished by, for example, administration of an activator molecule, by heat, or by expression of a protein activator of transcription for the promoter operatively linked to the gene encoding an A/3-containing protein. Many inducible expression systems which would be suitable for this purpose are known to those of skill in the art.
  • the brain tissue is prepared by the following method.
  • a brain from a transgenic test animal is dissected and the tissue is kept on ice throughout the homogenization procedure except as noted.
  • the brain tissue is homogenized in 10 volumes (w/v) of 5 M guanidine-HCl, 50 mM Tris-HCl, pH 8.5.
  • the sample is then gently mixed for 2 to 4 hours at room temperamre.
  • Homogenates are then diluted 1:10 in cold casein buffer #1 (0.25% casein/phosphate buffered saline (PBS) 0.05% sodium azide, pH 7.4, IX protease inhibitor cocktail) for a final 0.5 M guanidine concentration and kept on ice.
  • cold casein buffer #1 0.25% casein/phosphate buffered saline (PBS) 0.05% sodium azide, pH 7.4, IX protease inhibitor cocktail
  • 100X protease inhibitor cocktail is composed of 2 mg/ml aprotinin, 0.5 M EDTA, pH 8.0, 1 mg/ml leupeptin. Diluted homogenates are then spun in an Eppendorf microfuge at 14,000 rpm for 20 minutes at 4°C. If further dilutions are required, they can be made with cold guanidine buffer #2 (1 part guanidine buffer #1 to 9 parts casein buffer #1).
  • Antibody 266 (Seubert et al., Nature 359:325-327 (1992)) is dissolved at 10 ⁇ g/ml in buffer (0.23 g/L NaH 2 PO 4 -H 2 O, 26.2 g/L NaHPO 4 - 7H 2 O, 1 g/L sodium azide adjusted to pH 7.4) and 100 ⁇ l/well is coated onto 96-well immunoassay plates (Costar) and allowed to bind overnight.
  • the plate is then aspirated and blocked for at least 1 hour with a 0.25% human serum albumin solution in 25 g/L sucrose, 10.8 g/L Na 2 HPO 4 -7H 2 O, 1.0 g/L NaH 2 PO 4 -H 2 O, 0.5 g/L sodium azide adjusted to pH 7.4.
  • the 266 coated plate is then washed IX with wash buffer (PBS/0.05% Tween 20) using a Skatron plate washer. 100 ⁇ l/well of A/31-40 standards and brain tissue samples are added to the plate in triplicate and incubated overnight at 4°C.
  • Sl-40 standards are made from 0.0156, 0.0312, 0.0625, 0.125, 0.250, 0.500, and 1.000 ⁇ g/ml stocks in DMSO stored at -40° C as well as a DMSO only control for background determination.
  • a ⁇ standards consist of 1:100 dilution of each standard into guanidine buffer #3 (1 part BSA buffer to 9 parts guanidine buffer #1) followed by a 1:10 dilution into casein buffer #1 (Note: the final A/3 concentration range is 15.6 to 1000 pg/ml and the final guanidine concentration is 0.5 M).
  • BSA buffer consists of 1 % bovine serum albumin (BSA, immunoglobulin-free)/PBS/0.05% sodium azide.
  • casein buffer #2 (0.25% casein/PBS/0.05% Tween 20/pH 7.4) are then brought to room temperature (RT). The plates are then washed 3X with wash buffer. Next, 100 ⁇ l/well of 3D6-biotin at 0.5 ⁇ g/ml in casein buffer #2 is added to each well and incubated at 1 hour at RT.
  • Monoclonal antibody 3D6 was raised against the synthetic peptide DAEFRGGC (SEQ ID NO: 10) which was conjugated through the cysteine to sheep anti-mouse immunoglobulin.
  • the antibody does not recognize secreted APP but does recognize species that begin at A/3 position 1 (Asp).
  • the relative levels of mRNA encoding human A/3- containing protein mRNA encoding the endogenous APP of the transgenic animal be measured in the manner described by Bordonaro et al ,
  • yeast artificial chromosomes yeast artificial chromosomes
  • a human YAC library is commercially available (Clontech, Palo Alto, CA) with an average insert size of 250,000 base pairs (range of 180,000 to 500,000 base pairs).
  • a YAC clone of the Alzheimer's gene can be directly isolated by screening the library with the human APP770 cDNA. The inclusion of all of the essential gene regions in the clone can be confirmed by PCR analysis.
  • the YAC-APP clone shown in Figure 7a, can be established in embryonic stem (ES) cells by selecting for neomycin resistance encoded by the YAC vector.
  • ES cells bearing the YAC-APP clone can be used to produce transgenic mice by established methods described below under "Transgenic Mice” and "Embryonic Stem Cell Methods".
  • the YAC-APP gene bearing a mutation at amino acid 717 ( Figure 7b) can be produced through the generation of a YAC library using genomic DNA from a person affected by a mutation at amino acid 717. Such a clone can be identified and established in ES cells as described above. Genetic Alteration of the Mouse APP Gene.
  • Alzheimer's protein genes is approximately 85%. Within the A ⁇ -coding region, there are three amino acid differences between the two sequences. Amino acids Lys 670, Met671, and Val717, which can be mutated to alter APP processing, are conserved between mouse, rat, and man. Wild-type rodents do not develop Alzheimer's disease nor do they develop deposits or plaques in their central nervous system (CNS) analogous to those present in human Alzheimer's patients. Therefore, it is possible that the human but not the rodent form of A/3 is capable of causing disease.
  • CNS central nervous system
  • Homologous recombination can be used to convert the mouse Alzheimer's gene in situ to a gene encoding the human A ⁇ by gene replacement. This recombination is directed to a site downstream from the KPI and OX-2 domains, for example, within exon 9, so that the natural alternative splicing mechanisms appropriate to all cells within the transgenic animal can be employed in expressing the final gene product.
  • Both wild-type ( Figure 8a) and mutant ( Figure 8b) forms of human cDNA can be used to produce transgenic models expressing either the wild- type or mutant forms of APP.
  • the recombination vector can be constructed from a human APP cDNA (APP695, APP751, or APP770 form), either wild- type, mutant at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations. Cleavage of the recombination vector, for example, at the Xhol site within exon 9, promotes homologous recombination within the directly adjacent sequences (Capecchi (1989)).
  • the excised band is then placed in dialysis bags containing 0.3 M sodium acetate, pH 7.0.
  • D ⁇ A is electroeluted into the dialysis bags, extracted with phenol-chloroform (1:1), and precipitated by two volumes of ethanol.
  • the DNA is redissolved in 1 ml of low salt buffer (0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on an Elutip-DTM column.
  • the column is first primed with 3 ml of high salt buffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washing with 5 ml of low salt buffer.
  • the DNA solutions are passed through the column for three times to bind DNA to the column matrix.
  • DNA concentrations are measured by absorption at 260 nm in a UV spectrophotometer. For microinjection, DNA concentrations are adjusted to 3 ⁇ g/ml in 5 mM Tris, pH 7.4 and 0.1 mM EDTA. Other methods for purification of DNA for microinjection are also described in Hogan et al.
  • mice suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Germantown, NY), and Harlan Sprague Dawley (Indianapolis, IN). Many strains are suitable, but Swiss Webster (Taconic) female mice are preferred for embryo retrieval and transfer. B6D2F, (Taconic) males can be used for mating and vasectomized Swiss Webster studs can be used to stimulate pseudopregnancy. Vasectomized mice and rats can be obtained from the supplier.
  • mice six weeks of age are induced to superovulate with a 5 IU injection (0.1 cc, ip) of pregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours later by a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG; Sigma).
  • Females are placed with males immediately after hCG injection. Twenty-one hours after hCG injection, the mated females are sacrificed by CO 2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin (BSA; Sigma).
  • BSA bovine serum albumin
  • Embryos can be implanted at the two cell stage.
  • Randomly cycling adult female mice are paired with vasectomized males. Swiss Webster or other comparable strains can be used for this purpose.
  • Recipient females are mated at the same time as donor females.
  • the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body weight.
  • the oviducts are exposed by a single midline dorsal incision. An incision is then made through the body wall directly over the oviduct. The ovarian bursa is then torn with watchmakers forceps.
  • Embryos to be transferred are placed in DPBS (Dulbecco's phosphate buffered saline) and in the tip of a transfer pipet (about 10 to 12 embryos). The pipet tip is inserted into the infundibulum and the embryos transferred. After the transfer, the incision is closed by two sutures.
  • DPBS Dynamic Bisphosphate buffered saline
  • Donor females that have mated are sacrificed (CO 2 asphyxiation) and their oviducts removed, placed in DPBS (Dulbecco's phosphate buffered saline) with 0.5% BSA and the embryos collected. Cumulus cells surrounding the embryos are removed with hyaluronidase (1 mg/ml). The embryos are then washed and placed in EBSS (Earle's balanced salt solution) containing 0.5% BSA in a 37.5 °C incubator until the time of microinjection.
  • DPBS Dynabecco's phosphate buffered saline
  • hyaluronidase 1 mg/ml
  • the live embryos are moved to DPBS for transfer into foster mothers.
  • the foster mothers are anesthetized with ketamine (40 mg/kg, ip) and xylazine (5 mg/kg, ip).
  • a dorsal midline incision is made through the skin and the ovary and oviduct are exposed by an incision through the muscle layer directly over the ovary.
  • the ovarian bursa is torn, the embryos are picked up into the transfer pipet, and the tip of the transfer pipet is inserted into the infundibulum. Approximately 10 to 12 embryos are transferred into each rat oviduct through the infundibulum. The incision is then closed with sutures, and the foster mothers are housed singly.
  • Transfection is carried out bv one of several methods described in detail in Lovell-Badge, in Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, ed. E.J. Robertson, (IRL Press 1987), or in Potter et al, Proc. Natl. Acad. Sci. USA 81:7161 (1984).
  • Lipofection can be performed using reagents such as provided in commercially available kits, for example DOTAP (Boehringer-Mannheim) or lipofectin (BRL). Calcium phosphate/DNA precipitation, lipofection, direct injection, and electroporation are the preferred methods.
  • 0.5 X 10 6 ES cells are plated into tissue culmre dishes and transfected with a mixture of the linearized APP clone and 1 mg of pSV2neo DNA (Southern and Berg, J. Mol. Appl. Gen. 1:327-341 (1982)) precipitated in the presence of 50 mg lipofectin (BRL) in a final volume of 100 ⁇ l.
  • the cells are fed with selection medium containing 10% fetal bovine serum in DMEM supplemented with G418 (between 200 and 500 ⁇ g/ml). Colonies of cells resistant to G418 are isolated using cloning rings and expanded. DNA is extracted from drug resistant clones and Southern blots using an APP770 cDNA probe can be used to identify those clones carrying the APP sequences. PCR detection methods may also used to identify the clones of interest.
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination, described by Capecchi (1989). Direct injection results in a high efficiency of integration. Desired clones can be identified through PCR of DNA prepared from pools of injected ES cells. Positive cells within the pools can be identified by PCR subsequent to cell cloning (Zimmer and Gruss, Nature 338: 150-153 (1989). DNA introduction by electroporation is less efficient and requires a selection step.
  • Naturally cycling or superovulated female mice mated with males can be used to harvest embryos for the implantation of ES cells. It is desirable to use the C57BL/6 strain for this purpose when using mice. Embryos of the appropriate age are recovered approximately 3.5 days after successful mating. Mated females are sacrificed by CO 2 asphyxiation or cervical dislocation and embryos are flushed from excised uterine horns and placed in Dulbecco's modified essential medium plus 10% calf serum for injection with ES cells. Approximately 10 to 20 ES cells are injected into blastocysts using a glass microneedle with an internal diameter of approximately 20 ⁇ m.
  • Transgenic rodents can be identified by analyzing their DNA. For this purpose, tail samples (1 to 2 cm) can be removed from three week old animals. DNA from these or other samples can then be prepared and analyzed by Southern blot, PCR, or slot blot to detect transgenic founder (F 0 ) animals and their progeny (Fj and F 2 ).
  • the various F 0 , F,, and F 2 animals that carry a transgene can be analyzed by immunohistology for evidence of A ⁇ deposition, expression of APP or APP cleavage products, neuronal or neuritic abnormalities, and inflammatory responses in the brain.
  • Brains of mice and rats from each transgenic line are fixed and then sectioned. Sections are stained with antibodies reactive with the APP and/or the A ⁇ .
  • Secondary antibodies conjugated with fluorescein, rhodamine, horse radish peroxidase, or alkaline phosphatase are used to detect the primary antibody. These methods permit identification of amyloid plaques and other pathological lesions in specific areas of the brain.
  • Plaques ranging in size from 9 to > 50 ⁇ m characteristically occur in the brains of AD patients in the cerebral cortex, but also may be observed in deeper grey matter including the amygdaloid nucleus, corpus striamm and diencephalon. Sections can also be stained with other antibodies diagnostic of Alzheimer's plaques, recognizing antigens such as APP, Alz-50, tau, A2B5, neurofilaments, synaptophysin, MAP-2, ubiquitin, complement, neuron-specific enolase, and others that are characteristic of Alzheimer's pathology (Wolozin et al, Science 232:648 (1986); Hardy and Allsop, Trends in Pharm. Sci.
  • RNA messenger RNA can be isolated by the acid gua idinium thiocyanate- phenol: chloroform extraction method (Chomaczynski and Sacchi, Anal Biochem 162:156-159 (1987)) from cell lines and tissues of transgenic animals to determine expression levels by Northern blots, RNAse and nuclease protection assays.
  • Protein Protein
  • APP, A/3, and other fragments of APP can and have been detected by using polyclonal and monoclonal antibodies that are specific to the APP extra-cytoplasmic domain, A/3 region, A ⁇ 1 2 , A ⁇ , APP/3, FLAPP+APPa, and C-terminus of APP.
  • a variety of antibodies that are human sequence specific, such as 10D5 and 6C6, are very useful for this purpose (Games et al. (1995)).
  • Protein fractions can be isolated from tissue homogenates and cell lysates and subjected to Western blot analysis as described by, for example, Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor, NY, 1988); Brown et al, J. Neurochem. 40:299-308 (1983); and Tate-Ostroff et al, Proc Natl Acad Sci 86:745-749 (1989).
  • the protein fractions are denatured in Laemmli sample buffer and electrophoresed on SDS-Polyacrylamide gels.
  • the proteins are then transferred to nitrocellulose filters by electroblotting.
  • the filters are blocked, incubated with primary antibodies, and finally reacted with enzyme conjugated secondary antibodies. Subsequent incubation with the appropriate chromogenic substrate reveals the position of APP derived proteins.
  • Radioactive or enzymatically labeled nucleic acid probes can be used to detect mRNA in situ.
  • the probes are degraded or prepared to be approximately 100 nucleotides in length for better penetration of cells.
  • the hybridization procedure of Chou et al, J. Psych. Res. 24:27- 50 (1990), for fixed and paraffin embedded samples is briefly described below although similar procedures can be employed with samples sectioned as frozen material.
  • Paraffin slides for in situ hybridization are dewaxed in xylene and rehydrated in a graded series of ethanols and finally rinsed in phosphate buffered saline (PBS). The sections are post-fixed in fresh 4% paraformaldehyde.
  • the slides are washed with PBS twice for 5 minutes to remove paraformaldehyde. Then the sections are permeabilized by treatment with a 20 ⁇ g/ml proteinase K solution. The sections are re-fixed in 4% paraformaldehyde, and basic molecules that could give rise to background probe binding are acetylated in a 0.1 M triethanolamine, 0.3 M acetic anhydride solution for 10 minutes. The slides are washed in PBS, then dehydrated in a graded series of ethanols and air dried. Sections are hybridized with antisense probe, using sense probe as a control.
  • the animal must learn the platform's location relative to distal visual cues, and can be used to assess both reference and working memory.
  • a learning deficit in the water maze has been demonstrated with PDAPP transgenic mice.
  • An example of behavioral analysis for assessing the effect of transgenic expression of A/3-containing proteins is described in Example 9. Operant behavior studies of memory function: Memory function of the disclosed transgenic animals can be assessed by testing memory-related feeding behavior (Dunnett, "Operant delayed matching and non-matching position in rats" in Behavioral Neuroscience, Volume I: A Practical Approach (Sagal, ed., IRL Press, N.Y. , 1993) pages 123-136; Zornetzen, Behav. Neur. Biol. 36:49-60 (1982)).
  • Transgenic and non-transgenic mice are trained to earn food rewards in a two component operant procedure.
  • One component features a delayed spatial alternation schedule. Under this schedule, the mouse must remember over a variable time delay which lever it has pressed in the previous trial so that it can earn a reward by pressing the alternate lever on the current trial. This provides a measure of the animal's recent or "working" memory.
  • the second component features a discrimination spatial alternation schedule. Under this schedule, the mouse earns a reward by pressing whatever lever is illuminated. This discrimination behavior is an example of reference memory.
  • mice transgenic and non-transgenic
  • the disclosed transgenic mice will model the cognitive deficits of Alzheimer's disease with enhanced sensitivity to the memory-disrupting effects of cholinergic antagonists and impairment on "working" and reference memory tasks.
  • Dose-response challenges with the cholinergic antagonist can be conducted at various ages.
  • These memory behavioral tests can also be used to compare the effect of compounds on the behavioral impairment of the disclosed transgenic animals.
  • the two groups of mice are transgenic mice to which a test compound is admimstered and transgenic mice to which the compound is not administered.
  • Emotional reactivity and object recognition Various functions of the disclosed transgenic animals can be assessed by testing locomotor activity, emotional reactivity to a novel environment or to novel objects, and object recognition.
  • a first set of assessments are performed in the same animals at different ages (each animal is its own control) in order to test their performance in terms of locomotor activity, emotional reactivity to a novel environment or to novel objects, and object recognition, a form of memory which is severely impaired in AD patients.
  • transgemc and non-transgenic control mice are individually placed in a square open field with a central platform. For 30 minutes, horizontal and vertical activity, and crossings of the platform, are recorded by blocks of 5 minutes for each animal.
  • each animal is submitted to two trials with an intertrial of 1 hour.
  • two identical objects are placed in the open field and the animal is allowed 3 minutes of exploration.
  • one of the objects is replaced by a new object and the time spent by the animal in exploring the familiar and novel object is recorded during the next 3 minutes (Ennaceur and Delacour, Behav. Brain Res. 31:47-59 (1988)).
  • Animals are then tested for neophobic behavior, which is considered as an index of anxiety, in a free exploration situation, in which animals are given the opportunity to move freely between a familiar and a novel environment. Thereafter, the same animals are submitted to various learning tasks to investigate their learning and memory capacities.
  • Two additional groups can also be submitted to the same behavioral tests as above at 9 to 10 months old in order to determine whether behavioral screening performed at 2 to 3 months old influenced further learning and memory capacities. These memory behavioral tests can also be used to compare the effect of compounds on the behavioral impairment of the disclosed transgenic animals.
  • the two groups of mice are transgenic mice to which a test compound is administered, and transgenic mice to which the compound is not administered.
  • the above phenotypic characteristics of the disclosed transgenic animals can be used to identify those forms of the disclosed transgenic animals that are preferred as animal models. Additional phenotypic characteristics, and assays for measuring these characteristics, that can also be used to identify those forms of the disclosed transgenic animals that are preferred as animal models, are described in Example 6. These characteristics are preferably those that are similar to phenotypic characteristics observed in Alzheimer's disease. APP and A ⁇ markers which are also useful for identifying those forms of the disclosed transgenic animals that are preferred as animal models are described below. Any or all of the these markers or phenotypic characteristics can be used either alone or in combination to identify preferred forms of the disclosed transgenic animals.
  • the presence of plaques in brain tissue that can be stained with Congo red is a phenotypic characteristic which can identify a disclosed transgenic animal as preferred. It is intended that the levels of expression of certain APP-related proteins present in preferred transgenic animals (discussed above) is an independent characteristic for identifying preferred transgenic animals. Thus, the most preferred transgenic animals will exhibit both a disclosed expression level for one or more of the APP-related proteins and one or more of the phenotypic characteristics discussed above.
  • Especially preferred phenotypic characteristics are the presence of amyloid plaques that can be stained with Congo Red (Kelly (1984)), the presence of extracellular amyloid fibrils as identified by electron microscopy by 12 months of age, and the presence of type I dystrophic neurites as identified by electron microscopy by 12 months of age (composed of spherical neurites that contain synaptic proteins and APP; Dickson et al , Am J Pathol 132:86-101 (1988); Dickson et al, Acta Neuropath.
  • Example 6 Examples of the detection of these characteristics is provided in Example 6. It is most preferred that the transgenic animals have amyloid plaques that can be stained with Congo Red as of 14 months of age. Screening of Compounds for Treatment of Alzheimer's Disease.
  • transgenic animals or animal cells derived from transgenic animals, can be used to screen compounds for a potential effect in the treatment of Alzheimer's disease using standard methodology.
  • the compound is administered to the animals, or introduced into the culmre media of cells derived from these animals, over a period of time and in various dosages, then the animals or animal cells are examined for alterations in APP expression or processing, expression levels or localization of other AD markers, histopathology, and/or, in the case of animals, behavior using the procedures described above and in the examples below.
  • any improvement in behavioral tests, alteration in AD- associated markers, reduction in the severity of AD-related histopathology, reduction in the expression of A/3 or APP cleavage products, and/or changes in the presence, absence or levels of other compounds that are correlated with AD which are observed in treated animals, relative to untreated animals, is indicative of a compound useful for treating Alzheimer's disease.
  • the specific proteins, and the encoding transcripts, the enzymatic or biochemical activity, and/or histopathology of those proteins, that are associated with and characteristic of AD are referred to herein as markers. Expression or localization of these markers characteristic of AD has either been detected, or is expected to be present, in the disclosed transgenic animals.
  • markers useful for AD screening assays are selected based on detectable changes in these markers that are associated with AD. Many such markers have been identified in AD and have either been detected in the disclosed transgenic animals or are expected to be present in these animals. These markers fall into several categories based on their namre, location, or function. Preferred examples of markers useful in AD screening assays are described below, group as A/3-related markers, plaque-related markers, cytoskeletal and neuritic markers, inflammatory markers, and neuronal and neurotransmitter-related markers. A. A ⁇ -related Markers.
  • APP and A/3 can be directly measured and compared in treated and untreated transgenic animals both by immunohistochemistry and by quantitative ELISA measurements as described above and in the examples.
  • APP and A/3 Two forms of APP products are found, APP and A/3 (Haass and Selkoe, Cell 75:1039-1042 (1993)). They have been shown to be intrinsically associated with the pathology of AD in a time dependent manner. Therefore, preferred assays compare age-related changes in APP and A/3 expression in the transgenic mice. As described in Example 6, increases in A/3 have been demonstrated during aging of the PDAPP mouse.
  • a ⁇ markers known to increase in individuals with Alzheimer's disease are total A/3 (A/3 tot ), A/3 1-42 (Aj ⁇ M2 ; A ⁇ with amino acids 1-42), A/3 M0 (A/3 with amino acids 1-40), A/3 N3( ⁇ E) (A/3 N3 (pE)); A/3 X-42 (A ⁇ x ⁇ 2 ; A/3 forms ending at amino acid 42); A/3 X-40 (A/3 X ⁇ 0 ; A/3 forms ending at amino acid 40); insoluble A/3 (A/3 InsolUDle ); and soluble A/3 (A/3 Solub , e ; Kuo et al, J. Biol. Chem. 271(8): 4077-4081 (1996)).
  • A/3 N3 has pyroglutamic acid at position 3 (Saido, Neuron 14:457-466 (1995)).
  • A/3 X ⁇ 2 refers to any of the C-terminal forms of A ⁇ such as A/3 13 . 42 .
  • A/3 lnsoluble refers to forms of A/3 that are recovered as described in Gravina, J. Biol. Chem. 270:7013-7016 (1995).
  • APP/3 can also be specifically measured to assess the amount of /3-secretase activity (Seubert et al, Nature 361:260-263 (1993)).
  • Several of these A/3 forms and their association with Alzheimer's disease are described by Haass and Selkoe (1993).
  • A/3 tot A/3 M2 , and A ⁇ ⁇ are described in Example 6.
  • specific forms of A ⁇ can be assayed, either quantitatively or qualitatively using specific antibodies, as described below.
  • die positions correspond to the A/3 region of APP.
  • Amino acid 1 of A/3 corresponds to amino acid 672 of APP
  • amino acid 42 of A ⁇ corresponds to amino acid 714 of APP.
  • APP markers are also preferred as targets for assay measurement.
  • different forms of secreted APP can also be measured (Seubert et al., Nature 361:260-263 (1993)).
  • Other APP forms can also serve as targets for assays to assess the potential for compounds to affect Alzheimer's disease. These include FLAPP-hAPP ⁇ , full length APP, C-terminal fragments of APP, especially C100 (the last 100 amino acids of APP) and C57 to C60 (the last 57 to 60 amino acids of APP), and any forms of APP that include the region corresponding to A ⁇ lJKj .
  • APP forms are also preferred targets for assays to assess the potential for compounds to affect Alzheimer's disease.
  • the absolute level of APP and APP transcripts, the relative levels of the different APP forms and their cleavage products, and localization of APP expression or processing are all markers associated with Alzheimer's disease that can be used to measure the effect of treatment with potential therapeutic compounds.
  • the localization of APP to plaques and neuritic tissue is an especially preferred target for these assays.
  • Quantitative measurement can be accomplished using many standard assays. For example, transcript levels can be measured using RT-PCR and hybridization methods including RNase protection, Northern analysis, and R- dot analysis.
  • APP and A ⁇ levels can be assayed by ELISA, Western analysis, and by comparison of immunohistochemically stained tissue sections. Immunohistochemical staining can also be used to assay localization of APP and A ⁇ to particular tissues and cell types. Such assays were described above and specific examples are provided below.
  • B. Plaque-related Markers are described
  • plaque-related markers are apolipoprotein E, glycosylation end products, amyloid P component, advanced glycosylation end products (Smith et al. , Proc. Natl. Acad. Sci. USA 91:5710 (1994)), growth inhibitory factor, laminin, collagen type IV (Kalaria and Perry (1993); Ueda et al. (1993)), receptor for advanced glycosylation products (RAGE), and ubiquitin.
  • plaques and neuritic tissue can be used to detect specific components of plaques and neuritic tissue
  • location and extent of plaques can also be determined by using well known histochemical stains, such as Congo Red and thioflavin S, as described above and in some examples below.
  • cytoskeletal markers associated with AD have also been detected in transgenic PDAPP mice. These markers can be used in AD screening assays to determine the effect of compounds on AD. Many of the changes in cytoskeletal markers occur either in the neurofibrillary tangles or dystrophic neurites associated with plaques (Kosik et al. (1992); Lovestone and Anderton (1992); Brandan and Inestrosa (1993); Trojanowski et al. (1993); Masliah et al. (1993)).
  • cytoskeletal and neuritic markers that exhibit changes in and/or an association with AD. These markers can be detected, and changes can be determined, to measure the effect of compounds on the disclosed transgenic animals.
  • Spectrin exhibits increased breakdown in AD.
  • Tau and neurofilaments display an increase in hyperphosphorylation in AD, and levels of ubiquitin increase in AD.
  • Tau, ubiquitin, MAP-2, neurofilaments, heparin sulfate, and chrondroitin sulphate are localized to plaques and neuritic tissue in AD and in general change from the normal localization.
  • GAP43 levels are decreased in the hippocampus and abnormally phosphorylated tau and neurofilaments are present in PDAPP transgenic mice.
  • Alzheimer's disease is also known to stimulate an immunoinflammatory response, with a corresponding increase in inflammatory markers (Frederickson and Brunden (1994); McGeer et al. (1991); Wood et al. (1993)).
  • the following are preferred inflammatory markers that exhibit changes in and/or an association with AD. Detection of changes in these markers are useful in AD screening assays.
  • glial fibrillary acidic protein GFAP
  • Mac-1 glial fibrillary acidic protein
  • F4/80 glial fibrillary acidic protein
  • cytokines such as IL-l ⁇ and ⁇ , TNF ⁇ , IL-8, MIP-l ⁇
  • IL-l ⁇ and ⁇ IL-l ⁇
  • TNF ⁇ IL-8
  • MIP-l ⁇ cytokines
  • Complement markers such as C3d, Clq, C5, C4d, C4bp, and C5a-C9, are localized in plaques and neuritic tissue.
  • Major histocompatibility complex (MHC) glycoproteins such as HLA-DR and HLA-A, D,C increase in AD.
  • Microglial markers such as CR3 receptor, MHC I, MHC II, CD 31, CD 11 a, CDllb, CDllc, CD68, CD45RO, CD45RD, CD18, CD59, CR4, CD45, CD64, and CD44 (Akiyama et al, Brain Research 632:249-259 (1993)) increase in AD.
  • Additional inflammatory markers useful in AD screening assays include 2 macroglobulin receptor, Fibroblast growth factor (Tooyama et al , Neuroscience Letters 121: 155-158 (1991)), ICAM-1 (Akiyama et al , Acta Neuropathologica 85:628-634 (1993)), Lactotransferrin (Kawamata et al , American Journal of Pathology 142:1574-85 (1993)), Clq, C3d, C4d, C5b-9, Fc gamma RI, Fc gamma RII, CD8 (McGeer et al, Can J Neurol Sci 16:516-527 (1989)), LCA (CD45) (McGeer et al.
  • Fibroblast growth factor Tooyama et al , Neuroscience Letters 121: 155-158 (1991)
  • ICAM-1 Akiyama et al , Acta Neuropathologica 85:628-634 (1993)
  • Additional markers which are associated with inflammation or oxidative stress include 4-hydroxynonenal-protein conjugates (Uchida et al, Biochem. Biophys. Res. Comm. 212:1068-1073 (1995); Uchida and Stadtman, Methods in Enzymology 233:371-380 (1994); Yoritaka et al, Proc. Natl Acad. Sci. USA 93:2696-2701 (1996)), I B, NFKB (Kaltschmidt et al, Molecular Aspects of Medicine 14:171-190 (1993)), cPLA 2 (Stephenson et al , Neurobiology Dis.
  • AD neuronal and Neurotransmitter-related Markers. Changes in neuronal and neurotransmitter biochemistry have been associated with AD and in the disclosed PDAPP animals. In AD there is a profound reduction in cortical and hippocampal cholinergic innervation. This is evidenced by the dramatic loss of the synthetic enzyme choline acetyltransferase and decreased acetylcholinersterase, synaptosomal choline uptake (as measured by hemicholinium binding) and synthesis and release of acetylcholine (Rylett et al (1983); Sims et al.
  • markers can be used in AD screening assays to determine the effect of compounds on AD. There is also a loss of basal forebrain neurons and the galanin system becomes hypertrophic in AD.
  • Basal forebrain neurons are dependent on nerve growth factor (NGF).
  • NGF nerve growth factor
  • BDNF Brain-derived neurotrophic factor
  • Cat D,B, protein kinase C, and NADPH are localized in plaque and neuritic tissue in AD.
  • AD Activity and/or levels of nicotine receptors, 5-HT 2 receptor, NMDA receptor, ⁇ 2-adrenergic receptor, synaptophysin, p65, glutamine synthetase, glucose transporter, PPI kinase, drebrin, GAP43, cytochrome oxidase, heme oxygenase, calbindin, adenosine Al receptors, mono amine metabolites, choline acetyltransferase, acetylcholinesterase, and symptosomal choline uptake are all reduced in AD. Additional markers that are associated with AD or after treatment of cells with A/3 include (1) cPLA 2 (Stephenson et al, Neurobiology of Diseases 3:51-63 (1996)), which is upregulated in AD, (2) Heme oxygenase- 1
  • AD Markers F. Measuring the Amounts and Localization of AD Markers. Quantitative measurement can be accomplished using many standard assays. For example, transcript levels can be measured using RT-PCR and hybridization methods including RNase protection, Northern analysis, and R- dot analysis. Protein marker levels can be assayed by ELISA, Western analysis, and by comparison of immunohistochemically stained tissue sections. Immunohistochemical staining can also be used to assay localization of protein markers to particular tissues and cell types. The localization and the histopathological association of AD markers can be determined by histochemical detection methods such as antibody staining, laser scanning confocal imaging, and immunoelectron micrography. Examples of such techniques are described in Masliah et al (1993) and in Example 6 below.
  • activity of the receptors or enzymes can be measured.
  • the activity of neurotransmitter metabolizing enzymes such as choline acetyltransferase and acetylcholine esterase can be measured using standard radiometric enzyme activity assays.
  • the activity of certain neurotransmitter receptors can be determined by measuring phosphoinositol (PI) turnover. This involves measuring the accumulation of inositol after stimulation of the receptor with an agonist.
  • useful agonists include carbachol for cholinergic receptors and norepinephrine for glutaminergic receptors.
  • the number of receptors present in brain tissue can be assessed by quantitatively measuring ligand binding to the receptors.
  • the levels and turnover of receptor ligands and neurotransmitters can be determined by quantitative assays taken at various time points. Dopamine turnover can be measured using DOPAC and HVA. MOPEG sulfate can be used to measure norepinephrine turnover and 5-HIAA can be used to measure serotonin turnover. For example, norepinephrine levels have been shown to be reduced 20% in the hippocampus of 12 to 13 month old PDAPP transgenic mice relative to controls. Generally, the above assays can be performed as described in the literature, for example, in Rylett et al. (1983); Sims et al.
  • Cell culmres can be transfected generally in the manner described in International Patent Application No. 94/10569 and Citron et al (1995). Derived transgenic cells or transfected cell culmres can then be plated in Corning 96-well plates at 1.5 to 2.5 x 10 4 cells per well in Dulbecco's minimal essential media plus 10% fetal bovine serum.
  • the media are again removed and replaced with fresh media containing the compound to be tested as above and cells are incubated for an additional 2 to 16 hours.
  • plates are centrifuged in a Beckman GPR at 1200 rpm for five minutes at room temperamre to pellet cellular debris from the conditioned media. From each well, 100 ⁇ L of conditioned media or appropriate dilutions thereof are transferred into an ELISA plate precoated with antibody 266 (an antibody directed against amino acids 13 to 28 of A ⁇ ) as described in International Patent Application No. 94/10569 and stored at 4°C overnight.
  • An ELISA assay employing labelled antibody 6C6 (against amino acids 1 to 16 of A ⁇ ) can be run to measure the amount of A/3 produced. Different capture and detection antibodies can also be used.
  • Cytotoxic effects of the compounds are measured by a modification of the method of Hansen et al., J. Immun. Method. 119:203-210 (1989).
  • 25 ⁇ L of a 3,(4,5-dimethylthiazol- 2-yl)2,5-diphenyltetrazolium bromide (MTT) stock solution (5 mg/mL) is added to a final concentration of 1 mg/mL.
  • MTT 3,(4,5-dimethylthiazol- 2-yl)2,5-diphenyltetrazolium bromide
  • Example 1 Expression of pMTAPP-1 in NIH3T3 and PC12 Cells.
  • the clone pMTAPP-1 is an example of an APP770 expression construct as shown in Figure la where the promoter used is the metallothionine promoter.
  • Stable cell lines were derived by transfecting NIH3T3 and PC12 cell lines (ATCC #CCL92 and CRL1721). Five hundred thousand NIH3T3 or PC12 cells were plated into 100 mm dishes and transfected with a mixmre of 5 mg of the Sa l fragment and 1 mg of pSV2neo DNA (Southern and Berg (1982)) precipitated in the presence of 50 mg lipofectin (Gibco, BRL) in a final volume of 100 ⁇ l. Polylysine-coated plates were used for PC12 cells, which normally do not adhere well to tissue culmre dishes. The cells were fed with selection medium containing 10% fetal bovine serum in DMEM or RPMI and supplemented with G418.
  • Example 2 Expression of pEAPP-1 in PC12 Cells.
  • pEAPP-1 is an example of an APP770 expression construct as shown in Figure la where the promoter used is the 25 kb human APP gene promoter. DNA from this construct was transfected into PC 12 cells as described above. Certain clones of pEAPP-1 transfected cells exhibited a differentiation phenotype morphologically similar to that exhibited by PC 12 cells treated with nerve growth factor (NGF). PC 12 cells normally are fairly round and flat cells. Those transfected with pEAPP-1 have cytoplasmic extensions resembling neurites. PC 12 cells treated with NGF extend very long neuritic extensions. Thirteen PC 12 cell clones transfected with pEAPP-1 were selected and propagated. Eight of these cell clones exhibited the spontaneous differentiation phenotype with clones 1-8, 1-1, and 1-4 exhibiting the strongest phenotypes. Staining of pEAPP-1 transfected
  • PC 12 cells with antibody against the A ⁇ as described in Example 1 indicated that those cells exhibiting the differentiation were also expressing APP. Because PC 12 cells transfected with the pMTAPP-1 clone did not exhibit this phenotype even though the APP770 cDNA is expressed, these results suggest that expression of APP770 from the human promoter has novel properties regarding the physiology of the cell.
  • Example 3 Expression of pMTA4 in PC12 Cells.
  • pMTA4 is an example of the type of construct shown in Figure 4a where the promoter used is the metallothionine promoter. The protein encoded by this construct differs slightly from that depicted in Figure 4a.
  • An APP770 cDNA clone was digested with Aspl 8 which cleaves after position 57 (number system of Kang et al (1987)). The resulting 5' extension was filled in using the Klenow enzyme (Sambrook et al. (1989)). The same DNA preparation was also cleaved with EcoRI which also cuts after position 2020 and the resulting 5' extension was filled in using the Klenow enzyme (Sambrook et al (1989)).
  • mice were made by microinjecting pMTAPP-1 vector
  • pMTAPP-1 is an example of the type of construct shown in Figure la in which the APP770 coding sequence is operably linked to the metallothionine promoter.
  • the procedures for microinjection into mouse embryos are described in Manipulating the Mouse Embryo by Hogan et al. (1986). Only a brief description of the procedures is described below.
  • mice were obtained from Taconic Laboratories (German Town, New York). Swiss Webster female mice were used for embryo retrieval and implantation. B6D2F J males were used for mating and vasectomized Swiss webster studs were used to simulate pseudopregnancy. A. Embryo Recovery. Female mice, 4 to 8 weeks of age, were induced to superovulate with 5 IU of pregnant mare's serum gonadotropin (PMSG; Sigma) followed 48 hours later by 5 IU of human chorionic gonadotropin (hCG; Sigma). Females were placed with males immediately after hCG injection.
  • PMSG pregnant mare's serum gonadotropin
  • hCG human chorionic gonadotropin
  • Embryos were recovered from excised oviducts of mated females 21 hours after hCG in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin (BSA; Sigma). Surrounding cumulus cells were removed with hyaluronidase (1 mg/ml). Pronuclear embryos were then washed and placed in Earle's balanced salt solution containing 0.4% BSA (EBSS) in a 37.5 °C incubator with a humidified atmosphere at 7% CO 2 , 5% O 2 , and 88% N 2 until the time of injection. B. Microinjection.
  • BSA bovine serum albumin
  • Elutip-DTM purified Sail DNA was dissolved in 5 mM Tris (pH 7.4) and 0.1 mM EDTA at 3 ⁇ g/ml concentration for microinjection.
  • Microneedles and holding pipettes were pulled from Fisher coagulation mbes (Fisher) on a DKI model 720 pipette puller. Holding pipettes were then broken at approximately 70 ⁇ m (O.D.) and fire polished to an I.D. of about 30 ⁇ m on a Narishige microforge (model MF-83). Pipettes were mounted on Narishige micromampulators which were attached to a Nikon Diaphot microscope. The air-filled injection pipette was filled with DNA solution through the tip after breaking the tip against the holding pipette.
  • Embryos in groups of 30 to 40, were placed in 100 ⁇ l drops of EBBS under paraffin oil for micromanipulation. An embryo was oriented and held with the holding pipette. The injection pipette was then inserted into the male pronucleus (usually the larger one). If the pipette did not break through the membrane immediately the stage was tapped to assist in penetration. The nucleus was then injected and the injection was monitored by swelling of the nucleus. Following injection, the group of embryos was placed in EBSS until transfer to recipient females.
  • mice Randomly cycling adult female mice were paired witii vasectomized Swiss Webster males. Recipient females were mated at the same time as donor females. At the time of transfer, the females were anesthetized with avertin. The oviducts were exposed by a single midline dorsal incision. An incision was then made through the body wall directly over the oviduct. The ovarian bursa was then torn with watch makers forceps. Embryos to be transferred were placed in DPBS and in the tip of a transfer pipet (about 10 to 12 embryos). The pipet tip was inserted into the infundibulum and embryos were transferred. After the transfer, the incision was closed by two sutures.
  • tail samples about 1 cm long were excised for DNA analysis.
  • the tail samples were digested by incubating with shaking overnight at 55°C in the presence of 0.7 ml 5 mM Tris, pH 8.0, 100 mM EDTA, 0.5% SDS and 350 ⁇ g of proteinase K.
  • the digested material was extracted once with an equal volume of phenol and once with an equal volume of phenol -.chloroform (1:1 mixmre).
  • the supematants were mixed with 70 ⁇ l 3 M sodium acetate (pH 6.0) and the DNA was precipitated by adding equal volume of 100% ethanol.
  • the DNA was spun down in a microfuge, washed once with 70% ethanol, dried and dissolved in 100 ⁇ l TE buffer (10 mM Tris pH 8.0 and 1 mM EDTA).
  • transgenic mice A total of 671 pronuclear embryos were microinjected out of which 73 live and 6 dead pups were born. DNA analysis identified 9 transgenic mice (5 females and 4 males) which were bred to generate Fj and F 2 transgenics. These animals can be analyzed for expression of mRNA and protein of APP in different tissues and for analysis of behavioral and pathological abnormalities as described above. Transgenic mice with this construct express transgenic RNA.
  • Example 5 Construction of APP construct containing a combination cDNA/genomic coding sequence.
  • a cDNA/genomic APP construct containing introns 6, 7 and 8 was prepared by combining APP cDNA encoding exons 1-6 and 9-18 with genomic APP sequences encoding introns 6, 7 and 8, and exons 7 and 8 (see
  • DNA fragments containing the truncated introns were generated as follows: a BamHI site was engineered 143 bp into intron 6 nucleotide by PCR mutagenesis ("Mutagenesis by PCR” in PCR Technology: Current Innovations (Griffith and Griffith, eds., CRC Press, 1994) pages 69-83) and another BamHI site was engineered by PCR mutagenesis 263 bp prior to the beginning of exon 9. These sites were engineered into separate APP genomic DNA clones containing the junctions of exon 6 and intron 6, and intron 8 and exon 9, respectively, resulting in modified APP genomic DNA clones.
  • the entire cassette was assembled in the APP cDNA clone as follows ( Figure 11).
  • the 889 bp BamHI to Xcml fragment of APP cDNA contaimng exons 1 through 5 and part of exon 6 (including nucleotides 1 to 843 of SEQ ID NO: 5) was cloned into a vector containing BamHI and Xhol sites downstream from the insertion site to make APP770x-oligo-x.
  • APP770x- oligo-x was then cut with Xcml and BamHI. Then two fragments were obtained from the modified APP genomic DNA clone containing the junction of exon 6 and intron 6 described above by cutting with Xcml and .B ⁇ mHI.
  • APP770xE6E9x was then cut with JB ⁇ mHI and the 6.8 kb BamHI fragment of APP genomic DNA encoding the KPI and OX-2 domains (exons 7 and 8) was inserted at this site. This fragment starts at the .B ⁇ mHI site
  • This .B ⁇ mHI fragment was obtained from a lambda phage genomic clone encoding this portion of the APP gene, that was obtained from a Human Placental genomic library in the Lambda FDQI vector obtained from Stratagene.
  • This BamHI fragment originally contained an Xhol site which was destroyed by cutting, filling in, and religation. The locations of the deletions are diagramed in Figure 10.
  • the APP splicing cassette was cut out with Nrul and Xhol and used to replace the Nrul to Xhol cD ⁇ A fragment of APP cD ⁇ A bearing the Hardy mutation.
  • This mutant form of APP cD ⁇ A was produced by converting the G at nucleotide position 2145 to T by site directed mutagenesis. This changes the encoded amino acid from Val to Phe.
  • the resulting construct is a combination cD ⁇ A/genomic APP "minigene.
  • This APP minigene was operatively linked to the PDGF-B promoter to provide expression of the APP cD ⁇ A/genomic construct in mammalian cells.
  • the PDGF /3-chain 5' flanking sequence was inserted upstream of the ⁇ rul site at the beginning of the APP minigene.
  • This fragment includes 1.3 kb upstream of the transcription initiation site, where the PDGF-B promoter resides, and approximately 70 bp of 5' untranslated region, ending at the AH ⁇ II site (Higgins et al (1994)).
  • the late SV40 polyadenylation signal carried on a 240 bp B ⁇ mHI to Bcfl fragment, was added downstream of the APP minigene.
  • PDAPP This construct, combining the PDGF-B promoter, the APP splicing cassette, the Hardy mutation, and the SV40 polyadenylation signal is referred to as PDAPP ( Figure 9).
  • PDAPP Transgenic mice containing the PDAPP construct.
  • Transgenic mice were generated using the PDAPP construct described in Example 5. Transgenic mice were generated by microinjection using standard techniques as described above. PDAPP DNA was microinjected into the embryos at the two-cell stage. Plasmid sequences (pUC) were removed by S ⁇ cl and Notl digestion before microinjection. Seven founder mice were generated and line 109 was used for extensive analysis. Only heterozygous animals were used. Southern analysis of 104 animals from four generations showed that approximately 40 copies of the transgene were inserted at a single site and transmitted in a stable manner. Human APP messenger R ⁇ A was produced in several tissues of the transgenic mouse, but at especially high levels in brain.
  • R ⁇ ase protection assays revealed at least 20-fold more APP expression in the brains of line 109 animals than in the mouse lines expressing neuron-specific enolase ( ⁇ SE)-promoter-driven APP transgenes previously described by Quon et al. (1991), Mucke et al, Brain Res. 666:151-167 (1994), McConlogue et al , Neurobiol. Aging 15:S12 (1994), and Higgins et al , Ann Neurol 35:598-607 (1994).
  • ⁇ SE neuron-specific enolase
  • RT-PCR analysis demonstrated the presence of transcripts encoding the 695, 751 and 770 isoforms of human APP in transgenic animal brains but not in brains from non-transgenic littermates. The identities of the human APP RT-PCR bands from the transgenic mouse RNA were verified by subcloning and sequencing.
  • PDAPP mice expressed approximately 5-fold higher total APP mRNA levels than non- transgenic controls, and at least 20-fold higher human APP mRNA levels than most NSE-APP transgenic mice. While NSE-driven human APP expression does not affect the levels of murine APP mRNA, PDAPP transgenic mice showed a significant 30% decrease in murine APP transcripts. While the relative abundances of murine APP770: 751:695 mRNAs in non-transgenic mouse brains were roughly 1:1:35, the corresponding human APP mRNA levels in PDAPP transgenic mouse brains were 5:5:1.
  • holo-APP was performed by brain homogenization in 10 volumes of PBS containing 0.5 mM EDTA, 10 ⁇ g ml "1 leupeptin and 1 mM PMSF. Samples were spun at 12,000g for 10 min and the pellets resuspended in RIPA (150 mM NaCl, 50 mM Tris, ph 8.0, 20 mM EDTA, 1.0% deoxycholate, 1.0% Triton X-100, 0.1 % SDS, 1 mM PMSF and 10 ⁇ g ml "1 leupeptin).
  • RIPA 150 mM NaCl
  • 50 mM Tris 50 mM Tris, ph 8.0, 20 mM EDTA, 1.0% deoxycholate, 1.0% Triton X-100, 0.1 % SDS, 1 mM PMSF and 10 ⁇ g ml "1 leupeptin.
  • Immunoblot analysis of total APP expression (human and mouse) in transgenic mouse line 109 and control littermate brain tissue using C-terminal APP antibody anti-6 showed much higher levels of expression in the transgenic mice.
  • Immunoblot analysis of brain homogenates using either the holo-APP polyclonal antibody anti-6 or the human-specific APP monoclonal antibody 8E5 revealed human APP over- expression in the transgenic mouse at levels at least 3-fold higher in hippocampus than either endogenous mouse APP levels or those in AD brain.
  • a 4 kD ⁇ amyloid-immunoreactive peptide was isolated from the brains of the transgenic animals, which corresponds to the relative molecular mass of A/3. Brain levels of A/3 were at least 10-fold higher in line 109 animals than in the previously described human APP transgenic mice.
  • line- 109 animals greatly overexpressed human APP mRNA, holo-APP and A/3 in their brains.
  • mice Histopathology of PDAPP Transgenic Mice. Brains from 180 transgenic and 160 age-matched non-transgenic age- matched controls (4 to 20 months old) representing five generations of the line 109 pedigree were extensively examined histopathologically. Some mouse brains were removed and placed in alcohol fixative (Arai et al , Proc. Natl Acad. Sci. U.S.A. 87:2249-2253 (1990)) for 48 hours before paraffin embedding. Other mice were perfused with saline followed by 4% paraformaldehyde in 0.1 M sodium phosphate.
  • A/3 deposits of varying morphology were clearly evident as a result of using a variety of A/3 antibodies, including well characterized human-specific A/3 antibodies and antibodies specific for the free amino and carboxy termini of A ⁇ 1-42.
  • Antibody 9204 described by Saido et al, J. Biol. Chem. 289: 15253-15257 (1994), is specific to A ⁇ 1-5 and was used at a concentration of 7.0 ⁇ g ml "1 .
  • Antibody 277-2 specific for A/3 1-42, was prepared by immunizing New Zealand white rabbits with the peptide cysteine-aminoheptanoic acid- A/3 33-42 conjugated to cationized BSA ('Super Carriers'; Pierce) using a standard immunization protocol (500 ⁇ g per injection). Specific antibodies were affinity-purified from serum against the immunogen immobilized on agarose beads. Before incubation with antibody 277-2, sections were treated for 1 to 2 min with 80% formic acid. For detection, the antibody was reacted using the peroxidase rabbit IgG kit (Vector Labs).
  • the product was then visualized using DAB as the chromogen, Some sections were then incubated overnight at 4°C with a 1:500 dilution of polyclonal anti-GFAP (Sigma).
  • the GFAP antibody was reacted using the alkaline phosphatase anti-rabbit IgG kit and alkaline phosphatase substrate kit 1 (Vector Labs; used according to the manufacturer's recommendations). Additional sections were incubated overnight with the F480 antibody (Serotec) used at a 1:40 dilution to visualize microglial cells.
  • the mouse peroxidase kit (Vector Labs) was then used according to the manufacturer's recommendations.
  • Some sections were stained with thioflavin S using standard procedures (Dickson et al. , Acta Neuropath. 79:486-493 (1990)) and viewed with ultraviolet light through an FITC filter of maximum wavelength 440 nm.
  • paired helical filaments (PHF) have not yet been detected in PDAPP mice, the detection of abnormally phosphorylated neurofilaments and tau are thought to be associated with, and the initial step in, the formation of PHF in AD.
  • a ⁇ plaques were closely associated with distorted neurites that could be detected with human APP-specific antibodies and with anti- synaptophysin antibodies, suggesting that these neurites were derived in part from axonal sprouts, as observed in the AD brain.
  • the plaques compressed and distorted the surrounding neuropil, also as in the AD brain.
  • Synaptic and dendritic density were also reduced in the molecular layer of the hippocampal dentate gyrus of the transgenic mice. This was evident by reduced immunostaining for the presynaptic marker synaptophysin and the dendritic marker MAP-2 in AD brain (Masliah et al, Am. J. Path. 138:235- 246 (1991)).
  • mice were perfused with saline followed by 2.0% paraformaldehyde and 1.0% glutaraldehyde in cacodylate buffer. Forty ⁇ m thick vibratome sections were incubated with the R1280 antibody, and reacted using a peroxidase rabbit IgG kit (Vector Labs). Immunolabelled sections with A ⁇ deposits were then fixed in 1.0% ammonium tetraoxide and embedded in epon/araldite before viewing ultrathin sections with a Jeol CX100 electron microscope (Masliah et al. ,
  • Tables 3 and 4 present a summary of the above results, showing cytological and pathological similarities between AD and PDAPP mice. For every feature examined, with the exception of paired helical filaments, the PDAPP mice exhibited pathology characteristic of AD. These findings show that production of human APP in transgenic (TG) mice is sufficient to cause not only amyloid deposition, but also many of the complex subcellular degenerative changes associated with AD. Table 4. Pathology in Alzheimer's Disease and the PDAPP Mouse.
  • transgenic mice The most notable feature of these transgenic mice is their Alzheimer- like neuropathology, which includes extracellular A ⁇ deposition, dystrophic neuritic components, gliosis, and loss of synaptic density with regional specificity resembling that of AD. Plaque density increases with age in these transgenic mice, as it does in humans (Selkoe, Rev. Neurosi. 17:489-517 (1994)), implying a progressive A/3 deposition that exceeds its clearance, as also proposed for AD (Maggio et al, Proc. Natl. Acad. Sci. U.S.A. 89:5462- 5466 (1992)).
  • the PDAPP transgenic mice provide strong new evidence for the primacy of APP expression and A/3 deposition in AD neuropathology.
  • mice also provide a sufficiently robust AD model in which to test whether compounds that lower A/3 production and/or reduce its neurotoxicity in vitro can produce beneficial effects in an animal model prior to advancing such drugs into human trials.
  • Example 7 Construction APP transgenes expressing APP from the PDGF-B promoter.
  • PDAPP transgenic mice contain a splicing cassette that permits expression of all three major human APP isoforms, where expression is driven by the PDGF-B promoter, and which includes a mutation in amino acid 717, the site of familial AD mutations. It is expected that these features, and others described above, can be used independently to produce transgenic mice useful as models of Alzheimer's disease. Some specific examples of such constructs are described below. A. Construction of PDAPP-wt.
  • a wild type version of the cDNA/genomic clone PDAPP was constructed in which the mutation to amino acid 717 was replaced with the wild type. This was accomplished by replacing the 1448 bp Xhol to Spel fragment of PDAPP, which includes the part of the APP cDNA sequence that encodes the Hardy mutation in which Val717 is replaced by Phe, with the 1448 bp Xhol to Spel fragment of a wild type APP clone. This fragment corresponds to the region from position 1135 to 2588 of SEQ ID NO:5. None of the intron sequences of PDAPP are replaced or removed by this substimtion. This construct is referred to as PDAPP-wt. A schematic of PDAPP-wt and its construction is shown in Figure 12. B. Construction of PDAPP-SwHa.
  • Plasmid pNSE751.deita3'spl.sw contains cDNA of the human APP751 which includes the Swedish mutation of Lys to Asn and Met to Leu at amino acids 670 and 671, respectively.
  • a 563 bp EcoRI to Spel fragment from this plasmid was replaced with the corresponding 563 bp EcoRI to Spel fragment of PDAPP, which includes an identical part of the APP cDNA sequence with the exception of Phe717 of the Hardy mutation. This fragment corresponds to the region from position 2020 to 2588 of S ⁇ Q ID NO:5.
  • a construct encoding only APP695, but retaining the Hardy mutation, PDGF-B promoter, and vector sequences of PDAPP can be made. This can be accomplished by ligating the 6.6 kb Xhol to Nrul fragment from PDAPP, which contains the C-terminal part of the APP sequences, and the polyadenylation, pUC, and PDGF-B promoter sequences, to the 1.2 kb Xhol to Bell fragment of pCK695, which contains a hybrid splice signal and the remaining ⁇ -te ⁇ ninal portion of the APP sequences (on a 911 bp Xhol to Nrul fragment of APP695 cD ⁇ A).
  • the hybrid splice signal is the same as was described earlier and is also present in vector pohCK751, which is described by Dugan et al., J Biological Chem. 270: 10982-10989 (1995).
  • pCK695 is identical to pohCK751 except that the herpes simples virus replication and packaging sequences of pohCK751 were removed, and the plasmid encodes APP695 instead of APP751.
  • the PDGF-B promoter drives the expression of APP695 containing the mutation of Val717 to Phe.
  • the hybrid splice signal is included to potentially enhance expression. Additional vectors derived from this may be constructed which lack any splice signals, or into which other splice signals have been added to obtain this same function.
  • a construct encoding only APP751, but retaining the Hardy mutation, PDGF-B promoter, and vector sequences of PDAPP can be made. This can be accomplished by ligating the 6.65 kb Xhol to Kpnl fragment of PDAPP, including part of the APP sequences, the polyadenylation signals, pUC and PDGF-B promoter sequences to the 1.0 kb Kpnl to Xhol fragment containing the remainder of the human APP751 cD ⁇ A sequences (nucleotides 57 to 1084 of SEQ ID ⁇ O:3) to make the intermediate plasmid PDAPP ⁇ s ⁇ 751 v .
  • the 1.0 kb Kpnl to Xhol fragment encoding a portion of human APP751 can be obtained from the plasmid poCK751, which is identical to pohCK751 except that the herpes simplex viral sequences were removed.
  • the first intron from PDAPP which is intron ⁇ 6, is then inserted into PDAPP ⁇ sp751 v . F to make PDAPP751 V-F -
  • the 2,758 bp AspllS to S al fragment of PDAPP containing exons 2 through 6, intron ⁇ 6, and part of exon 7, is ligated to the 6,736 bp fragment obtained by complete digestion of PDAPP ⁇ sp751 v-F with AspllS and partial digestion with Seal.
  • This 6,736 bp fragment provides the remaining additional APP sequences (part of exon 1, the rest of exon 7, and exons 9 through 18), polyadenylation signals, pUC and PDGF-B promoter sequences.
  • the resulting construct is referred to as PDAPP751 V . F .
  • the PDGF-B promoter drives the expression of APP751 containing the mutation of Val717 to Phe.
  • One splice signal (derived from intron 6) is included to potentially enhance expression. Additional vectors derived from this may be constructed which lack any splice signals, or into which other splice signals have been added to obtain this same function.
  • a construct encoding only APP770, but retaining the Hardy mutation, PDGF-B promoter, and vector sequences of PDAPP can be made. This can be accomplished by replacing the Kpnl to Xhol fragment of PDAPP751 V . F containing APP exons 2-7 and a part of exon 9, with the Kpnl to Xhol fragment of APP770 cDNA, which contains exons 2-8 and a part of exon 9. This fragment corresponds to nucleotides 57 to 1140 of SEQ ID NO:5. The resulting construct is referred to as PDAPP770 V-F .
  • PDGF-B promoter drives the expression of APP770 containing the mutation of Val717 to Phe.
  • PDAPP770 V-F contains the same intron sequences present in PDAPP751 V . F . Additional vectors derived from this may be constructed into which a splice signals have been added to obtain enhanced expression.
  • Example 8 Expression Levels of APP Expression Products in Brain Tissue of PDAPP Mice.
  • the PDAPP mouse line described in Example 6 was examined for the levels of several derivatives of the APP in hippocampal, cortical, and cerebellar brain regions of mice of various ages. Levels of APP cleaved at the beta-secretase site (APP/3) and APP containing at least 12 amino acids of
  • A/3 (FLAPP+APP ⁇ ; a mixmre of APP ⁇ and full length APP (FLAPP)) were found to be nearly constant within a given brain region at all ages evaluated.
  • the hippocampus expressed the highest level of all APP forms.
  • guanidine extractable levels of A/3 showed remarkable age-dependent increases in a manner that mirrored the amyloid plaque deposition observed immunohistochemically.
  • A/3 levels in hippocampus increased 17-fold by 8 months of age and 106-fold by 1 year of age, compared to that found in 4 month old animals.
  • A/3 constitutes approximately 1 % of the total protein in hippocampus.
  • the cerebral cortex also showed large increases in A/3 with age.
  • the mean level of A/3 in cerebellum across all age groups was comparatively low and unchanging.
  • A/3-containing proteins were measured through the use of ELISAs configured with antibodies specific to A/3, A/3 2 , APP cleaved at the /3-secretase site (Seubert et al. (1993)), and APP containing the first 12 amino acids of A/3 (FLAPP +APP ⁇ ; a mixmre of full length APP and - secretase cleaved APP (Esch et al)). Striking similarities in both the regional variation and depositing form of A/3 are noted between the mouse model and the human AD condition.
  • the heterozygote transgenic (Line 109, Games et al. ; Rockenstein et al.) and non-transgenic animals were anesthetized with Nembutol (1:5 solution in 0.9% saline) and perfused intracardially with ice cold 0.9% saline.
  • the brain was removed and one hemisphere was prepared for immunohistochemical analysis, while four brain regions (cerebellum, hippocampus, thalamus, and cortex) were dissected from the other hemisphere and used for A/3 and APP measures.
  • each brain region was homogenized in 10 volumes of ice cold guanidine buffer (5.0 M guanidine-HCl, 50 mM Tris- Cl, pH 8.0) using a motorized pestle (Kontes). The homogenates were gently mixed on a Nutator for three to four hours at room temperamre, then either assayed directly or stored at -20 °C prior to quantitation of A ⁇ and APP. Preliminary experiments showed the analytes were stable to this storage condition. 2. A ⁇ Measurements.
  • the brain homogenates were further diluted 1:10 with ice-cold casein buffer (0.25% casein, phosphate buffered saline (PBS), 0.05% sodium azide, 20 ⁇ g/ml aprotinin, 5 mM EDTA pH 8.0, 10 ⁇ g/ml leupeptin), reducing the final concentration of guanidine to 0.5 M, before centrifugation (16,000 x g for 20 minutes at 4°C).
  • the A/3 standards (1-40 or 1-42 amino acids) were prepared such that the final composition included 0.5 M guanidine in the presence of 0.1 % bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the "total" A ⁇ sandwich ELISA consists of two monoclonal antibodies (mAb) to A/3.
  • the capture antibody, 266, is specific to amino acids 13-28 of A/3 (Seubert et ⁇ /. (1992)); while the antibody 3D6, which is specific to amino acids 1-5 of A ⁇ , was biotinylated and served as the reporter antibody.
  • the 3D6 biotinylation procedure employs the manufacturer's (Pierce) protocol for NHS-biotin labeling of immunoglobulins except 100 mM sodium bicarbonate, pH 8.5 buffer was used.
  • the 3D6 antibody does not recognize secreted APP or full-length APP but detects only A/3 species with amino terminal aspartic acid.
  • the assay has a lower limit of sensitivity of approximately 50 pg/ml (11.4 pM) and showed no cross-reactivity to the endogenous murine A/3 peptide at concentrations up to 1 ng/ml.
  • the configuration of the A3 W2 -specific sandwich ELISA employs the mAb 21F12, which was generated against amino acids 33-42 of A/3.
  • the antibody shows less than 0.4% cross-reactivity with A/3 M0 in either ELISA or competitive radioimmunoassay (RIA).
  • Biotinylated 3D6 is also the reporter antibody in this assay which has a lower limit of sensitivity of approximately 125 pg/ml (28.4 pM).
  • the 266 and 21F12 mAbs were coated at 10 ⁇ g/ml into 96-well immunoassay plates (Costar) overnight at room temperamre. The plates were then aspirated and blocked with 0.25% human serum albumin in PBS buffer for at least 1 hour at room temperamre, then stored desiccated at 4°C until use. The plates were rehydrated with wash buffer prior to use. The samples and standards were added to the plates and incubated at room temperamre for 1 hour. The plates were washed at least 3 times with wash buffer (Tris buffered saline, 0.05% Tween 20) between each step of the assay.
  • wash buffer Tris buffered saline, 0.05% Tween 20
  • biotinylated 3D6 diluted to 0.5 ⁇ g/ml in casein assay buffer (0.25% casein, PBS, 0.05% Tween 20, pH 7.4), was incubated in the well for 1 hour at room temperamre.
  • Avidin-HRP Vector, Burlingame, CA
  • diluted 1:4000 in casein assay buffer was added to the wells and incubated for 1 hour at room temperamre.
  • the colorimetric substrate 100 ⁇ l
  • Slow TMB-ELISA Pierce
  • Reaction product was quantified using a Molecular Devices Vmax measuring the difference in absorbance at 450 nm and 650 nm. 3. APP ELISAs.
  • the capture antibody for both the FLAPP+APP ⁇ and APP/3 assays is 8E5, a monoclonal antibody raised to a bacterially expressed fusion protein corresponding to human APP amino acids 444-592 (Games et al).
  • the reporter mAb (2H3) for the FLAPP+APP ⁇ assay was generated against amino acids 1-12 of A/3.
  • the lower limit of sensitivity for the 8E5/2H3 assay is approximately 11 ng/ml (150 pM).
  • the polyclonal antibody 192 was used as the reporter. This antibody has the same specificity as antibody 92 (Seubert et al. (1993)), that is, it is specific to the carboxy-terminus of the /3-secretase cleavage site of APP.
  • the lower limit of sensitivity for the /3-secretase 8E5/192 assay is approximately 43 ng/ml (600 pM).
  • the 8E5 mAb was coated onto 96-well Costar plates as described above for 266.
  • Purified recombinant secreted APP ⁇ (the APP751 form) and APP596 were the reference standards used for the FLAPP +APP ⁇ and APP/3 assays, respectively.
  • APP was purified as described previously (Esch et al.) and APP concentrations were determined by amino acid analysis.
  • the 5 M guanidine brain homogenate samples were diluted 1:10 in specimen diluent for a final buffer composition of 0.5 M NaCl, 0.1 % NP-40, 0.5 M guanidine.
  • the APP standards for the respective assays were diluted into buffer of the same final composition as for the samples.
  • the immunogens for 3D6, 21F12, and 2H3 were separately conjugated to sheep anti-mouse immunoglobulin (Jackson Immunoresearch Labs) using maleimidohexanoyl-N-hydroxysuccinimide (Pierce).
  • A/J mice (Jackson Laboratories) were given intraperitoneal injections (IP) of 100 ⁇ g of the appropriate immunogen emulsified in Freund's complete adjuvant (Sigma) and two subsequent IP injections of 100 ⁇ g immunogen were given on a biweekly basis in Freund's incomplete adjuvant (Sigma).
  • the highest titer mouse of a given immunogen was injected intravenously and intraperitoneally with 50-100 ⁇ g of immunogen in PBS.
  • the spleen was removed, splenocytes were isolated and fused with SP2/0-Agl4 mouse myeloma cells.
  • the hybridoma supematants were screened for high affinity monoclonal antibodies by RIA as previously described (Seubert et al. (1992)). Purified monoclonal antibodies were prepared from ascites.
  • the tissue from one brain hemisphere of each mouse was drop-fixed in 4% paraformaldehyde and post-fixed for three days.
  • the tissue was mounted coronally and 40 ⁇ m sections were collected using a vibratome.
  • the sections were stored in anti-freeze at -20 °C prior to staining. Every sixth section, from the posterior part of the cortex through the hippocampus, was immunostained with biotinylated 3D6 at 4°C, overnight.
  • the sections were then incubated with horseradish peroxidase avidin-biotin complex (Vector) and developed using 3,3'-diaminobenzidine (DAB) as the chromogen.
  • Vector horseradish peroxidase avidin-biotin complex
  • DAB 3,3'-diaminobenzidine
  • the FLAPP +APP ⁇ assay recognizes secreted APP including the first 12 amino acids of A/3. Since the reporter antibody (2H3 ⁇ -12 ) is not specific to the alpha clip site occurring between A/3 amino acids 16 and 17 (Esch et al), this assay also recognizes full length APP. Preliminary experiments using immobilized APP antibodies to the cytoplasmic tail of full length APP to deplete the mixture suggest that approximately 30 to 40% of the FLAPP +APP ⁇ is full length.
  • the APP/3 assay recognizes only the APP clipped immediately amino-terminal to the A ⁇ region due to the specificity of the polyclonal reporter antibody 192 (Seubert et al. (1993)).
  • the specific namre of the A/3 immunoreactivity was further characterized as follows. Guanidine homogenates of brain (excluding cerebellum and brain stem) were subjected to size exclusion chromatography (Superose 12) and the resulting fractions analyzed using the total A ⁇ assay. Comparisons were made of 2, 4, and 12 month transgenic mouse brain homogenates and a non-transgenic mouse brain homogenate to which A ⁇ had been spiked at a level roughly equal to that found in the 12 month old transgenic mice. The elution profiles of the transgenic brain homogenate were similar in that the peak fractions of A/3 immunoreactivity occurred in the same position, a single broad symmetric peak which was coincident with the immunoreactive peak of spiked A/3 1-40 .
  • the A/3 M2 ELISA employs a capture antibody that recognizes A/3 M2 but not A/3 M0 -
  • the A/3 M2 assay is not affected by the full length or carboxy-terminal forms of APP containing the A/3 region in the homogenates as shown by similar immunodepletion studies.
  • Total A ⁇ and APP Measures Table 5 shows the levels of total A ⁇ , FLAPP+APP ⁇ , and APP/3 in the hippocampus, cortex, cerebellum, and thalamus of transgenic mice as a function of age. Each data point represents the mean value for each age group. The relative levels of FLAPP +APP ⁇ and APP/3 in all four brain regions remain relatively constant over time.
  • the hippocampus expresses the highest levels of FLAPP+APP ⁇ and APP/3 followed by the thalamus, cortex, and cerebellum, respectively. In the hippocampus, the levels of
  • FLAPP+APP ⁇ are approximately 3.5 to 4.5-fold higher than APP/3 at all ages.
  • the mean value of all ages for FLAPP+APP ⁇ and APP/3 assays in the hippocampus were 674 (+465) pmoles/gram and 175 ( ⁇ 11) pmoles/gram, respectively. From this it can be estimated that the pool of brain APP consists of approximately 50% APP ⁇ , 30% full length APP, and 20% APP/3.
  • A/3 levels increased dramatically with age in the hippocampus and cortex. However, no such increase was noted in the cerebellum of the PDAPP transgenic mice, and only a moderate increase was seen in thalamus (Table 5). The increase of A/3 is greater in the hippocampus relative to the cortex, which also correlates with the 3D6 immunohistochemical results (see discussion below). Compared to the cortex levels of 4 month old mice, A ⁇ levels increase 10-fold by 8 months of age and 41-fold at 12 months old (660 + 380 pmoles A/3/gram tissue at age 12 months).
  • a ⁇ constitutes approximately 1 % of protein in hippocampus of the PDAPP mice.
  • Concentrations of A/3 M2 in the cortex of transgenic mice were evaluated at different ages. As shown in Table 6, the percentage of A/3 which is A/3 M2 in the cortex of transgemc mice, also increases with age. The ELISA data suggest that A/3 M2 is preferentially depositing in the transgenic mice, and that the deposits detected by mAb 3D6 immunostaining are primarily A/3 M2 .
  • Transgenic animals with A ⁇ values representing the mean A/3 value of the age group were used for 3D6 immunostaining.
  • a progression of A ⁇ deposition is seen in the 4, 8, 10, and 12 months old animals.
  • transgenic brains contained small, rare punctate deposits, 20 ⁇ m in diameter, that were only infrequently observed in the hippocampus and frontal and cingulate cortex.
  • these regions contained a number of thioflavin-positive A/3 aggregates that formed plaques as large as 150 ⁇ m in diameter.
  • many large A/3 deposits were found throughout the frontal and cingulate cortex, and the molecular layers of the hippocampus.
  • a ⁇ amyloidosis is an established diagnostic criteria of Alzheimer's disease (Mirra et al , Neurology 41:479-486 (1991)) and is consistently seen in higher cortical areas as well as the hippocampal formation of the brain in affected subjects.
  • a ⁇ amyloidosis is a relatively early event in the pathogenesis of AD that subsequently leads to neuronal dysfunction and dementia through a complex cascade of events (Mann et al , Neurodegeneration 1:201-215 (1992); Morris et al., Neurology 46:707-719 (1996)).
  • Various pathways of APP processing have been described (reviewed in Schenk et al, J. Med. Chem.
  • A/3 amyloid deposition seen in the PDAPP mouse brain is highly age and region specific. Amyloid deposition begins at around 7 months of age, and by 12 months of age, amyloid deposition is very profound throughout the hippocampus and in the rostral region of the cortex. The age dependent increases in amyloid deposition correlate well with the dramatic rise in A ⁇ levels in these brain regions as measured by ELISA assay. An increase in A/3 is measurable by 7 months of age and by 10 months the hippocampus as 2180 pmoles/g of A/3, a concentration equivalent to that of my cytoskeletal proteins and comparable to the levels found in the cortex of human AD brain (Gravina et al, J. Biol. Chem. 270:7013-7016 (1995)).
  • brain A/3 levels reflects amyloid burden and therefore direct immunoassay measurement of brain A/3 levels can be used to test for compounds that reduce amyloid plaque burden.
  • overproduction of A ⁇ is almost certainly an accelerating factor not only in A ⁇ deposition but in subsequent neuropathology (Citron et al. , Mann et al. , Miller et al. , Archives of Biochem. Biophys.
  • Example 9 Behavioral Differences in PDAPP Transgenic Mice. Alzheimer's disease is characterized by cognitive deficits including memory loss, and impairment of memory functions. To determine if the disclosed transgenic mice exhibit similar deficits, transgenic (TG) and non- transgenic (nTG) mice were evaluated for task performance in three types of maze apparatus used to test working and reference memory; the Y maze, the radial arm maze (RAM), and the water maze.
  • TG transgenic
  • nTG non- transgenic mice were evaluated for task performance in three types of maze apparatus used to test working and reference memory; the Y maze, the radial arm maze (RAM), and the water maze.
  • the transgenic mice tested represent the fifth generation derived from the PDAPP mice described in Example 6.
  • the Y maze and the radial arm maze are used to assess spontaneous alternation which is a function of working memory.
  • the mouse is placed in the stem of a Y maze twice, each time allowing a choice entry into one of the arms. Entering both arms is a successful alternation, requiring memory of the previously entered arm, while entering the same arm on both trials is a failure.
  • Chance performance is 50% alternation, that is, 50% of the mice alternate.
  • the mouse is placed at the center of a maze with multiple arms radiating from the center.
  • a radial eight- arm maze was used. Alternation performance is measured by allowing only eight entries, with the number of different arms entered being the measure of performance. The number of different-arm entries can be compared to the number of different-arm entries expected by chance, which is 5.25 (Spetch and Wilkie, "A program that stimulates random choices in radial arm mazes and similar choice situations" Behavior Research Methods & Instrumentation 12:377-378 (1980)). Performance above chance, that is, above 5.25, requires memory of the previously entered arms.
  • the water maze used for the tests described below consists of a pool of water in which a submerged platform is placed.
  • This hidden platform can be found by swimming mice either by chance (first trial) or through memory of positional clues visible from the tank (subsequent trials).
  • Subject mice were trained in the hidden platform task according to standard procedures. Briefly, mice were first pretrained in a small pool (47 cm diameter, 20 cm platform), which teaches them how to navigate in water, that the platform is the goal, that there is no other escape, and that to find it they must resist their natural inclination to stay along the sides of the pool. They were then trained to find a single platform position in the hidden platform task using a larger pool and smaller platform (71 cm pool, 9 cm platform). During the HP task, visual cues were located inside the pool
  • mice assigned to the characterization cohort study were tested on the behavioral tasks described above over 3 days during the week or two before euthanasia. Their transgenic status was not known to the tester. Non- transgenic littermates were used for comparison. Each morning the subject mice were run in the Y maze and RAM as described above. They were then tested for general strength on the inclined plane (INP) test. For this, mice were placed in a 10-cm-wide runway lined with ridged plastic and elevated with the head up at 35°.
  • IPP inclined plane
  • mice were tested in the water maze as described above. Briefly, mice were pretrained in a small pool to climb on a large submerged platform as their only means of escape from the water. They were then given six blocks of four trials each to learn the location of a small platform in a large pool. For analysis, all four trials within each block were averaged. The exception was the first hidden platform block, for which only the last three trials were averaged. The first trial was analyzed separately, because it is the only one for which platform location could not be known, and thus did not relate to spatial learning. It is thus used as a control for non-spatial factors, such as motivation and swimming speed. The performance effects between blocks were analyzed as a repeated measure for the hidden platform task. Standard analysis of variance (ANOVA) calculations were used to assess the significance of the results.
  • ANOVA Standard analysis of variance
  • nTG mice retain a better memory of the platform location than TG mice.
  • Example 10 Detection and Measurement of Alzheimer's Disease Markers.
  • Glial fibrillary acidic protein (GFAP), a marker which increases in AD brain tissue, was measured in the following manner. Tissue extracts were prepared from hippocampi of control and PDAPP transgenic mice, as described in Example 6, aged 14 months. Tissue was sonicated in 10 volumes (v/w) of 10 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF, 10 ⁇ g/ml leupeptin, 5 ⁇ g/ml calpain inhibitor 1. Protein determinations were made on the extract and SDS-PAGE sample buffer added before boiling the samples for 5 minutes.
  • Gliosis is one of the changes that is associated with the neuropathology of Alzheimer's disease.
  • the isoquinoline carboxamide PK 11195 has been shown to be a preferential marker of the peripheral benzodiazepine sites associated with gliosis. These sites have been shown to be enhanced in several diseases and animal models associated with neuronal damage and activated necroglia including stroke (Stephenson et al. , J. Neuroscience 15:5263-5274 (1991)) and Alzheimer's disease (Diorio et al , Neurobiology of Aging 12:255-258 (1991)).
  • PDAPP mice were euthanised by cervical dislocation and the brains rapidly dissected on ice.
  • Homogenates (10 mg/ml wet weight) of cerebral cortex, hippocampus and cerebellum were prepared in 50 mM Tris HCl, pH 7.4. 0.3, 1.0 and 3.0 nM [ ⁇ ] PK 11195 was incubated with these brain regions for 60 minutes at 23 °C followed by rapid filtration over Whatman GF/B filters using a Brandell cell harvester. Non-specific binding was determined using 1 ⁇ M unlabelled PK 11195. Quantitation was performed by liquid scintillation spectrometry.
  • PDAPP mice were euthanised using carbon dioxide, the brains removed and snap frozen in methyl butane/dry ice.
  • the brains were sectioned in the coronal plane through the hippocampus. Twenty micron thick sections were mounted on glass slides and stored at -20 °C. Sections were incubated at 1 hour at 23 °C in 170 mM Tris-HCl, pH 7.4 containing 1 nM [ 3 H] PK 11195. Non-specific binding was determined using 1 ⁇ M unlabelled PK 11195. Incubations were terminated by rinsing sections twice for 5 minutes in ice-cold incubation buffer followed by a brief wash in ice-cold distilled water. Following rapid drying, sections were exposed to tritium Hyperfilm (Amersham International) for up to 5 weeks.
  • the pattern of labeling corresponded to microglial cell or astrocytic clumps associated with plaques, rather than the more widespread pattern of astrocytosis or microgliosis in the hippocampal and cortical parenchyma.
  • the non-transgenic mouse did not show this labeling pattern.
  • C. Detection and Measurement of Cholinergic Nerve Terminals A population of cholinergic neurones projecting to the forebrain have been shown to be selectively decreased in the postmortem brains of patients diagnosed with Alzheimer's disease. Hemicholinium-3 is a potent inhibitor of high affinity choline uptake and has been shown to be a good marker of cholinergic nerve terminals (Pascual et al. , J Neurochem 54:792-800 (1990)).
  • the total number of high affinity choline uptake sites in PDAPP transgenic animals, which are described in Example 6, has been measured using both crude whole-brain preparations and homogenates from selective brain regions using the selective ligand [ 3 H]-Hemicholinium-3 ([ 3 H]HCh-3).
  • [ 3 H]-Hemicholinium-3 binding was determined using a modification of the methods described in Pascual et al. Mice were euthanised by asphyxiation with carbon dioxide and the brains rapidly removed and dissected on ice. The cortex, cerebellum, striamm and hippocampus were homogenized in 5 ml of 10 mM phosphate buffer without NaCl. Samples were spun at 17,000 x g for 10 minutes, and the pellets washed twice in 5 ml 10 mM PO 4 buffer. The final pellet was resuspended in 5 ml IX phosphate buffered saline (PBS) to produce a protein concentration of 0.5 mg/ml. Brain regions were assayed in triplicate for high affinity choline uptake sites by the addition of [ 3 H]HCh-3 (3 nM final concentration). Following a 20 minute incubation, assays were terminated by rapid filtration through
  • Non specific binding was determined in the presence of 100 mM ouabain and the absence of ATP. Assays were terminated by rapid filtration over Whatman GF/B filters. Tubes were washed with ice cold 50 mM Tris HCl, pH 7.4, 15 mM KCl, 5 mM MgCl 2 . Filters were transferred into scintillation vials, and specific binding estimated by liquid scintillation spectrometry.
  • the difference in Na/K-ATPase activity between transgenic and non- transgenic tissue is significant (p ⁇ 0.05) in the case of 12 month old cerebellum, and is highly sigmficant (p ⁇ 0.01) in the case of 12 month old hippocampus.
  • the difference in Mg-ATPase activity between transgenic and non-transgenic tissue is significant (p ⁇ 0.05) in the case of 8 and 12 month old cortex, and is highly significant (p ⁇ 0.01) in the case of 12 month old hippocampus.
  • tissue pre-treatment for hybridization with probe for example, paraffin embedded sections
  • post- hybridization washes depend on the method used, examples of which are described in the references cited above. Known and appropriate precautions against RNase contamination should be employed and are also discussed in the above references.
  • Tissue Preparation Freshly dissected whole brains, or sub-regions of interest, from transgenic or control mice at various developmental stages, or post-natal ages, are snap frozen in isopentane pre-equilibrated to -70 °C. If desired, the animals may be perfused with PBS to eliminate circulating cells from brain prior to dissection. The brains are removed following 15 to 20 seconds immersion in isopentane, wrapped in aluminum foil, labeled appropriately, and stored at -80 °C for sectioning. It should be noted that although the signal from in situ hybridization to cryostat sectioned tissues is more sensitive than to paraffin embedded sections, it is dependent upon the time from dissection to hybridization.
  • Frozen tissue is preferably analyzed by hybridization with probe within six weeks. RNA integrity in tissues declines beyond this time. Thus, if longer time periods between dissection and analysis are anticipated, the tissue should be fixed (see, for example, Lu and Gillett) before long term storage at -80 °C.
  • Probe-On-Plus glass slides Prior to sectioning, Probe-On-Plus glass slides (Fisher Scientific, Pittsburgh, PA) can be made RNAase free by overnight soaking in absolute ethanol, air dried briefly in a dust free environment, and baked at 180°C for a minimum of 4 hours. After cooling to room temperamre, the slides are coated with 0.01 % poly-lysine (prepared in DEPC treated H 2 O) for approximately 5 seconds, and air dried in a dust free area. The coated slides can be stored for up to one month before use in a slide box with silica gel or drierite pellets.
  • the frozen brain stored at -80 °C is transferred to a cryostat at -20 °C, mounted onto a sectioning block, embedded in OCT. ® , and allowed to equilibrate.
  • the tissue is then cut into 7 to 14 ⁇ m thick sections using a sterilized microtome knife (treated with 70% EtOH in DEPC H 2 O), and thaw mounted onto poly-lysine coated slides.
  • the slides are kept at - 20 °C until the sectioning is complete.
  • the sections are fixed and dehydrated by immersing the slides sequentially in the solutions noted below.
  • the fixed sections are stored immersed in the 95 % EtOH/DEPC H 2 O solution at 4°C until use. If the sections are not fixed immediately, they may be stored at -80 °C in the presence of drierite until use. In this case the sections are allowed to equilibrate to room temperamre prior to the fixation/dehydration steps. 2. Probe Design and Preparation.
  • the sequence of the mouse BDNF mRNA/cDNA (accession #55573) is available from the Genbank database of Nucleic acid sequences (NCBI, Bethesda, MD).
  • Anti-sense oligodeoxynucleotide probes against BDNF were designed using the primer select module of the DNAstarTM software package (Lasergene Inc., Madison, WI). Numerous other software packages, such Oligo ® (NBI, Madison, MN), offer similar capabilities, and are also suitable.
  • Candidate probes of 45 to 55 nucleotides length, approximately 50% G+C content, and hybridizing to the pre-cursor or mature peptide encoding regions of the BDNF mRNA were synthesized on an ABI 380B DNA synthesizer.
  • sense oligonucleotides corresponding to each probe were also synthesized.
  • the BDNF probes synthesized correspond to BDNF nucleotide positions 47 to 94 (probes 2710 & 2711), 158 to 203 (probes 2712 & 2713), 576 to 624 (probes 2714 & 2715), and 644 to 692 (probes 2716 & 2717).
  • the even numbered oligonucleotides are probes for the sense strand, and the odd numbered oligonucleotides are probes for the anti-sense strand.
  • the probes are gel purified on denaturing acrylamide gels and reconstimted in H 2 O using standard protocols (Sambrook et al).
  • the probes (30 to 35 ng, 2 pmoles) are labeled by 3' homopolymeric tailing using terminal deoxynucleotidyl transferase (Promega, Madison WI) and 35 S-dATP (1000 Ci/mmol, Amersham Inc.) according to the enzyme manufacturer's recommendation.
  • the radiolabeled probes are purified by column chromatography on size exclusion mini-spin columns (Biospin-6,
  • Tissue Hybridization and Post Hybridization Washes In preparation for hybridization, the desired number of slides are removed from storage under alcohol, and allowed to air dry thoroughly in the slide rack (approximately 1 hour). Meanwhile, the probe is heat denatured in a boiling H 2 O bath for 2 to 5 minutes, quick chilled in an ice/H 2 O bath, and diluted in hybridization buffer (10% dextran sulfate, 50% deionized formamide, 4X SSC, 5X Dehardts, 100 ⁇ g/ml sheared salmon sperm DNA, 100 ⁇ g/ml polyadenylic acid) to a final concentration of 5 X 10 3 to 10 X 10 3 cpm/ ⁇ l. DTT is added to a 10 mM final concentration.
  • hybridization buffer 10% dextran sulfate, 50% deionized formamide, 4X SSC, 5X Dehardts, 100 ⁇ g/ml sheared salmon sperm DNA, 100 ⁇ g/ml polya
  • hybridization 100 ⁇ l of diluted probe in hybridization buffer (corresponding to 0.5 X 10 6 to 1.0 X 10 6 cpm probe) is carefully applied to each section being hybridized with probe. The solution is gently spread over the section with a pipet tip to cover the entire section(s) on each slide. The slides containing probe are then placed in humidified hybridization chambers at 42 °C for hybridization overnight with the probe.
  • the hybridization chambers can be covered utility boxes, or acrylic boxes, with raised platforms to accommodate slides. The boxes are lined with filter paper (or paper towels), saturated in 4X SSC, 50% formamide, and humidified by pre- incubating them with closed lids in a 42° C incubator for 1 to 3 hours before the slides are placed inside them.
  • cover slips can be placed on the sections after the hybridization buffer is applied, this is not necessary provided the hybridization chambers are adequately humidified during the procedure. If pre-hybridization is used to obtain a lower background, the sections may be incubated at 42 °C under 50 ⁇ l hybridization buffer (minus probe) per section for 1 to 2 hours. After this time, an equal volume of hybridization buffer containing probe at twice the concentration described above is applied to each section, and hybridization is carried out as described above. For washes, the slides are transferred from the hybridization chamber to a slide holder. The slides can be placed in the slide holder every 4th or 5th slot so as to allow adequate flow of wash solution over the surface of each section. This placement can significantly lowers background on the sections.
  • a moderate flow rate of wash solution over the surface of the sections promotes removal of unhybridized probe, and consequently reduces background. This is best accomplished during the 55 °C wash steps by suspending the slides in the slide holder, above a magnetic stir bar.
  • the stir bar is preferably placed approximately one inch under the slide holder. This can be done by hanging the slide holder(s) from pipets straddling the wash chamber.
  • a large beaker, or a 4 to 6 inch deep Pyrex baking dish makes a suitable wash chamber.
  • the wash chamber is placed on a hot-plate stirrer, the temperature setting of which is precalibrated to maintain the wash solution at 55 °C during the procedure.
  • the sections are air dried thoroughly at room temperamre for 2 hours, followed by 30 minutes at 55 °C.
  • the dried sections on the slides are placed in autoradiographic cassette and exposed to X-ray film (Hyperfilm, /3-max, Amersham Inc., Arlington Heights, IL) at 4 °C for 2 to 3 days to estimate the exposure time required under emulsion.
  • the X-ray film is developed according to the manufacturers recommendation.
  • the sections are coated with emulsion (Amersham LM-1, #RPN40) by dipping in emulsion at 42 °C under appropriate safelight conditions.
  • the emulsion coated slides are air dried on a cooled surface for approximately 30 minutes, and transferred to a plastic slide box containing a drying agent (drierite pellets).
  • the seams of the box can be sealed with black tape, and the box wrapped in several layers of aluminum foil to ensure a light-tight enclosure.
  • the boxes are transferred to 4°C for autoradiographic exposure for 2 to 6 weeks.
  • the box Prior to developing the emulsion coated slides, the box is removed from the refrigerator and allowed to equilibrate to room temperamre for approximately 1 hour.
  • the slides are then developed according to the manufacmrers instructions, air dried for 1 to 2 hours, and if desired, counterstained with the appropriate counterstain.
  • NAME Pabst, Patrea L.
  • GAG ACA CCT GGG GAT GAG AAT GAA CAT GCC CAT TTC CAG AAA GCC AAA 960 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gin Lys Ala Lys 305 310 315 320
  • GGT GCA ATC ATT GGA CTC ATG GTG GGC GGT GTT GTC ATA GCG ACA GTG 1920 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635 640
  • AAG AAG AAA CAG TAC ACA TCC ATT CAT CAT GGT GTG GTG GAG GTT GAC 2160 Lys Lys Lys Gin Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp 705 710 715 720
  • Gin Glu Lys Val Glu Ser Leu Glu Gin Glu Ala Ala Asn Glu Arg Gin 420 425 430

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US6175057B1 (en) 1997-10-08 2001-01-16 The Regents Of The University Of California Transgenic mouse model of alzheimer's disease and cerebral amyloid angiopathy
US6455757B1 (en) 1997-10-08 2002-09-24 The Regents Of The University Of California Transgenic mice expressing human APP and TGF-β demonstrate cerebrovascular amyloid deposits
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US6374130B1 (en) * 1999-04-06 2002-04-16 Eric M. Reiman Methods for tracking the progression of Alzheimer's disease identifying treatment using transgenic mice
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