WO1996040895A1

WO1996040895A1 - Method for identifying alzheimer's disease therapeutics using transgenic animal models

Info

Publication number: WO1996040895A1
Application number: PCT/US1996/009679
Authority: WO
Inventors: Lisa Claire Mcconlogue; Peter A. Seubert
Original assignee: Athena Neurosciences, Inc.
Priority date: 1995-06-07
Filing date: 1996-06-07
Publication date: 1996-12-19
Also published as: JP2001517065A; AU6264996A; CA2221986A1; EP0832205A1

Abstract

The construction of transgenic animal models for testing potential treatments for Alzheimer's disease are described. The models are characterized by pathology similar to that observed in Alzheimer's disease, based on expression of all three forms of the β-amyloid precursor protein (APP), APP695, APP751, and APP770, as well as various point mutations based on naturally occurring mutations, such as the London and Indiana familial Alzheimer's disease (FAD) mutations at amino acid 717, predicted mutations in the APP gene, and truncated forms of APP that contain the Aβ region. The APP gene constructs are prepared using the human platelet derived growth factor B(PDGF-B) chain gene promoter, or other promoters able to express A$(b) or mutant forms APP at a high level in transgenic animal brain tissue. Animal cells can be isolated from the transgenic animals or prepared using the same constructs with standard techniques such as lipofection or electroporation. 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 of APP and β-amyloid peptide, neuropathology, and behavioral alterations.

Description

METHOD FOR IDENTIFYING ALZHEIMER'S DISEASE

THERAPEUTICS USING TRANSGENIC ANIMAL MODELS

Background of the Invention

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.

Alzheimer's disease (AD) is a degenerative disorder of the brain first described by Alios Alzheimer in 1907 after examining one of his patients who suffered drastic reduction in cognitive abilities and had generalized dementia (The early story of Alzheimer's Disease, edited by Bick et al. (Raven Press, New York 1987)). It is the leading cause of dementia in elderly persons. 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. The impact of AD on society and on the national economy is enormous. It is expected that the demented elderly population in the United States will increase by 41 % by the year 2000. It is expensive for the health care systems that must provide institutional and ancillary care for the AD patients at an estimated annual cost of $40 billion (Jorm (1987); Fisher, "Alzheimer's Disease", New York Times, August 23, 1989, page Dl, edited by Reisberg (The Free Press, New York & London 1983)). These factors imply action must be taken to generate effective treatments for AD.

At a macroscopic level, the brains of AD patients are usually smaller, sometimes weighing less than 1,000 grams. At a microscopic level, 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. Loss of neurons in the cholinergic system leads to a consistent deficit in cholinergic presynaptic markers in AD (Fisher (1983); Alzheimer's Disease and Related Disorders, Research and Development edited by Kelly (Charles C. Thomas, Springfield, IL. 1984)). In fact, AD is defined by the neuropathology of the brain.

AD is associated with neuritic plaques measuring up to 200 μ 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.

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 predisposition for AD. First, molecular analysis has 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. , Nature 376:775-778 (1995); Levy-Lahad et al. , Science 269:973-977 (1995); Sherrington et al., Nature 375:754-760 (1995)). Another loci associated with AD resides on chromosome 19 and encodes a variant form of apolipoprotein E (Corder, Science 261:921-923 (1993).

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 β peptide (Aβ; sometimes also referred to in the literature as β-amyloid peptide or β peptide) (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885- 890 (1984)), which is also the primary protein constituent in cerebrovascular amyloid deposits. The amyloid is a filamentous material that is arranged in beta-pleated sheets. AjS 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. 84:4190- 4194 (1987); Tanzi et al., Nature 331:528-530 (1988)) and genomic APP DNA (Lemaire et al., Nucl. Acids Res. 17:517-522 (1989); Yoshikai et al., Gene 87, 257-263 (1990)). A number of forms of APP cDNA have been identified, including the three most abundant forms, APP695, APP751, and APP770. These forms arise from a single precursor RNA by alternate splicing. The gene spans more than 175 kb with 18 exons (Yoshikai et al. (1990)). APP contains an extracellular domain, a transmembrane region and a cytoplasmic domain. Aβ consists of up to 28 amino acids just outside the hydrophobic transmembrane domain and up to 15 residues of this transmembrane domain. Thus, Aβ is a cleavage product derived from APP which is normally found in brain and other tissues such as heart, kidney and spleen. However, Aβ deposits are usually found in abundance only in the brain.

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)). 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. Unless otherwise noted, all forms of APP and fragments of APP, including all forms of Aβ, referred to herein are based on the human APP amino acid sequence. APP is post-translationally modified by the removal of the leader sequence and by the addition of sulfate and sugar groups.

Van Broeckhaven et al., Science 248: 1120-1122 (1990), have demonstrated that the APP gene is tightly linked to hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) in two Dutch families. This was confirmed by the finding of a point mutation in the APP coding region in two Dutch patients (Levy et al., Science 248: 1124-1128 (1990)). The mutation substituted a glutamine for glutamic acid at position 22 of the Aβ (position 618 of APP695, or position 693 of APP770). In addition, 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 and are absent in families with late-onset AD. This mutation at amino acid 717 increases the production of the Aβ,... form of Aβ 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. (1992)). This mutation to amino acids 670 and 671 increases the production of total Aβ from APP (Citron et al, Nature 360:622-674 (1992)). There are no robust animal models to study AD, although aging nonhuman primates seem to develop amyloid plaques of AjS in brain parenchyma and in the walls of some meningeal and cortical vessels. Although aged primates and canines can serve as animal models, they are expensive to maintain, need lengthy study periods, and are quite variable in the extent of pathology that develops.

There are no spontaneous animal mutations with sufficient similarities to AD to be useful as experimental models. Various models have been proposed in which some 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)). Flood et al., Proc. Natl. Acad. Sci. 88:3363-3366 (1986), reported amnestic effects in mice of four synthetic peptides homologous to the Aβ. Because none of these share with AD either common symptoms, biochemistry or pathogenesis, they are not likely to yield much useful information on etiology or treatment.

Several 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 0-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 Aβ (Wirak et al., Science 253:323-325 (1991); Sandhu et al., J. Biol. Chem. 266:21331-21334 (1991); Kawabata et al , Nature 354:476-478 (1991)) have been produced. Results obtained in the different studies appear to depend upon the source of promoter and the protein coding sequence used. For example, Wirak et al , Science 253:323-325 (1991), found that in transgenic mice expressing a form of the Aβ, intracellular accumulation of "amyloid¬ like" material, reactive with antibodies prepared against Aβ were observed but did not find other histopathological disease symptoms. The intracellular nature of the antibody-reactive material and the lack of other symptoms suggest that this particular transgenic animal is not a faithful model system for Alzheimer's disease. Later studies have shown that similar staining is seen in non-transgenic control mice and Wirak et al, Science 253:323-325 (1991) was partially retracted in a comment in Science 255:143-145 (1992). Thus, the staining seen by Wirak et al. appears to be artifactual. Kawabata et al. (1991) report the production of amyloid plaques, neurofibrillary tangles, and neuronal cell death in their transgenic animals. In each of these studies, Aβ or a fragment containing Aβ was expressed. Wirak et al. (1991), used the human APP promoter while Kawabata et al. (1991) used the human thy-1 promoter. However, Kawabata et al. (1991) was later retracted by Kawabata et al, Nature 356:23 (1992) and Kawabata et al , Nature 356:265 (1992). In 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 Aβ 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)).

Transgenic 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). Such mice have only sparse AjS 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 Aβ 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).

Transgenic 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.

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. Similarly, Fukuchi et al. discusses the failure of prior transgenic animal models to display most of the characteristics known to be associated with AD. For example, the transgenic mouse reported by Quon et al is reported to produce Aβ immunoreactive deposits that stain only infrequently with thioflavin S and not at all with Congo Red, in contrast to the staining pattern of AD Aβ deposits.

It is therefore an object of the present invention to provide an animal model for Alzheimer's disease that is constructed using transgenic technology.

It is a further object of the present invention to provide transgenic animals characterized by certain genetic abnormalities in the expression of the amyloid precursor protein.

It is a further object of the present invention to provide transgenic animals exhibiting one or more histopathologies similar to those of Alzheimer's disease. It is a further object of the present invention to provide transgenic animals expressing one or more AjS-containing proteins at high levels in brain tissue.

It is a further object of the present invention to provide a method of screening potential drugs for the treatment of Alzheimer's disease using transgenic animal models.

Summary of the Invention The construction of 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. 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 β 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. Neuron-specific expression of constructs can be achieved by using the rat neuron specific enolase promoter.

The 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.

The constructs disclosed herein generally encode all or a contiguous portion of one of the three forms of APP: APP695, APP751, or APP770, preferably an AjS-containing protein, as described herein. Examples of Aβ- 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 AjS- 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 jS-secretase or -secretase cleavage, respectfully; APP cDNA truncated to encode amino acids 646 to 770 of APP; APP cDNA truncated to encode amino acids 646 to 770 of APP and including at least one intron; the APP leader sequence followed by the Aβ region (amino acids 672 to 714 of APP) plus the remaining carboxy terminal 56 amino acids of APP; the APP leader sequence followed by the Aβ region plus the remaining carboxy terminal 56 amino acids with the addition of a mutation at amino acid 717; the APP leader sequence followed by the Aβ region; the Aβ region plus the remaining carboxy terminal 56 amino acids of APP; the Aj3 region plus the remaining carboxy terminal 56 amino acids of APP with the addition of a mutation at amino acid 717; a combination cDNA/genomic APP gene construct; a combination cDNA/genomic APP gene construct with the addition of a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a combination cDNA/genomic APP gene construct truncated at amino acid 671 or 685; and an APP cDNA construct containing at least amino acids 672 to 722 of APP. These protein coding sequences are operably linked to leader sequences specifying the transport and secretion of the encoded Aβ 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 Aβ in transgenic animal brain tissue. A preferred promoter is the human platelet derived growth factor β 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 these mutations. These constructs 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 amounts of APP, Aβ, and neuropathology in the animals, as well as by behavioral alterations.

Brief Description of the Drawings The boxed portions of the drawings indicate the amino acid coding poπions 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 8 indicates the presence of a long DNA sequence. 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 Aβ 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 9 portion of the gene. As a result of this recombination event, sequences in the resident mouse chromosomal APP gene beyond the recombination point in exon 9 are replaced by the analogous human sequences.

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

PDAPP construct. The sizes of original introns 6, 7 and 8, as well as the sizes of the final introns are indicated on the diagram. The locations of the deletions in introns 6 and 8 present in the PDAPP construct are also indicated.

Figure 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„ vector and the plasmids and intermediate constructs used to make the PDAPP695., vector.

Figure 15 is a diagram of the PDAPP75 , vector and the plasmids and intermediate constructs used to make the PDAPP75 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. Dmitry Goldgaber, NIH. 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.

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 Aβ. 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 Aβ. 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 Aβ. 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 Aβ. These sequences are derived from Ponte et al. (1988).

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 AjS. 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 AjS. 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. According to this numbering convention, for example, 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). Unless otherwise noted, all forms of APP and fragments of APP, including all forms of Aβ, referred to herein are based on the human APP amino acid sequence. For example, Aβ refers to the human Aβ, APP refers to human APP, and APP770 refers to human APP770. As used herein, the term 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. As such, the term 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.

Characterization of phage and cosmid clones of human genomic DNA clones listed in Table 1 below originally established a minimum size of at least 100 kb for the Alzheimer's gene. There are a total of 18 exons in the APP gene (Lemaire et al, Nucl Acid Res 17:517-522 (1989); Yoshikai et al. (1990); Yoshikai et al, Nucleic Acids Res 102:291-292 (1991)). Yoshikai et al. (1990) describes the sequences of the exon- intron boundaries of the APP gene. These results taken together indicate that the minimum size of the Alzheimer's gene is 175 kb.

Table 1. Alzheimer's Cosmid and Lambda Clones.

Name of Insert

Library Clone Size (kb) Assigned APP Region

1 GPAPP47A 35 25 kb » promoter & 9 kb intron 1

Cosmid 2 GPAAP36A 35 12 kb promoter & 22 kb intron 1

3 GAPP30A 30-35 5' coding region

4 GAPP43A 30-35 exons ; 9, 10 and 11

1 GAPP6A 12 exon 6

2 GAPP6B 18 exons 4 and 5

3 GAPP20A 20 exon 6

4 GAPP20B 17 exons 4 and 5

Lambda 5 GAPP28A 18 exons 4 and 5

6 GAPP3A 14 exon 6

7 GAPP4A 19 exon 6

8 GAPP10A 16 exons 9, 10 and 11

9 GAPP16A 21 exon 6

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. (1990) and 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.

Starting Ending Following nucleotide nucleotide Intron

Exon 1 -146 57 Intron 1

Exon 2 58 225 Intron 2

Exon 3 226 355 Intron 3

Exon 4 356 468 Intron 4

Exon 5 469 662 Intron 5

Exon 6 663 865 Intron 6

Exon 7 866 1033 Intron 7

Exon 8 1034 1090 Intron 8

Exon 9 1091 1224 Intron 9

Exon 10 1225 1299 Intron 10

Exon 11 1300 1458 Intron 11

Exon 12 1459 1587 Intron 12

Exon 13 1588 1687 Intron 13

Exon 14 1688 1909 Intron 14

Exon 15 1910 1963 Intron 15

Exon 16 1964 2064 Intron 16

Exon 17 2065 2211 Intron 17

Exon 18 2212 3432

APP Gene Mutations.

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-Hariin 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)). In one form of this mutation, 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 AjS 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 Aβ. Additional mutations in APP at amino acids 669, 670, and 671 have been shown to reduce the amount of AjS 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 Aβ.

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 Aj3 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 He, 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. It is believed that the mutations affect the expression and/or processing of APP, shifting the balance toward Alzheimer's pathology. 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 He, or deletion of the codon. A preferred substitution for Lys670 is Asn, and a preferred substitution for Met671 is Leu. These mutations predispose individuals expressing the mutant proteins to develop Alzheimer's disease. The other listed mutations to amino acids 669, 670, and 671 are known to reduce the amount of Aβ processed from APP (Citron et al. (1995)). It is believed that these mutations affect processing of APP leading to a change in Aβ production. Truncated forms of APP can also be expressed from transgene constructs. For example, 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. Nucleic Acid Constructs Encoding AS-containing Proteins.

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 Aβ- containing protein. As used herein, an AjS-containing protein is 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, where the protein includes all or a portion of amino acids 672 to 714 of human APP. Preferred AjS-containing proteins include amino acids 672 to 714 of human APP. Preferred forms of such AjS- 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,

670, 671, 690, 692, and/or 717, where each of these AjS-containing proteins includes amino acids 672 to 714 of human APP. Preferred forms of the above AjS-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, 671, 690, 692, 717; APP695; 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.

In the constructs disclosed herein, the DNA encoding the AjS- 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 an amino acid sequence comprising amino acids 672 to 714 of APP770; APP cDNA truncated to encode amino acids 646 to 770 of APP; a combination cDNA/genomic DNA hybrid APP gene construct; a combination cDNA/genomic DNA hybrid APP gene construct bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; or a combination cDNA/genomic DNA hybrid APP gene construct truncated at amino acid 671 or 685.

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 APP695 cDNA encoding an APP amino acid sequence, the amino acid sequence comprising amino acids 672 to 714 of APP770; APP cDNA truncated to encode amino acids 646 to 770 of APP; a combination cDNA/genomic DNA hybrid APP gene construct; a combination cDNA/genomic DNA hybrid APP gene construct bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, and a combination of these mutations; and a combination cDNA/genomic DNA hybrid APP gene construct truncated at amino acid 671 or 685. Construction of Transgenes.

Construction of various 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 8 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. A 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. For example, an APP770 cDNA clone (SΕQ ID NO: 5) can be digested with Aspl 18 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 Bglll, 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 AjS, followed by the AjS, 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 Aβ. 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. Combination cDNA/Genomic DNA Clones.

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. Such sequences have been identified in APP genomic DNA by Yoshikai et al. (1990). The disclosed 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.

Other 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 PstI 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). It is preferred that the heterologous intron be placed between the promoter and the region encoding the APP. 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. First, 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. Second, 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. These two exon/intron junction fragments can then be released from their respective plasmid vectors by cleavage with Xhol and either BamHI or Bglll, 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 itømHI-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. In the genomic DNA, 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. After cleavage with Xhol, 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 mechanism. 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 AjS-containing proteins. The ability to regulate expression of the gene encoding an AjS-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 Aβ-containing protein in cultured cells is believed to be useful in evaluating expression and processing of the different AjS-containing gene products and may provide the basis for cell cultured drug screens. A preferred promoter is the human platelet derived growth factor jS (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 Aβ. 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, Aβ,.. 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 APPα combined (FLAPP +APPα) to a level of at least 150 pmoles/g brain tissue, APPS to a level of at least 42 pmoles/g brain tissue, and mRNA encoding human AjS-containing protein to a level at least twice that of mRNA encoding the endogenous APP of the transgenic animal. Aβ.. is the total of all forms of Aβ. Aβ,.. is a form of A/3 having amino acids 1 to 42 of Aβ (corresponding to amino acids 672 to 714 of APP). FLAPP -f-APPα: refers to APP forms containing the first 12 amino acids of the AjS region (corresponding to amino acids 672 to 684 of APP). Thus, FLAPP - APPo: represents a mixture of full length forms of APP and APP cleaved at the α-secretase site (Esch et al , Science 248: 1122-1124 (1990)). APP/3 is APP cleaved at the jS-secretase site (Seubert et al, Nature 361:260-263 (1993)). It is intended that 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. Thus, 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. Where expression is tissue-specific, it is understood that if the expression level is sufficient in the specific brain tissue, the promoter is considered preferred even though the expression level in brain tissue as a whole may not, and need not, reach a threshold level. It is preferred that this level of expression is observed in hippocampal and/or cortical brain tissue. The promoter can mediate expression of the above expression products to the levels described above either constitutively 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.

It is preferred that, in making the above measurements, 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 temperature. 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. 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). It is preferred that the following assay be used to identify preferred promoters for their ability to mediate expression of AjS to the levels described above. Antibody 266 (Seubert et al, Nature 359:325-327 (1992)) is dissolved at 10 μg/ml in buffer (0.23 g/L NaHPO.-HO, 26.2 g/L NaHPO.- 7HO, 1 g/L sodium azide adjusted to pH 7.4) and 100 μl/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 NaHPO.-7HO, 1.0 g/L NaHPO.-HO, 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 μl/well of Aβ 1-40 standards and brain tissue samples are added to the plate in triplicate and incubated overnight at 4°C. A/31-40 standards are made from 0.0156, 0.0312, 0.0625, 0.125, 0.250, 0.500, and 1.000 μg/ml stocks in DMSO stored at -40° C as well as a DMSO only control for background determination. Aj3 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 Aβ 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. The plates and 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 μl/well of 3D6-biotin at 0.5 μg/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 Aβ position 1 (Asp). For biotinylating 3D6, follow Pierce's NHS-Biotin protocol for labeling IgG (cat. #20217X) except use 100 mM sodium bicarbonate, pH 8.5 and 24 mg NHS- biotin per ml of DMSO.

The plates are then again washed 3X with wash buffer. Then, 100 μl/well of horseradish peroxidase (HRP)-avidin (Vector Labs, cat. # A-2004) diluted 1:4000 in casein buffer #2 is added to each well and incubated for 1 hour at RT. The plates are washed 4X with wash buffer and then 100 μl/well of TMB substrate (Slow TMB-ELISA (Pierce cat. # 34024)) at RT is added to each well and incubated for 15 minutes at RT. Finally, 25 μl/well of 2 N HSO. is added to each well to stop the enzymatic reaction, and the plate is read at 450 nm to 650 nm using the Molecular Devices Vmax reader.

It is preferred that the relative levels of mRNA encoding human AjS- containing protein mRNA encoding the endogenous APP of the transgenic animal be measured in the manner described by Bordonaro et al. , Biotechniques 16:428-430 (1994), and Rockenstein et al, J. Biol. Chem. 270:28257-28267 (1995). Preferred methods for measuring the expression level of Aβ,.„ FLAPP +APPQ;, and APP_/3 are described in Example 8. Yeast Artificial Chromosomes.

The constructs shown in Figure 7 can be constructed as follows. Large segments of human genomic DNA, when cloned into certain vectors, can be propagated as autonomously-replicating units in the yeast cell. Such vector-borne segments are referred to as yeast artificial chromosomes (YAC; Burke et al. Science 236:806 (1987)). 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.

The nucleotide sequence homology between the human and murine Alzheimer's protein genes is approximately 85%. Within the A|8-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 AjS is capable of causing disease. Homologous recombination (Capecchi, Science 244: 1288-1292 (1989)) can be used to convert the mouse Alzheimer's gene in situ to a gene encoding the human AjS 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 endogenous APP gene resulting from this event would be normal up to the point of recombination, within exon 9 in this example, and would consist of the human cDNA sequence thereafter. Preparation of Constructs for Transfections and Microinjections.

DNA clones for microinjection are cleaved with enzymes appropriate for removing the bacterial plasmid sequences, such as Sail and Notl, and the DΝA fragments electrophoresed on 1 % agarose gels in TBE buffer (Sambrook et al. (1989)). The DΝA bands are visualized by staining with ethidium bromide, and the band containing the APP expression sequences is excised. The excised band is then placed in dialysis bags containing 0.3 M sodium acetate, pH 7.0. DΝA is electroeluted into the dialysis bags, extracted with phenol-chloroform (1:1), and precipitated by two volumes of ethanol. The DΝA is redissolved in 1 ml of low salt buffer (0.2 M ΝaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on an Elutip-D™ column. The column is first primed with 3 ml of high salt buffer (1 M ΝaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washing with 5 ml of low salt buffer. The DΝA solutions are passed through the column for three times to bind DΝA to the column matrix. After one wash with 3 ml of low salt buffer, the DΝA is eluted with 0.4 ml of high salt buffer and precipitated by two volumes of ethanol. DΝA concentrations are measured by absorption at 260 nm in a UV spectrophotometer. For microinjection, DΝA concentrations are adjusted to 3 μg/ml in 5 mM Tris, pH 7.4 and 0.1 mM EDTA. Other methods for purification of DΝA for microinjection are also described in Hogan et al. , Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory, Cold Spring Harbor, ΝY, 1986); in Palmiter et al, Nature 300:611 (1982); in The Qiagenologist, Application Protocols, 3rd edition, published by Qiagen, Inc., Chatsworth, CA.; and in Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, ΝY, 1989). Construction of Transgenic Animals. A. Animal Sources. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA),

Taconic (Germantown, ΝY), 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. B. Microinjection Procedures.

The procedures for manipulation of the rodent embryo and for microinjection of DNA are described in detail in Hogan et al. , Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1986), the teachings of which are generally known and are incoφorated herein.

C. Transgenic Mice.

Female 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, 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). Surrounding cumulus cells are removed with hyaluronidase (1 mg/ml). Pronuclear embryos are then washed and placed in Earle's balanced salt solution containing 0.5% BSA (EBSS) in a 37.5 °C incubator with a humidified atmosphere at 5 % CO_;, 95 % air until the time of injection. 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 puφose. Recipient females are mated at the same time as donor females. At the time of embryo transfer, 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. D. Transgenic Rats.

The procedure for generating transgenic rats is similar to that of mice (Hammer et al, Cell 63:1099-112 (1990)). Thirty day-old female rats are given a subcutaneous injection of 20 IU of PMSG (0.1 cc) and 48 hours later each female placed with a proven male. At the same time, 40-80 day old females are placed in cages with vasectomized males. These will provide the foster mothers for embryo transfer. The next morning females are checked for vaginal plugs. Females who have mated with vasectomized males are held aside until the time of transfer. Donor females that have mated are sacrificed (CO= 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. Once the embryos are injected, 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. E. Embryonic Stem (ES) Cell Methods. 1. Introduction of cDNA into ES cells.

Methods for the culturing of ES cells and the subsequent production of transgenic animals, the introduction of DNA into ES cells by a variety of methods such as electroporation, calcium phosphate/DNA precipitation, and direct injection are described in detail in Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, ed. E.J. Robertson, (IRL Press 1987), the teachings of which are generally known and are incoφorated herein. Selection of the desired clone of transgene-containing ES cells can be accomplished through one of several means. For random gene integration, an APP clone is co-precipitated with a gene encoding neomycin resistance. Transfection is carried out by one of several methods described in detail in Lo veil-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. In these procedures, 0.5 X 10^* ES cells are plated into tissue culture 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 μl. The cells are fed with selection medium containing 10% fetal bovine serum in DMEM supplemented with G418 (between 200 and 500 μg/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. Methods for positive selection of the recombination event (for example, neo resistance) and dual positive-negative selection (for example, neo resistance and gancyclovir resistance) and the subsequent identification of the desired clones by PCR have been described by Joyner et al, Nature 338: 153-156 (1989), and Capecchi (1989), the teachings of which are generally known and are incoφorated herein.

2. Embryo Recovery and ES cell Injection.

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 puφose when using mice. Embryos of the appropriate age are recovered approximately 3.5 days after successful mating. Mated females are sacrificed by CO, 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 μm.

3. Transfer of Embryos to Pseudopregnant Females. Randomly cycling adult female mice are paired with vasectomized males. Mouse strains such as Swiss Webster, ICR or others can be used for this puφose. Recipient females are mated such that they will be at 2.5 to 3.5 days post-mating when required for implantation with blastocysts containing

ES cells. At the time of embryo transfer, the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body weight. The ovaries are exposed by making an incision in the body wall directly over the oviduct and the ovary and uterus are externalized.

A hole is made in the uterine horn with a 25 gauge needle through which the blastocysts are transferred. After the transfer, the ovary and uterus are pushed back into the body and the incision is closed by two sutures. This procedure is repeated on the opposite side if additional transfers are to be made.

Identification, Characterization, and Utilization of Transgenic Mice and Rats. Transgenic rodents can be identified by analyzing their DNA. For this puφose, 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.) animals and their progeny (F, and F=).

A. Pathological Studies.

The various F„, F„ and F, animals that carry a transgene can be analyzed by immunohistology for evidence of Aβ 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 Aβ. 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 μm 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, coφus striatum 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. 12:383-388 (1991); Selkoe, Ann. Rev. Neurosci. 12:463-490 (1989); Arai et al , Proc. Natl Acad. Sci. USA 87:2249-2253 (1990); Majocha et al. , Amer. Assoc. Neuropathology Abs 99:22 (1988); Masters et al, Proc. Natl. Acad. Sci. 82:4245-4249 (1985); Majocha et al , Can J Biochem Cell Biol 63:577-584 (1985)). Staining with thioflavin S and Congo Red can also be carried out to analyze the presence of amyloid and co-localization of Aβ deposits within neuritic plaques and NFTs.

B. Analysis of APP and Aβ Expression. 1. mRNA.

Messenger RNA can be isolated by the acid guanidinium 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.

2. Protein.

APP, Aβ, 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, Aβ region, AjS,., A0„, APPjS, FLAPP+APPα, and C-terminus of APP. A variety of antibodies that are human sequence specific, such as 10D5 and 6C6, are very useful for this puφose (Games et al. (1995)).

3. Western Blot Analysis. 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 86:145-1 '49 (1989). Briefly, 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. C. Pathological and Behavioral Studies.

1. Pathological Studies. Immunohistology and thioflavin S staining are conducted as described elsewhere herein. In situ Hybridizations: 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% paraf ormaldehyde. The slides are washed with PBS twice for 5 minutes to remove paraf ormaldehyde. Then the sections are permeabilized by treatment with a 20 μg/ml proteinase K solution. The sections are re-fixed in 4% paraf ormaldehyde, 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. After appropriate washing, bound radioactive probes are detected by autoradiography or enzymatically labeled probes are detected through reaction with the appropriate chromogenic substrates. 2. Behavioral Studies. Behavioral tests designed to assess learning and memory deficits are employed. An example of such as test is the Morris water maze (Morris, Learn Motivat. 12:239-260 (1981)). In this procedure, the animal is placed in a circular pool filled with water, with an escape platform submerged just below the surface of the water. A visible marker is placed on the platform so that the animal can find it by navigating toward a proximal visual cue. Alternatively, a more complex form of the test in which there are no local cues to mark the platform's location will be given to the animals. In this form, 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 AjS-containing proteins is described in Example 9. The procedures applied to test transgenic mice are similar for transgenic rats. D. Preferred Characteristics.

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 AjS 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. For example, 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 (the presence of which identifies the animal as a preferred transgenic animal) 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. 79:486-493 (1990); Masliah et al. , J Neuropathol Exp Neurol 52: 135-142 (1993);

Masliah et al, Acta Neuropathol 87: 135-142 (1994); Wang and Munoz, J Neuropathol Exp Neurol 54:548-556 (1995)). 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. The 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. In such AD screening assays, the compound is administered to the animals, or introduced into the culture 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, histopathology, and/or, in the case of animals, behavior using the procedures described above and in the examples below.. In general, any improvement in behavioral tests, reduction in the severity of histopathology characteristic of AD present in the transgenic animals, and/or reduction in the levels of AjS or APP cleavage products observed in treated animals, relative to untreated animals, is indicative of a compound useful for treating Alzheimer's disease.

Expression of the various forms of APP and Aj3 can be directly measured and compared in treated and untreated transgenic animals both by immunohistochemistry and by quantitative ELISA or immunoblot measurements as described above and in the examples. Currently, it is known that two forms of APP products are found, APP and Aβ (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 AjS expression in the transgenic mice. As described in Example 6, increases in Aj3 have been demonstrated during aging of the PDAPP mouse.

Targets for expression measurements can be selected based on changes in the expression or location of specific markers in Alzheimer's disease. Preferred targets for assay measurement are AS markers known to increase in individuals with Alzheimer's disease are total Aj3 (Aj3„), AjS 1-42 (Aj3,,_;; Aβ with amino acids 1-42), A_/3,., (Aβ with amino acids 1-40), Aj3 N3(pE) (A/3„(ρE)); Aβ X-42 (Aft.; Aβ forms ending at amino acid 42); Aβ X-40 (A/ ; A/S forms ending at amino acid 40); insoluble AS (Aj8_); and soluble AjS (Aj3_; Kuo et al, J. Biol Chem. 271(8): 4077-4081 (1996)). Aj3„(pE) has pyroglutamic acid at position 3 (Saido, Neuron 14:457-466 (1995)). AjS,. refers to any of the C-terminal forms of AjS such as Aj8,„. Aβ_^. refers to forms of AjS that are recovered as described in Gravina, J. Biol. Chem. 270:7013-7016 (1995). APPjS can also be specifically measured to assess the amount of /3-secretase activity (Seubert et al, Nature 361:260-263 (1993)). Several of these AS forms and their association with Alzheimer's disease are described by Haass and Selkoe (1993). Detection and measurement of Aβ., Aj3,., and Aj3,„ are described in Example 6. Generally, specific forms of AjS can be assayed, either quantitatively or qualitatively using specific antibodies, as described below. When referring to amino acid positions in forms of AjS, the positions correspond to the AjS region of APP. Amino acid 1 of AjS corresponds to amino acid 672 of APP, and amino acid 42 of AjS corresponds to amino acid 714 of APP.

Also preferred as targets for assay measurement are APP markers. For example, different forms of secreted APP (termed APPα and APPS) 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 +APPα:, 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/ . 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 AjS 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/3 to particular tissues and cell types. Such assays were described above and specific examples are provided below. A. Screening assays using cultured cells. Screening assays for determining the therapeutic potential of compounds can also be performed using cells derived from animals transgenic for the disclosed APP constructs and cell cultures stably transfected with the disclosed constructs. For example, such assays can be performed on cultured cells in the following manner. Cell cultures 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 cultures can then be plated in Corning 96-well plates at 1.5 to 2.5 x 10" cells per well in Dulbecco's minimal essential media plus 10% fetal bovine serum. Following overnight incubation at 37 °C in an incubator equilibrated with 10% carbon dioxide, media are removed and replaced with media containing a compound to be tested for a two hour pretreatment period and cells were incubated as above. Stocks containing the compound to be tested are first prepared in 100% dimethy lsulfoxide such that at the final concentration of compound used in the treatment, the concentration of dimethylsulfoxide does not exceed 0.5 %, preferably about 0.1 % .

At the end of the pretreatment period, 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. After treatment, plates are centrifuged in a Beckman GPR at 1200 φm for five minutes at room temperature to pellet cellular debris from the conditioned media. From each well, 100 μL 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 Aβ) 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 AjS) can be run to measure the amount of Aβ 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). To the cells remaining in the tissue culture plate, 25 μL 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. Cells are incubated at 37 °C for one hour, and cellular activity is stopped by the addition of an equal volume of MTT lysis buffer (20% w/v sodium dodecylsulfate in 50% dimethylformamide, pH 4.7). Complete extraction is achieved by overnight shaking at room temperature. The difference in the OD- , and the OD„_ is measured in a Molecular Device's UV„ microplate reader, or equivalent, as an indicator of the cellular viability.

The results of the AjS ELISA are fit to a standard curve and expressed as ng/mL Aβ. In order to normalize for cytotoxicity, these results are divided by the MTT results and expressed as a percentage of the results from a control assay run without the compound.

B. A_/8 toxicity assays.

The effect of potential therapeutic compounds on the toxicity of AjS to neuronal cells can be determined in the following manner. Primary Rat Cortical Cell Cultures: Primary rat cortical cultures are established from 18 day rat fetuses. Cortical tissue is dissociated by incubation in a trypsin/EDTA solution (0.05% trypsin + 0.53 mM EDTA in HBSS; GIBCO Laboratories, Grand Island, New York, USA) for 20 minutes at 37 °C. The trypsin is then inactivated by resuspending the cells in serum- containing medium (DMEM/FBS): Dulbecco's Modified Eagles' Medium (DMEM) containing 4.5 g/L glucose, 1 mM sodium pyruvate, 1 mM glutamine, 100 Units/mL penicillin, 100 μg/mL streptomycin, and supplemented with 10% heat-inactivated fetal bovine serum (GIBCO Laboratories, Grand Island, New York, USA). Cells are then pelleted by centrifugation and resuspended in a chemically-defined medium (DMEM/B27:DMEM containing B27 supplement; GIBCO Laboratories, Grand Island, New York, USA) in place of FBS. Polyethyleneimine (PEI; Sigma Chemical Company, St. Louis, Missouri, USA)-coated 6.4 mm (96- well) dishes are rinsed once with DMEM/FBS, and then seeded at 0.75 to 1.25 x 10" cells per well in 0.1 mL DMEM/B27. Cultures are maintained in a HO-saturated incubator with an atmosphere of 90%air/10%CO, at 37°C. Cell viability is visually assessed by phase contrast microscopy and quantified by measuring the reduction of alamarBlue™ (Alamar Biosciences, Inc., Sacramento, California, USA) as described below. Serum replacement with B27 supplement yields nearly pure neuronal cultures as judged by immunocytochemistry for glial fibrillary acidic protein and neuron-specific enolase (Brewer et al, J. Neuro. Sci. Res. 35(5):567-577 (1993)). This procedure can also be used to prepare cell cultures from the disclosed transgenic animals.

All publications cited herein are hereby incoφorated by reference. The information contained in these publications for which the publications are cited is generally known.

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 PC 12 cells were plated into 100 mm dishes and transfected with a mixture of 5 mg of the Sail 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 μl. Poly ly sine-coated plates were used for PC 12 cells, which normally do not adhere well to tissue culture dishes. The cells were fed with selection medium containing 10% fetal bovine serum in DMEM or RPMI and supplemented with G418. Five hundred mg/ml (biological weight) and 250 mg/ml of G418 were used to select colonies form NIH3T3 and PC 12 cells, respectively. Fifteen days after transfection, colonies of cells resistant to G418 were isolated by cloning rings and expanded in T flasks. The presence of APP cDNA in the cells was detected by PCR using the procedure of Mullis and Faloona, Methods

Enzymol. 155:335-350 (1987), the teachings of which are generally known and are incoφorated herein.

Expression of APP in 25 colonies from each cell line was analyzed by immunostaining (Majocha et al (1988)). Cells were grown to subconfluence and fixed in a solution containing 4% paraf ormaldehyde, 0.12 M NaCI, and 20 mm NaPO., pH 7.0. They were incubated overnight with a primary monoclonal antibody against a synthetic A/3 sequence (Masters et al. (1985); Glenner and Wong) provided by Dr. Ronald Majocha, Massachusetts General Hospital, Boston, MA, followed by a generalized anti-mouse antibody conjugated to biotin (Jackson ImmunoResearch Labs, PA). Immunostaining was then performed by adding avidin-horseradish peroxidase (HRP) (Vector Labs, Burlingame, CA) and diaminobenzidine as the chromogen (Majocha et al. (1985)). The results indicated that the pMTAPP-1 vector was expressing APP in both NIH3T3 and PC12 cells. 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 moφhologically 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/3 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)). Self-ligation of this molecule results in an expression clone in which the truncated protein thus encoded contains the leader sequence, followed by a shortened version of the A/3 starting with the sequence Phe-Arg-Val-Gly-Ser-of the A/3 followed by the 56 terminal amino acids of APP. DNA from this construct was transfected into PC 12 cells as described above.

Example 4: Generation of Transgenic Mice expressing APP under the control of the MT-1 promoter.

Transgenic mice were made by microinjecting pMTAPP-1 vector DNA into pronuclear embryos. 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, 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. 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, 5% O„ and 88% N, until the time of injection. B. Microinjection.

Elutip-D™ purified Sail DNA was dissolved in 5 mM Tris (pH 7.4) and 0.1 mM EDTA at 3 μg/ml concentration for microinjection. Microneedles and holding pipettes were pulled from Fisher coagulation tubes (Fisher) on a DKI model 720 pipette puller. Holding pipettes were then broken at approximately 70 μm (O.D.) and fire polished to an I. D. of about 30 μm on a Narishige microforge (model MF-83). Pipettes were mounted on Narishige micromanipulators 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 μl 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. C. Transfer.

Randomly cycling adult female mice were paired with 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.

D. Analysis Of Mice For Transgene Integration.

At three weeks of age or older, 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 μg 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 mixture). The supernatants were mixed with 70 μl 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 μl TE buffer (10 mM Tris pH 8.0 and 1 mM EDTA).

Ten to twenty microliters of DNA from each sample was digested with BamHI, electrophoresed on 1 % agarose gels, blotted onto nitrocellulose paper, and hybridized with "P- labeled APP cDNA fragment. Transgenic animals were identified by autoradiography of the hybridized nitrocellulose filters. The DNAs were also analyzed by PCR carried out by synthetic primers to generate an 800 bp fragment of APP DNA.

A total of 671 pronuclear embryos were microinjected out of which 73 live and 6 dead pups were bom. DNA analysis identified 9 transgenic mice (5 females and 4 males) which were bred to generate F, and F_: transgenics. These animals can be analyzed for expression of mRNA and 48 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

Figure 6). In order to create a splicing cassette small enough for convenient insertion in a pUC vector, two deletions in intronic sequences were made. A deletion was made in intron 6 from position 143 of intron 6 to the BamHI site located upstream of the beginning of exon 7 (1658 bp before the beginning of exon 7). Another deletion was made in intron 8 from the first BamHI site in intron 8 to a site at 263 bp before the beginning of exon 9. To avoid confusion, these truncated forms of APP introns 6 and 8 are referred to herein as intron Δ6 and intron Δ8. BamHI sites were engineered at the sites of these deletions, so that they are marked by the presence of BamHI sites. In this construct, referred to as PDAPP, exons 7 and 8 and intron 7 are intact genomic sequences, except that the unique Xhol site in intron 7 was destroyed.

DNA fragments contaimng 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 containing 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 BamHI. The resulting 34 bp fragment from the Xcml in exon 6 to the Xcml in intron 6, and 131 bp fragment from the Xcml in intron 6 to the artificially created BamHI site at position 143 bp of intron 6 were ligated into APP770x-oligo-x in a three-way ligation step to make APP770x-E6oligo-x. The orientation of the fragments was confirmed by sequencing. APP770x-E6oligo-x was then cut with BamHI and Xhol. Then the 313 bp BamHI and Xhol fragment from the modified APP genomic DNA clone containing the junction of intron 8 and exon 9 was ligated into APP770x-E6oligo-x to make APP770xE6E9x.

APP770xE6E9x was then cut with BamHI 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 BamHI site

1658 bp upstream of the start of exon 7 and extends to the first BamHI site in intron 8. This BamHI 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 FIXII 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. This clone, containing exons 1-8 and part of 9, and introns 6, 7 and 8, was termed the "APP splicing cassette. " The APP splicing cassette was cut out with Nr«I and Xhol and used to replace the Nrul to Xhol cDΝA fragment of APP cDΝA bearing the Hardy mutation. This mutant form of APP cDΝA 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 cDΝA/genomic APP "minigene. " Sequencing of the 6.8 kb BamHI fragment containing APP exons 7 and 8 derived from the APP genomic clone used to generate this construct showed that intron 7 is 2.6 kb long, and that the first BamHI site in intron 8, the upstream site of the deletion in intron 8 engineered into the APP minigene construct, is 2329 bp downstream from the end of exon 8. This does not coincide with the restriction map of the APP gene published by Yoshikai et al. (1990) and Yoshikai et al. (1991). Comparison of their map to our sequence indicates that Yoshikai et al. switched the order of two

EcoRI fragments in their restriction mapping. The 1.60 kb EcoRI fragment containing exon 8 is actually upstream of the 1.48 kb EcoRI fragment and the 1.48 kb EcoRI fragment Yoshikai et al. mapped in intron 7 is actually in intron 8. We have confirmed this location for the EcoRI fragment containing exon 8 by sizing of PCR generated fragments from human DNA.

This APP minigene was operatively linked to the PDGF-B promoter to provide expression of the APP cDNA/genomic construct in mammalian cells. The PDGF /3-chain 5' flanking sequence was inserted upstream of the Nrul 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 Aurll site (Higgins et al. (1994)). The late SV40 polyadenylation signal, carried on a 240 bp BamHI to Bcfl fragment, was added downstream of the APP minigene. 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). Example 6: 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 Sacl 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 RΝA was produced in several tissues of the transgenic mouse, but at especially high levels in brain. RNase 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 (NSE)-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). A. Expression of APP Transcripts and Protein. RNA was isolated from brain tissue as described by Chomaczynski and Sacchi, Analyt. Biochem. 162:156-159 (1987), and subjected to RT-PCR as described by Wang et al, Proc. Natl. Acad. Sci. U.S.A. 86:9717-9721 (1989), using human-specific APP primers (5'-CCGATGATGACGAGGACGAT-3\ SEQ ID NO:7; 5'-TGAACACGTGACGAGGCCGA-3\ SEQ ID NO:8) using 40 cycles of 1 minute at 94°C, 40 seconds at 60°C, and 50 seconds at 72°C. 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. The relative levels and alternative splicing of APP transcripts in brains of PDAPP transgenic mice, NSE- APP transgenic mice, non-transgenic mice, and humans with and without AD were compared in RNase protection assays (Rockenstein et al , J. Biol Chem. 270:28257-28267 (1995). 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. Analysis of holo-APP was performed by brain homogenization in 10 volumes of PBS containing 0.5 mM EDTA, 10 μg ml" leupeptin and 1 mM PMSF. Samples were spun at 12,000g for 10 min and the pellets resuspended in RIPA (150 mM NaCI, 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 μg ml' leupeptin). Samples (each containing 30 μg total protein) were analyzed by SDS-PAGE, transferred to Immobilon membranes and reacted with either the holo-APP antibody, anti-6 (anti Bx 6), described by Oltersdorf et al, J. Biol. Chem. 285:4492-4497 (1990), or 8E5 monoclonal antibody. 8E5 was prepared against a bacterial fusion protein encompassing human APP residues 444-692 (Oltersdorf et al. (1990)) and is human-specific, showing essentially no crossreactivity against mouse APP. 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. For immunoblot analysis of A/3, a 9-month-old mouse brain was homogenized in 5 ml 6 M guanidine HCl, 50 mM Tris, pH 7.5. The homogenate was centrifuged at 100,000g for 15 min and the supernatant was dialyzed against HO overnight adjusted to PBS with 1 mM PMSF and 25 μg ml leupeptin. This material was immunoprecipitated with antibody 266 resin, and immunoblotted with the human-specific A/3 antibody, 6C6, as described by Seubert et al, Nature 359:325-327 (1992). Using this human- specific AS antibody (6C6), a 4 kD /3 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. Embryonic day 16 cortical cell cultures from transgenic animals constitutively secreted human AjS, including a substantial fraction of /3 1-42 (5 ng ml' total A/3; 0.7 ng ml' Aβ 1-42), as detected in media by human-specific A/3 enzyme-linked immunosorbent assays, as described by Seubert et al. (1992) and McConlogue et al. (1994), and as described in Example 8. Thus, line- 109 animals greatly overexpressed human APP mRNA, holo-APP and A/3 in their brains.

B. 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% paraf ormaldehyde in 0.1 M sodium phosphate. For paraffin embedded brains, 6 μm coronal or parasaggital sections from transgenic and non- transgenic mice were placed adjacent to each other on poly-L-lysine coated slides. The sections were deparaffinized, rehydrated and treated with 0.03% HO, for 30 min before overnight incubation at 4°C with a 1: 1,000 dilution of the AjS antibody, R1280 (Tamaoka et al , Proc. Natl Acad. Sci. U.S.A. 89: 1345-1349 (1992)). For absoφtion studies, synthetic human A/3 1-40 peptide (Games et al , Neurobiol. Aging 13:569-576 (1992)) in 10% aqueous dimethy lsulphoxide was added to a final concentration of 7.0 μM to the diluted antibody and incubated for 2 hours at 37 °C. The diluent was applied to the sections and processed under the same conditions as the standard antibody solution. Peroxidase rabbit IgG kit (Vector Labs) was then used as recommended, with 3,3'-diaminobenzidine (DAB) as the chromogen. Similarly fixed human AD brain was processed simultaneously under identical conditions.

Before 4 months of age, no obvious Aβ deposition was detected. However, by approximately 4 months of age, the transgenic animals began to exhibit deposits of human A/3 in the hippocampus, coφus callosum and cerebral cortex. These A/3 plaques increased with age, and by eight months many deposits of 30 to 200 μm were seen. As the animals aged beyond 9 months, the density of the plaques increased until the A/3-staining pattern resembled that of AD. Vascular amyloid, another feature of AD pathology, developed in older mice. Robust pathology was also seen in another transgenic line generated from the PDAPP vector (line 35). A/3 deposits of varying moφhology were clearly evident as a result of using a variety of A/3 antibodies, including well characterized human-specific Aβ antibodies and antibodies specific for the free amino and carboxy termini of AjS 1-42. Antibody 9204, described by Saido et al, J. Biol. Chem. 289:15253-15257 (1994), is specific to A/3 1-5 and was used at a concentration of 7.0 μg ml'. Antibody 277-2, specific for Aβ 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 μg 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. Serial sections demonstrated many plaques were positively stained with both the 9204 and 277-2 antibodies. The forms of the A/3 deposition ranged from diffuse irregular types to compacted plaques with cores. Roughly spherical, and wispy, irregular deposits, were labelled with antibody 9204 specific for the free amino terminus of A/3. Astrocytic gliosis associated with A/3 deposition was evident after double immunolabelling with antibodies to glial fibrillary acidic protein (GFAP) and human A/3. A compacted A/3 core and 'halo' was evident in several plaques. Non- transgenic littermates showed none of these neuropathological changes. Immunostaining was fully absorbable with the relevant synthetic peptide, and was apparent using a variety of processing conditions, including fixation with paraf ormaldehyde and Trojanowski methods. Many plaques were stained with thioflavin S, and some were also stained using the Bielschowsky silver method and were birefringent with Congo Red, indicating the true amyloid nature of these deposits.

The majority of plaques were intimately surrounded by GFAP-positive reactive astrocytes, similar to the gliosis found in AD plaques. The neocortices of the transgenic mice contained diffusely activated microglial cells, as defined by their amoeboid appearance, shortened processes, and staining with Mac-1 antibody. Staining by antibodies recognizing phosphorylated neurofilaments and phosphorylated tau indicated that aberrant phosphorylation occurred in PDAPP brain that was similar to AD. These phosphorylations are seen in AD and are thought to preclude formation of neurofibrillary tangles. Although 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. Clear evidence for neuritic pathology was apparent using both conventional and confocal immunomicroscopy. Forty μm thick vibratome sections were incubated overnight at 4°C with R1280 (1: 1,000) in combination with polyclonal anti-synaptophysin (1:150; Dako) or 8E5 (7.0 μg 1). Some sections were incubated with anti-synaptophysin or monoclonal anti-MAP 2 (1:20, Boehringer-Mannheim), and then reacted with a goat anti- rabbit biotinylated antibody (1 : 100) followed by a mixmre of FITC- conjugated horse anti-mouse IgG (1:75) and avidin D Texas red (1: 100) (Vector Labs). The double-immunolabelled sections were viewed on a Zeiss Axiovert 35 microscope with attached laser confocal scanning system MRC 600 (Bio-Rad). The Texas red channel collected images of the R1280 or synaptophysm labelling, and the FITC channel collected synaptophysin, 8E5, or MAP 2 labelling. Optical z-sections 0.5 μm in thickness were collected from each region, similar to the image processing and storage described by Masliah et al, J. Neuropath. Exp Neurol. 52:619-632 (1993).

Many Aβ 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)).

Confirmation of the presence of extracellular AS was obtained using immunoelectron microscopy. For irnmunoelectron microscopy, mice were perfused with saline followed by 2.0% paraformaldehyde and 1.0% glutaraldehyde in cacodylate buffer. Forty μm thick vibratome sections were incubated with the R1280 antibody, and reacted using a peroxidase rabbit IgG kit (Vector Labs). Immunolabelled sections with A/3 deposits were then fixed in 1.0% ammonium tetraoxide and embedded in epon/araldite before viewing ultrathin sections with a Jeol CXI 00 electron microscope (Masliah et al, Acta Neuropath. 81:428-433 (1991)). Table 3. Ultrastructural Similarities and Differences Between AD and PDAPP Transgenic Plaques.

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.

Alzheimer's PDAF

Disease

A/3 Deposition into Plaques

Diffuse + +

Neuritic + +

Vascular + +

Brain Region Specificity + +

Neuritic Dystrophy + +

Synaptic Loss + +

Inflammatory Response

Astrocytosis + +

Microgliosis + +

Cytoskeletal Alterations

Phosphorylated Neurofilaments + +

Phosphorylated Tau + +

PHF/Tangles + -(?)

The most notable feature of these transgenic mice is their Alzheimer- like neuropathology, which includes extracellular Aβ 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 Aβ 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. Such 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 substitution. 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.

Another version of the cDNA/genomic clone PDAPP was constructed in which the Swedish mutant at amino acids 670 and 671 was introduced. 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 SΕQ ID NO:5. This results in pNSΕ.deita3'spl.sw/ha, which contains both the Swedish mutation at amino acids 670 and 671, and the Hardy mutation at amino acid 717. The 1448 bp Xhol to Spel fragment of PDAPP was then replaced with the 1448 bp Xhol to Spel fragment of pNSE752.deita3'spl.sw/ha, which contains both the Swedish mutation and the Hardy mutation, to form PDAPP-Sw/Ha. A schematic of PDAPP-Sw/Ha and its construction is shown in Figure 13.

C. Construction of PDAPP695.,.

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 N-terminal portion of the APP sequences (on a 911 bp Xhol to Nrul fragment of APP695 cDNA). 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 heφes simples virus replication and packaging sequences of pohCK751 were removed, and the plasmid encodes APP695 instead of APP751. In this vector 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. D. Construction of PDAPP751...

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 cDNA sequences (nucleotides 57 to 1084 of SEQ ID NO: 3) to make the intermediate plasmid PDAPPδsp75 . 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 heφes simplex viral sequences were removed.

To introduce splicing sequences, the first intron from PDAPP, which is intron Δ6, is then inserted into PDAPPδsp751„ to make PDAPP75 ,. To accomplish this, the 2,758 bp Aspll8 to Seal fragment of PDAPP containing exons 2 through 6, intron Δ6, and part of exon 7, is ligated to the 6,736 bp fragment obtained by complete digestion of PDAPPδsp751»_F t Aspl\8 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 PDAPP75L,.

In this vector 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.

E. Construction of PDAPP770„.

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 PDAPP75 , 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 refeπed to as PDAPP770„.

In this vector the PDGF-B promoter drives the expression of APP770 containing the mutation of Val717 to Phe. PDAPP770„ contains the same intron sequences present in PDAPP751_vf. 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 +APPα; a mixmre of APPα 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. In contrast, guanidine extractable levels of A/3 showed remarkable age-dependent increases in a manner that mirrored the amyloid plaque deposition observed immunohistochemically. Specifically, 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. At 1 year of age A/3 constitutes approximately 1% of the total protein in hippocampus. The cerebral cortex also showed large increases in A/3 with age. In contrast, the mean level of A/3 in cerebellum across all age groups was comparatively low and unchanging.

Further analysis of the A/3 in these brains using an ELISA specific for A/3,, showed that this longer version made up 27% of the 19 pmoles/g of the A/3 present in the brains of young animals; this percentage increased to 97% of the 690 pmoles/g in 12 month old animals. The selective deposition of A/3,, and the spacial distribution of the A/3 deposits are further evidence that the pathological processes ongoing in the PDAPP transgenic mice parallel the human Alzheimer's diseased condition.

Levels of A/3-containing proteins were measured through the use of ELISAs configured with antibodies specific to A/3, A/3,., APP cleaved at the j3-secretase site (Seubert et al. (1993)), and APP containing the first 12 amino acids of A/3 (FLAPP -HAPPα; 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 results also show that, because of the magnitude and temporal predictability of A/3 deposition, the PDAPP mouse is a practical model in which to test agents that either inhibit the processing of APP to AjS or retard A/3 amyloidosis. A. Materials and Methods. 1. Brain Tissue Preparation.

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 AjS and APP measures.

To prepare tissue for ELISAs, 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 temperature, then either assayed directly or stored at -20 °C prior to quantitation of AjS and APP. Preliminary experiments showed the analytes were stable to this storage condition. 2. Aβ 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 μg/ml aprotinin, 5 mM EDTA pH 8.0, 10 μg/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 AS 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).

The "total" Aβ sandwich ELISA consists of two monoclonal antibodies (mAb) to A/3. The capture antibody, 266, is specific to amino acids 13-28 of AjS (Seubert et al. (1992)); while the antibody 3D6, which is specific to amino acids 1-5 of A/3, 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 AjS 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 A/3,.-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 Aβ„ 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 μg/ml into 96-well immunoassay plates (Costar) overnight at room temperature. The plates were then aspirated and blocked with 0.25% human semm albumin in PBS buffer for at least 1 hour at room temperature, 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 temperature 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.

The biotinylated 3D6, diluted to 0.5 μg/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 temperature. Avidin-HRP (Vector, Burlingame, CA), diluted 1 :4000 in casein assay buffer, was added to the wells and incubated for 1 hour at room temperature. The colorimetric substrate (100 μl), Slow TMB-ELISA (Pierce), was added and allowed to react for 15 minutes, after which the enzymatic reaction is stopped with 25 μl of 2 N HSO.. Reaction product was quantified using a Molecular Devices Vmax measuring the difference in absorbance at 450 nm and 650 nm. 3. APP ELISAs.

Two different APP assays were utilized. The first recognizes APPo; and full length forms of APP (FLAPP +APPα), while the second recognizes APPjS (APP ending at the methionine preceding the A/3 domain (Seubert et al. (1993)). The capture antibody for both the FLAPP +APPα 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 +APPα; assay was generated against amino acids 1-12 of Aβ. The lower limit of sensitivity for the 8E5/2H3 assay is approximately 11 ng/ml (150 pM). For the APPjS assay, 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).

For both APP assays, the 8E5 mAb was coated onto 96-well Costar plates as described above for 266. Purified recombinant secreted APPα (the AP 7 1 form) and APP596 were the reference standards used for the FLAPP + APPα 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 NaCI, 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 APP standards and samples were added to the plate and incubated for 1.5 hours at room temperature. The plates were thoroughly washed between each step of the assay with wash buffer. Reporter antibodies 2H3 and 192 were biotinylated following the same procedure as for 3D6 and were incubated with samples for 1 hour at room temperature. Streptavidin- alkaline phosphatase (Boehringer Mannheim), diluted 1:1000 in specimen diluent, was incubated in the wells for 1 hour at room temperature. The fluorescent substrate 4-methyl-umbellipheryl-phosphate, was added, and the plates read on a Cytofluor™ 2350 (Millipore) at 365 nm excitation and 450 nm emission. 4. Monoclonal Antibody Production.

The immunogens for 3D6, 21F12, and 2H3 were separately conjugated to sheep anti-mouse immunoglobulin (Jackson Immunoresearch Labs) using maleimidohexanoyl-N-hydroxysucciriimide (Pierce). A/J mice (Jackson Laboratories) were given intraperitoneal injections (IP) of 100 μg of the appropriate immunogen emulsified in Freund's complete adjuvant (Sigma) and two subsequent IP injections of 100 μg immunogen were given on a biweekly basis in Freund's incomplete adjuvant (Sigma). Two to three weeks after the third boost, the highest titer mouse of a given immunogen was injected intravenously and intraperitoneally with 50-100 μg of immunogen in PBS. Three days post injection, the spleen was removed, splenocytes were isolated and fused with SP2/0-Agl4 mouse myeloma cells. The hybridoma supernatants were screened for high affinity monoclonal antibodies by RIA as previously described (Seubert et al. (1992)). Purified monoclonal antibodies were prepared from ascites.

5. Immunohistochemistry.

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 μm 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 chromagen.

B. Results.

1. Aβ and APP Assays. The FLAPP -hAPPα assay recognizes secreted APP including the first 12 amino acids of A/3. Since the reporter antibody (2H3,„) 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+APPG! is full length. The APPjS assay recognizes only the APP clipped immediately amino-terminal to the A/3 region due to the specificity of the polyclonal reporter antibody 192 (Seubert et al (1993)). The specific nature 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/3 assay. Comparisons were made of 2, 4, and 12 month transgenic mouse brain homogenates and a non-transgenic mouse brain homogenate to which A/3,., 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,.. Attempts were then made to immunodeplete the A/3 immunoreactivity using resin bound antibodies against AjS (mAb 266 against A/3,,,), the secreted forms of APP (mAb 8E5 against APP_ of the APP695 form), the carboxy-terminus of APP (mAb 13G8 against APP_ of the APP695 form), or heparin agarose. Only the 266 resin captured A/3 immunoreactivity, demonstrating that full length APP or carboxy-terminal fragments of APP are not contributing to the Aβ measurement. The A/3,. ELISA employs a capture antibody that recognizes A/3,, but not A/3,.. The A_/3,, assay, like the total A/3 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. 2. Total A3 and APP Measures. Table 5 shows the levels of total A/3, FLAPP +APPα, 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 +APPα and APP/3 in all four brain regions remain relatively constant over time. The hippocampus expresses the highest levels of FLAPP - -APPα and APP/3 followed by the thalamus, cortex. and cerebellum, respectively. In the hippocampus, the levels of

FLAPP -t-APPα are approximately 3.5 to 4.5-fold higher than APP/3 at all ages. The mean value of all ages for FLAPP + APPα 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% APPα, 30% full length APP, and 20% APP/3.

TABLE 5. PDAPP Transgene Cohort Animal Data Total AjS & APP Measures in pmoles/gram of Brain Tissue.

AGE IN Aβ & APP CEREBELLUM HIPPOCAMPUS CORTEX THALAMUS MONTHS FORM

2 AjS 4.03 ±1.08 35.41 ±6.38 14.25 6.41 ±1.59 (n=8) (n=8) ±2.27 (n=8) (n=8)

2 FLAPP+ ND ND ND ND APPα

2 APP/S ND ND ND ND

4 Aβ 4.10 ±0.61 38.08 ±6.51 15.95 7.60 ±1.52 (n=14) (n=14) ±2.60 (n=14) (n=14)

4 FLAPP+ 395 ±120 703 ±106 446 ±70 6.37 ±166 APPα (n=14) (n = 14) (n=14) (n = 14)

4 APPjS 78 ±38 (n=14) 198 ±30 (n-14) 126 ±23 70 ±17 (n = 14) (n = 14)

6 AjS 4.55 ±1.38 87.48 ±30.33 30.19 8.34 ±2.40 (n = 10) (n = 10) ±8.33 (n = 10) (n=10)

6 FLAPP+ 403 ±77 694 ±107 506 ±97 670 ±156 APPα (n=10) (n = 10) (n=10) (n = 10)

6 APPjS 51 ±87 (n=10) 194 ±35 (n = 10) 129 ±25 56 ±33 (n = 10) (n = 10)

6.5 Aβ 5.42 ±1.08 133.63 ±57.10 33.27 8.83 ±1.19 (n=10) (n=10) ±12.19 (n = 10) (n=10)

6.5 FLAPP+ 346 ±74 580 ±115 436 ±63 553 ±123 APPα (n = 10) (n = 10) (n = 10) (n = 10)

6.5 APPjS 27 ±77 (n = 10) 169 ±41 (n = 10) 108 ±16 58 ±22 (n=10) (n = 10)

7 AjS 4.44 ±0.56 200.77 ±94.68 60.55 8.94 ±1.19 (n=10) (n=10) ±27.13 (n=10) (n = 10) 7 FLAPP+ 378 ±70 656 ±73 (n = 10) 469 ±62 604 ±107 APPα (n = 10) (n=10) (n = 10)

7 APP_/S 56 ±52(n=10) 176 ±27 (n = 10) 101 ±20 56 ±28 (n = 10) (n = 10)

7.5 A/S 5.14 ±1.39 461.35 ±345.95 81.839 10.84 ±5.22 (n=10) (n=10) ±53.00 (n=10) (n = 10)

7.5 FLAPP+ 362 ±54 554 ±77 (n=10) 409 ±44 503 ±80 APPα (n=10) (n=10) (n=10)

7.5 APP/S, 20 ±58 (n=10) 168 ±27 (n=10) 118 ±21 57 ±22 (n=10) (n=10)

8 AjS 4.42 ±0.73 635.52 ±302.45 128.68 10.87 ±3.39 (n=13) (n=13) ±62.80 (n=13) (n=13)

8 FLAPP+ 386 ±52 660 ± 102 494 ±87 672 ±150 APPα (n=13) (n=13) (n = 13) (n=13)

8 APP/J 64 ±77 (n=13) 174 ±27 (π = 13) 102 ±26 57 ±30 (n = 13) (n=13)

8.5 Aβ 5.54 ±1.11 633.11 ±363.14 118.39 13.96 ±7.34 (n=10) (n = 10) ±59.91 (n = 10) (n=10)

8.5 FLAPP+ 439 ±79 764 ±114 558 ±80 750 ±132 APPα (n = 10) (n = 10) (n = 10) (n = 10)

8.5 APPjS 28 ±59(n = 10) 185 ±34 (n = 10) 108 ±42 47 ±28 (n = 10) (n = 10)

9 AjS 5.52 ±1.11 1512.39 254.83 19.46 ±8.99 (n = 10) ±624.286 ± 105.927 (n=10)

(n = 10) (n = 10)

9 FLAPP+ 500 ±112 763 ±125 549 ±78 815 ±167 APPα (n=10) (n=10) (n=10) (n=10)

9 APP_/S 4 ±83 (n = 10) 169 ±25 (n=10) 121 ±32 49 ±26 (n = 10) (n = 10)

10 AjS 4.04 ±1.02 2182.21 343.49 15.46 (n = ll) ±1194.49 ±165.531 ±13.38

(n = ll) (n = ll) (n=ll)

10 FLAPP+ 452 ±130 678 ±93 (n=ll) 491 ±102 693 ±166 APPα (n=ll) (n=ll) (n = ll)

10 APPjS 52 ±32 (n=ll) 159 ±22 (n = ll) 87 ±15 46 ±10 (n = ll) (n=ll)

12 AjS 3.26 ±0.35 4356.23 691.17 18.08 (n=9) ±1666.44 (n=9) ±360.93 ±13.50

(n=9) (n=9)

12 FLAPP+ 385 ±166 638 ±272 444 ±171 708 ±278 APPα (n=10) (n=10) (n = 10) (n = 10) 12 APP_/S 41 ±29 (n = 10) 134 ±47 (n=10) 76 ±31 35 ± 19 (n = 10) (n = 10)

ND = not determined

In contrast to APP levels, 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 Aβ 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, AjS 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). The corresponding increases in A/3 observed in hippocampus are even more impressive, as the 8 month value is 15 times that at 4 months old and increases to 106-fold at 12 months old (4,040 ± 1750 pmoles A/3/g tissue at 12 months). At 12 months of age, A/3 constimtes approximately 1 % of protein in hippocampus of the PDAPP mice.

To see if the dramatic rise in brain A/3 concentration is due to amyloid deposition, we next visualized A/3 deposits immunohistochemically, using the opposite hemisphere of the same mice used for A/3 measurements. Notably, a parallel increase in A/3 plaque burden and AjS level exists. These findings strongly argue that the rise in brain AjS concentration determined by ELISA is due to the age-dependent amyloidosis. 3. AS,. Measures in Transgenic Mouse Brain.

Concentrations of A/3,, 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,. in the cortex of transgenic mice, also increases with age. The ELISA data suggest that A/3,, is preferentially depositing in the transgenic mice, and that the deposits detected by mAb 3D6 immunostaining are primarily A/3,.. Table 6. AS,. Levels in the Cortex of Transgenic Brain. Age (months) A/3,, (pmoles/g) 4 . 4.71

8 75.65 10 247.43 12 614.53

4. Aβ Immunostaining in PDAPP Transgenic Brain.

Transgenic animals with A/3 values representing the mean A/3 value of the age group were used for 3D6 immunostaining. A progression of A/3 deposition is seen in the 4, 8, 10, and 12 months old animals. At four months of age, transgenic brains contained small, rare punctate deposits, 20 μm in diameter, that were only infrequently observed in the hippocampus and frontal and cingulate cortex. By eight months of age, these regions contained a number of thioflavin-positive A/3 aggregates that formed plaques as large as 150 μm in diameter. At ten months of age, many large Aβ deposits were found throughout the frontal and cingulate cortex, and the molecular layers of the hippocampus. The outer molecular layer of the dentate gyms receiving perforant pathway afferents from the entorhinal cortex was clearly delineated by A/3 deposition. This general pattern was more pronounced by heavier A/3 deposition at one year of age. The anatomical localization of AjS deposition is remarkably similar to that seen in Alzheimer's disease. C. Discussion.

AS 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. It is believed that A/3 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. 38:4141-4154 (1995)) including the major α-secretase pathway where cleavage of APP occurs with A/3 (Esch et al) and the amyloidogenic or /3-secretase pathway where cleavage of APP occurs at the N-terminus of A/3 (Seubert et al. (1993)). Further cleavage of APP leads to the constimtive production of A/3 forms including those ending at position 40 (A/3„) or 42 (A/3,.). ELISAs that detect specific APP products arising from these individual pathways in the PDAPP mouse brain allow determination of whether differential processing of APP contributes to the regional or temporal specificity of amyloid formation and deposition.

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/3 levels in these brain regions as measured by ELISA assay. An increase in Aβ 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)). AjS levels in the cerebellum, an unaffected brain region, still are at 4 pmoles/g ~ essentially unchanged relative to the levels at 4 months of age - again correlating with amyloid deposition measured by histological analyses. These results indicate that in aged PDAPP mice, brain Aβ 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.

In the PDAPP mouse, individuals suffering Down's Syndrome, and individuals with certain forms of FAD, oveφroduction of AjS is almost certainly an accelerating factor not only in AjS deposition but in subsequent neuropathology (Citron et al. , Mann et al. , Miller et al. , Archives of Biochem. Biophys. 301:41-52 (1993)). A comparison of the AjS measurements seen in the PDAPP mouse with those reported for AD brain tissue reveals several striking similarities. For example, in the PDAPP mouse, the relative levels of Aβ peptide in hippocampus from young (2 months of age) versus old (10 months of age) mice is nearly a hundred fold. Similar findings were noted by Gravina et al. in comparing control brain tissue relative to that of AD. The rise in brain A/3 levels in the PDAPP mouse is rather pronounced between the ages of six to nine months of age. Again, this timecourse parallels, in an accelerated manner, that seen in Down's Syndrome brain tissue, where amyloid deposition begins at approximately 30 years of age and increases substantially until approximately age 60 (Mann).

In summary, the above results show that a reproducible increase in measurable Aβ occurs in the brain tissue of the PDAPP mice and that this increase correlates with the severity of amyloid deposition. These findings indicate that these mice can be used to identify agents or compounds that pharmacologically reduce A/3 peptide production or affect its deposition. 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. 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. For the Y maze task, 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.

For the radial arm maze task, the mouse is placed at the center of a maze with multiple arms radiating from the center. In the testing described below, 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 (HP) 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

(intramaze cues; black pieces of cardboard - circle, plus, or horizontal lines - located in three quadrants at the top of the wall, which was 38 cm high above water level), and various room cues were visible outside the pool (extramaze cues). The 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 mn 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 . The angle was then increased until the mouse slid off, and the angle was recorded. This was repeated three times each day. The average scores for the three days were calculated for each mouse for the Y maze (0= repeat, 1 = alternate), RAM (number of different arms and time to finish, 10 minute limit), and INP (average of all nine trials). General activity was also rated on the first day of testing. Each mouse was observed in the cage, and picked up and held. A mouse that remained calmly in the hand was scored 1 , with progressively greater activity and reaction to handling scored up to 4.

Following the above tests each day, 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 leam 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.

Results in the RAM show that TG performed significantly worse than nTG across all ages (Group effect: p =0.00006). The time to finish was also significantly different between TG and nTG mice (Group effect: p=0.005). The correlation between the time to finish and the number of arms chosen was small (R= -0.15, p= 0.245 in each group). This suggests that the consistent impairment in the RAM is not accounted for by the increased time to complete the task taken by TG mice. Results in the Y maze were also significantly different for TG and nTG mice (Group effect: p=0.011).

Validation studies performed on non-transgenic mice indicate that the Y maze is a less sensitive measure than the RAM.

Measures of strength (INP) and activity indicate no differences between TG and nTG mice. These are considered very rough measures, with only large differences being detectable. There was, however, a decrease in the activity score for all mice over time (Age effect: p= 0.070). There was a difference in body weight, with TG weighing 8% less than controls (Group effect: p =0.0003), primarily in female TG mice. However, this does not seem to have an effect on the results, as shown by the lack of any difference in strength (see above) or swimming speed (see below) between TG and nTG mice. Results of the hidden platform task, considered here a test of reference memory, show a consistent difference between TG and nTG mice. ANOVA reveals that the effects of transgenic status (Group effect: p=0.00016) and trial blocks (Block effect: p< 0.00001) are significant. The effect of transgenic status on performance is accounted for by slower performance by TG mice across all trial blocks and ages. Analysis of Trial 1 reveals an effect of transgenic status (Group effect: p=0.018), suggesting a difference in performance before learning has occurred. However, an analysis of covariance, with trial 1 as the covariate, still yields a significant deficit in TG mice (p=0.00051). It was also possible that some physical differences between TG and nTG mice, rather than cognitive differences, could have been responsible for some of the performance differences seen in the water maze tasks. However, no significant difference in strength or activity was observed (see above). Another possibility considered was the effect of swimming speed on performance since a slower swimmer with equivalent cognitive ability would take longer to reach the platform. To test this, video tracking was used in the hidden platform task to measure the distance travelled to reach the platform (a measure of the amount of searching done by the mice which is related to cognitive ability), the swimming speed (a measure of physical ability unrelated to cognitive ability), and the amount of time need to find the platform (a measure of the combination of both the distance travelled and the swimming speed). This was done in older and younger mice than reported above, using three trials per block and no pretraining. The time needed to find the platform was significantly different in TG and nTG mice (Group effect: p< 0.0005), with the TG mice taking longer. However, the swimming speed was not significantly different between TG and nTG mice (Group effect: p=0.879). Thus, the difference in time needed to find the platform is likely to be due to a cognitive difference between TG and nTG mice. This is confirmed by measures of the distance travelled to find the platform. The TG mice travelled significantly further than the nTG mice before reaching the platform (Group effect: p < 0.0005). These results indicate that the differences seen between TG and nTG mice in the time to reach the platform in the water maze tasks are due to differences in cognitive ability.

To test whether nTG mice retain a better memory of the platform location than TG mice, a probe trial was given immediately following hidden platform training in which the platform was removed. Video tracking was used to determine the number of crossings of the former platform location made by the mice relative to crossings of non-platform locations. There was a significant difference seen between the relative crossings of TG and nTG mice (Group effect: p=0.006). This is evidence that the nTG mice remember the former location of the platform better than TG mice.

It was also possible that the difference observed between TG and nTG mice in the time needed to reach the platform could have been influenced by differences in perception of the cues or motivational differences. To test this, TG and nTG mice were subjected to visible platform tasks in the water maze. For these tasks, a platform was placed in the pool so that it was visible above the water. Three different platforms were tested, a dark platform 25 mm above the surface (most visible), a gray platform 25 mm above the surface, and a dark platform 5 mm above the surface (both less visible). The results show no difference in the time to find the most visible platform between TG and nTG mice (Group effect: p=0.403). There was not any greater decrease in performance in TG mice when less visible platforms were used, suggesting that their vision was as good as nTG mice. These results indicate that perceptual and motivational differences do not influence the time to reach the platform in the water maze tasks described above.

Performance differences between TG and nTG mice were shown for RAM, Y maze, and water maze cognitive tasks in mice aged 4 to 8 months (2 to 12 months for the water maze). All of these differences indicate, and are consistent with, cognitive deficits in the transgenic mice as a group. The various tasks combined to test working memory and reference memory, both of which are implicated in cognitive impairment observed in Alzheimer's victims.

Modifications and variations of the making and testing of transgenic animal models for testing of Alzheimer's disease will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Athena Neurosciences, Inc. (ii) TITLE OF INVENTION: Method For Identifying Alzheimer's Disease

Therapeutics Using Transgenic Animal Models (iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Patrea L. Pabst

(B) STREET: 2800 One Atlantic Center

1201 West Peachtree Street

(C) CITY: Atlanta

(D) STATE: GA

(E) COUNTRY: USA

(F) ZIP: 30309-3450 (v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

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(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/486,538

(B) FILING DATE: June 7, 1995

(C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Pabst, Patrea L.

(B) REGISTRATION NUMBER: 31,284

(C) REFERENCE/DOCKET NUMBER: ANS100CIP (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (404) -873-8794

(B) TELEFAX: (404) -873-8795

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2085 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1-2085

(D) OTHER INFORMATION: /function= "coding region for APP695." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATG CTG CCC GGT TTG GCA CTG CTC CTG CTG GCC GCC TGG ACG GCT CGG 48 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15

GCG CTG GAG GTA CCC ACT GAT GGT AAT GCT GGC CTG CTG GCT GAA CCC 96 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30

CAG ATT GCC ATG TTC TGT GGC AGA CTG AAC ATG CAC ATG AAT GTC CAG 144 Gin lie Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gin 35 40 45

AAT GGG AAG TGG GAT TCA GAT CCA TCA GGG ACC AAA ACC TGC ATT GAT 192 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys lie Asp 50 55 60

ACC AAG GAA GGC ATC CTG CAG TAT TGC CAA GAA GTC TAC CCT GAA CTG 240 Thr Lys Glu Gly lie Leu Gin Tyr Cys Gin Glu Val Tyr Pro Glu Leu 65 70 75 80

CAG ATC ACC AAT GTG GTA GAA GCC AAC CAA CCA GTG ACC ATC CAG AAC 288 Gin lie Thr Asn Val Val Glu Ala Asn Gin Pro Val Thr lie Gin Asn 85 90 95

TGG TGC AAG CGG GGC CGC AAG CAG TGC AAG ACC CAT CCC CAC TTT GTG 336 Trp Cys Lys Arg Gly Arg Lys Gin Cys Lys Thr His Pro His Phe Val 100 105 110

ATT CCC TAC CGC TGC TTA GTT GGT GAG TTT GTA AGT GAT GCC CTT CTC 384 lie Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125

GTT CCT GAC AAG TGC AAA TTC TTA CAC CAG GAG AGG ATG GAT GTT TGC 432 Val Pro Asp Lys Cys Lys Phe Leu His Gin Glu Arg Met Asp Val Cys 130 135 140

GAA ACT CAT CTT CAC TGG CAC ACC GTC GCC AAA GAG ACA TGC AGT GAG 480 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160

AAG AGT ACC AAC TTG CAT GAC TAC GGC ATG TTG CTG CCC TGC GGA ATT 528 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly lie 165 170 175

GAC AAG TTC CGA GGG GTA GAG TTT GTG TGT TGC CCA CTG GCT GAA GAA 576 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190

AGT GAC AAT GTG GAT TCT GCT GAT GCG GAG GAG GAT GAC TCG GAT GTC 624 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205

TGG TGG GGC GGA GCA GAC ACA GAC TAT GCA GAT GGG AGT GAA GAC AAA 672 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220

GTA GTA GAA GTA GCA GAG GAG GAA GAA GTG GCT GAG GTG GAA GAA GAA 720 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240

GAA GCC GAT GAT GAC GAG GAC GAT GAG GAT GGT GAT GAG GTA GAG GAA 768 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255

GAG GCT GAG GAA CCC TAC GAA GAA GCC ACA GAG AGA ACC ACC AGC ATT 816 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser lie 260 265 270

GCC ACC ACC ACC ACC ACC ACC ACA GAG TCT GTG GAA GAG GTG GTT CGA 864 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285

GTT CCT ACA ACA GCA GCC AGT ACC CCT GAT GCC GTT GAC AAG TAT CTC 912 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300

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

GAG AGG CTT GAG GCC AAG CAC CGA GAG AGA ATG TCC CAG GTC ATG AGA 1008 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gin Val Met Arg 325 330 335

GAA TGG GAA GAG GCA GAA CGT CAA GCA AAG AAC TTG CCT AAA GCT GAT 1056 Glu Trp Glu Glu Ala Glu Arg Gin Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350

AAG AAG GCA GTT ATC CAG CAT TTC CAG GAG AAA GTG GAA TCT TTG GAA 1104 Lys Lys Ala Val lie Gin His Phe Gin Glu Lys Val Glu Ser Leu Glu 355 360 365

CAG GAA GCA GCC AAC GAG AGA CAG CAG CTG GTG GAG ACA CAC ATG GCC 1152 Gin Glu Ala Ala Asn Glu Arg Gin Gin Leu Val Glu Thr His Met Ala 370 375 380

AGA GTG GAA GCC ATG CTC AAT GAC CGC CGC CGC CTG GCC CTG GAG AAC 1200 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400

TAC ATC ACC GCT CTG CAG GCT GTT CCT CCT CGG CCT CGT CAC GTG TTC 1248 Tyr lie Thr Ala Leu Gin Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415

AAT ATG CTA AAG AAG TAT GTC CGC GCA GAA CAG AAG GAC AGA CAG CAC 1296 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gin Lys Asp Arg Gin His 420 425 430

ACC CTA AAG CAT TTC GAG CAT GTG CGC ATG GTG GAT CCC AAG AAA GCC 1344 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445

GCT CAG ATC CGG TCC CAG GTT ATG ACA CAC CTC CGT GTG ATT TAT GAG 1392 Ala Gin lie Arg Ser Gin Val Met Thr His Leu Arg Val lie Tyr Glu 450 455 460

CGC ATG AAT CAG TCT CTC TCC CTG CTC TAC AAC GTG CCT GCA GTG GCC 1440 Arg Met Asn Gin Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480

GAG GAG ATT CAG GAT GAA GTT GAT GAG CTG CTT CAG AAA GAG CAA AAC 1488 Glu Glu lie Gin Asp Glu Val Asp Glu Leu Leu Gin Lys Glu Gin Asn 485 490 495

TAT TCA GAT GAC GTC TTG GCC AAC ATG ATT AGT GAA CCA AGG ATC AGT 1536 Tyr Ser Asp Asp Val Leu Ala Asn Met lie Ser Glu Pro Arg lie Ser 500 505 510

TAC GGA AAC GAT GCT CTC ATG CCA TCT TTG ACC GAA ACG AAA ACC ACC 1584 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525

GTG GAG CTC CTT CCC AGC CTG GAC GAT CTC CAG CCG TGG CAT TCT TTT 1632 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gin 530 535 540

GTG AAT GGA GAG TTC GGG GCT GAC TCT GTG CCA GCC AAC ACA GAA AAC 1680 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560

GAA GTT GAG CCT GTT GAT GCC CGC CCT GCT GCC GAC CGA GGA CTG ACC 1728 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575

ACT CGA CCA GGT TCT GGG TTG ACA AAT ATC AAG ACG GAG GAG ATC TCT 1776 Thr Arg Pro Gly Ser Gly Leu Thr Asn lie Lys Thr Glu Glu lie Ser 580 585 590

GAA GTG AAG ATG GAT GCA GAA TTC CGA CAT GAC TCA GGA TAT GAA GTT 1824 Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 CAT CAT CAA AAA TTG GTG TTC TTT GCA GAA GAT GTG GGT TCA AAC AAA 1872 His His Gin Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620

GGT GCA ATC ATT GGA CTC ATG GTG GGC GGT GTT GTC ATA GCG ACA GTG 1920 Gly Ala He He Gly Leu Met Val Gly Gly Val Val He Ala Thr Val 625 630 635 640

ATC GTC ATC ACC TTG GTG ATG CTG AAG AAG AAA CAG TAC ACA TCC ATT 1968 He Val He Thr Leu Val Met Leu Lys Lys Lys Gin Tyr Thr Ser He 645 650 655

CAT CAT GGT GTG GTG GAG GTT GAC GCC GCT GTC ACC CCA GAG GAG CGC 2016 His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670

CAC CTG TCC AAG ATG CAG CAG AAC GGC TAC GAA AAT CCA ACC TAC AAG 2064 His Leu Ser Lys Met Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685

TTC TTT GAG CAG ATG CAG AAC 2085

Phe Phe Glu Gin Met Gin Asn 690 695

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 695 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15

Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30

Gin He Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gin 35 40 45

Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys He Asp 50 55 60

Thr Lys Glu Gly He Leu Gin Tyr Cys Gin Glu Val Tyr Pro Glu Leu 65 70 75 80

Gin He Thr Asn Val Val Glu Ala Asn Gin Pro Val Thr He Gin Asn 85 90 95

Trp Cys Lys Arg Gly Arg Lys Gin Cys Lys Thr His Pro His Phe Val 100 105 110

He Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125

Val Pro Asp Lys Cys Lys Phe Leu His Gin Glu Arg Met Asp Val Cys 130 135 140

Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160

Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly He 165 170 175

Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205

Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220

Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240

Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255

Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser He 260 265 270

Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285

Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300

Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gin Lys Ala Lys 305 310 315 320

Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gin Val Met Arg 325 330 335

Glu Trp Glu Glu Ala Glu Arg Gin Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350

Lys Lys Ala Val He Gin His Phe Gin Glu Lys Val Glu Ser Leu Glu 355 360 365

Gin Glu Ala Ala Asn Glu Arg Gin Gin Leu Val Glu Thr His Met Ala 370 375 380

Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400

Tyr He Thr Ala Leu Gin Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415

Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gin Lys Asp Arg Gin His 420 425 430

Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445

Ala Gin He Arg Ser Gin Val Met Thr His Leu Arg Val He Tyr Glu 450 455 460

Arg Met Asn Gin Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480

Glu Glu He Gin Asp Glu Val Asp Glu Leu Leu Gin Lys Glu Gin Asn 485 490 495

Tyr Ser Asp Asp Val Leu Ala Asn Met He Ser Glu Pro Arg He Ser 500 505 510

Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525

Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gin 530 535 540

Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560

Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575

Thr Arg Pro Gly Ser Gly Leu Thr Asn He Lys Thr Glu Glu He Ser 580 585 590

Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605

His His Gin Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620

Gly Ala He He Gly Leu Met Val Gly Gly Val Val He Ala Thr Val 625 630 635 640

He Val He Thr Leu Val Met Leu Lys Lys Lys Gin Tyr Thr Ser He 645 650 655

His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670

His Leu Ser Lys Met Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685

Phe Phe Glu Gin Met Gin Asn 690 695

(2) INFORMATION FOR SEQ ID NO:3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2253 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1-2253

(D) OTHER INFORMATION: /function= "coding region for APP751." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

CAG ATT GCC ATG TTC TGT GGC AGA CTG AAC ATG CAC ATG AAT GTC CAG 144 Gin He Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gin 35 40 45

AAT GGG AAG TGG GAT TCA GAT CCA TCA GGG ACC AAA ACC TGC ATT GAT 192 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys He Asp 50 55 60

ACC AAG GAA GGC ATC CTG CAG TAT TGC CAA GAA GTC TAC CCT GAA CTG 240 Thr Lys Glu Gly He Leu Gin Tyr Cys Gin Glu Val Tyr Pro Glu Leu 65 70 75 80

CAG ATC ACC AAT GTG GTA GAA GCC AAC CAA CCA GTG ACC ATC CAG AAC 288 Gin He Thr Asn Val Val Glu Ala Asn Gin Pro Val Thr He Gin Asn 85 90 95 TGG TGC AAG CGG GGC CGC AAG CAG TGC AAG ACC CAT CCC CAC TTT GTG 336 Trp Cys Lys Arg Gly Arg Lys Gin Cys Lys Thr His Pro His Phe Val 100 105 110

ATT CCC TAC CGC TGC TTA GTT GGT GAG TTT GTA AGT GAT GCC CTT CTC 384 He Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125

AAG AGT ACC AAC TTG CAT GAC TAC GGC ATG TTG CTG CCC TGC GGA ATT 528 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly He 165 170 175

GAG GCT GAG GAA CCC TAC GAA GAA GCC ACA GAG AGA ACC ACC AGC ATT 816 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser He 260 265 270

GAG GTG TGC TCT GAA CAA GCC GAG ACG GGG CCG TGC CGA GCA ATG ATC 912 Glu Val Cys Ser Glu Gin Ala Glu Thr Gly Pro Cys Arg Ala Met He 290 295 300

TCC CGC TGG TAC TTT GAT GTG ACT GAA GGG AAG TGT GCC CCA TTC TTT 960 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320

TAC GGC GGA TGT GGC GGC AAC CGG AAC AAC TTT GAC ACA GAA GAG TAC 1008 Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335

TGC ATG GCC GTG TGT GGC AGC GCC ATT CCT ACA ACA GCA GCC AGT ACC 1056 Cys Met Ala Val Cys Gly Ser Ala He Pro Thr Thr Ala Ala Ser Thr 340 345 350

CCT GAT GCC GTT GAC AAG TAT CTC GAG ACA CCT GGG GAT GAG AAT GAA 1104 Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu 355 360 365 CAT GCC CAT TTC CAG AAA GCC AAA GAG AGG CTT GAG GCC AAG CAC CGA 1152 His Ala His Phe Gin Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg 370 375 380

GAG AGA ATG TCC CAG GTC ATG AGA GAA TGG GAA GAG GCA GAA CGT CAA 1200 Glu Arg Met Ser Gin Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gin 385 390 395 400

GCA AAG AAC TTG CCT AAA GCT GAT AAG AAG GCA GTT ATC CAG CAT TTC 1248 Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val He Gin His Phe 405 410 415

CAG GAG AAA GTG GAA TCT TTG GAA CAG GAA GCA GCC AAC GAG AGA CAG 1296 Gin Glu Lys Val Glu Ser Leu Glu Gin Glu Ala Ala Asn Glu Arg Gin 420 425 430

CAG CTG GTG GAG ACA CAC ATG GCC AGA GTG GAA GCC ATG CTC AAT GAC 1344 Gin Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp 435 440 445

CGC CGC CGC CTG GCC CTG GAG AAC TAC ATC ACC GCT CTG CAG GCT GTT 1392 Arg Arg Arg Leu Ala Leu Glu Asn Tyr He Thr Ala Leu Gin Ala Val 450 455 460

CCT CCT CGG CCT CGT CAC GTG TTC AAT ATG CTA AAG AAG TAT GTC CGC 1440 Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg 465 470 475 480

GCA GAA CAG AAG GAC AGA CAG CAC ACC CTA AAG CAT TTC GAG CAT GTG 1488 Ala Glu Gin Lys Asp Arg Gin His Thr Leu Lys His Phe Glu His Val 485 490 495

CGC ATG GTG GAT CCC AAG AAA GCC GCT CAG ATC CGG TCC CAG GTT ATG 1536 Arg Met Val Asp Pro Lys Lys Ala Ala Gin He Arg Ser Gin Val Met 500 505 510

ACA CAC CTC CGT GTG ATT TAT GAG CGC ATG AAT CAG TCT CTC TCC CTG 1584 Thr His Leu Arg Val He Tyr Glu Arg Met Asn Gin Ser Leu Ser Leu 515 520 525

CTC TAC AAC GTG CCT GCA GTG GCC GAG GAG ATT CAG GAT GAA GTT GAT 1632 Leu Tyr Asn Val Pro Ala Val Ala Glu Glu He Gin Asp Glu Val Asp 530 535 540

GAG CTG CTT CAG AAA GAG CAA AAC TAT TCA GAT GAC GTC TTG GCC AAC 1680 Glu Leu Leu Gin Lys Glu Gin Asn Tyr Ser Asp Asp Val Leu Ala Asn 545 550 555 560

ATG ATT AGT GAA CCA AGG ATC AGT TAC GGA AAC GAT GCT CTC ATG CCA 1728 Met He Ser Glu Pro Arg He Ser Tyr Gly Asn Asp Ala Leu Met Pro 565 570 575

TCT TTG ACC GAA ACG AAA ACC ACC GTG GAG CTC CTT CCC GTG AAT GGA 1776 Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly 580 585 590

GAG TTC AGC CTG GAC GAT CTC CAG CCG TGG CAT TCT TTT GGG GCT GAC 1824 Glu Phe Ser Leu Asp Asp Leu Gin Pro Trp His Ser Phe Gly Ala Asp 595 600 605

TCT GTG CCA GCC AAC ACA GAA AAC GAA GTT GAG CCT GTT GAT GCC CGC 1872 Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg 610 615 620

CCT GCT GCC GAC CGA GGA CTG ACC ACT CGA CCA GGT TCT GGG TTG ACA 1920 Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr 625 630 635 640 AAT ATC AAG ACG GAG GAG ATC TCT GAA GTG AAG ATG GAT GCA GAA TTC 1968 Asn He Lys Thr Glu Glu He Ser Glu Val Lys Met Asp Ala Glu Phe 645 650 655

CGA CAT GAC TCA GGA TAT GAA GTT CAT CAT CAA AAA TTG GTG TTC TTT 2016 Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu Val Phe Phe 660 665 670

GCA GAA GAT GTG GGT TCA AAC AAA GGT GCA ATC ATT GGA CTC ATG GTG 2064 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He Gly Leu Met Val 675 680 685

GGC GGT GTT GTC ATA GCG ACA GTG ATC GTC ATC ACC TTG GTG ATG CTG 2112 Gly Gly Val Val He Ala Thr Val He Val He Thr Leu Val Met Leu 690 695 700

AAG AAG AAA CAG TAC ACA TCC ATT CAT CAT GGT GTG GTG GAG GTT GAC 2160 Lys Lys Lys Gin Tyr Thr Ser He His His Gly Val Val Glu Val Asp 705 710 715 720

GCC GCT GTC ACC CCA GAG GAG CGC CAC CTG TCC AAG ATG CAG CAG AAC 2208 Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gin Gin Asn 725 730 735

GGC TAC GAA AAT CCA ACC TAC AAG TTC TTT GAG CAG ATG CAG AAC 2253

Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin Met Gin Asn 740 745 750

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 751 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15

Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30

Gin He Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gin 35 40 45

Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys He Asp 50 55 60

Thr Lys Glu Gly He Leu Gin Tyr Cys Gin Glu Val Tyr Pro Glu Leu 65 70 75 80

Gin He Thr Asn Val Val Glu Ala Asn Gin Pro Val Thr He Gin Asn 85 90 95

Trp Cys Lys Arg Gly Arg Lys Gin Cys Lys Thr His Pro His Phe Val 100 105 110

He Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125

Val Pro Asp Lys Cys Lys Phe Leu His Gin Glu Arg Met Asp Val Cys 130 135 140

Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160

Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly He 165 170 175

Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190

Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205

Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220

Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240

Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255

Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser He 260 265 270

Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285

Glu Val Cys Ser Glu Gin Ala Glu Thr Gly Pro Cys Arg Ala Met He 290 295 300

Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320

Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335

Cys Met Ala Val Cys Gly Ser Ala He Pro Thr Thr Ala Ala Ser Thr 340 345 350

Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu 355 360 365

His Ala His Phe Gin Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg 370 375 380

Glu Arg Met Ser Gin Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gin 385 390 395 400

Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val He Gin His Phe 405 410 415

Gin Glu Lys Val Glu Ser Leu Glu Gin Glu Ala Ala Asn Glu Arg Gin 420 425 430

Gin Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp 435 440 445

Arg Arg Arg Leu Ala Leu Glu Asn Tyr He Thr Ala Leu Gin Ala Val 450 455 460

Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg 465 470 475 480

Ala Glu Gin Lys Asp Arg Gin His Thr Leu Lys His Phe Glu His Val 485 490 495

Arg Met Val Asp Pro Lys Lys Ala Ala Gin He Arg Ser Gin Val Met 500 505 510

Thr His Leu Arg Val He Tyr Glu Arg Met Asn Gin Ser Leu Ser Leu 515 520 525 Leu Tyr Asn Val Pro Ala Val Ala Glu Glu He Gin Asp Glu Val Asp 530 535 540

Glu Leu Leu Gin Lys Glu Gin Asn Tyr Ser Asp Asp Val Leu Ala Asn 545 550 555 560

Met He Ser Glu Pro Arg He Ser Tyr Gly Asn Asp Ala Leu Met Pro 565 570 575

Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly 580 585 590

Glu Phe Ser Leu Asp Asp Leu Gin Pro Trp His Ser Phe Gly Ala Asp 595 600 605

Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg 610 615 620

Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr 625 630 635 640

Asn He Lys Thr Glu Glu He Ser Glu Val Lys Met Asp Ala Glu Phe 645 650 655

Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu Val Phe Phe 660 665 670

Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He Gly Leu Met Val 675 680 685

Gly Gly Val Val He Ala Thr Val He Val He Thr Leu Val Met Leu 690 695 700

Lys Lys Lys Gin Tyr Thr Ser He His His Gly Val Val Glu Val Asp 705 710 715 720

Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gin Gin Asn 725 730 735

Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin Met Gin Asn 740 745 750

(2) INFORMATION FOR SEQ ID NO:5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2310 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1-2310

(D) OTHER INFORMATION: /function= "coding region for APP770." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 :

CAG ATC ACC AAT GTG GTA GAA GCC AAC CAA CCA GTG ACC ATC CAG AAC 288 Gin He Thr Asn Val Val Glu Ala Asn Gin Pro Val Thr He Gin Asn 85 90 95

TAC GGC GGA TGT GGC GGC AAC CGG AAC AAC TTT GAC ACA GAA GAG TAC 1008

Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335

TGC ATG GCC GTG TGT GGC AGC GCC ATG TCC CAA AGT TTA CTC AAG ACT 1056

Cys Met Ala Val Cys Gly Ser Ala Met Ser Gin Ser Leu Leu Lys Thr 340 345 350

ACC CAG GAA CCT CTT GCC CGA GAT CCT GTT AAA CTT CCT ACA ACA GCA 1104

Thr Gin Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala

355 360 365

GCC AGT ACC CCT GAT GCC GTT GAC AAG TAT CTC GAG ACA CCT GGG GAT 1152

Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380

GAG AAT GAA CAT GCC CAT TTC CAG AAA GCC AAA GAG AGG CTT GAG GCC 1200

Glu Asn Glu His Ala His Phe Gin Lys Ala Lys Glu Arg Leu Glu Ala 385 390 395 400

AAG CAC CGA GAG AGA ATG TCC CAG GTC ATG AGA GAA TGG GAA GAG GCA 1248

Lys His Arg Glu Arg Met Ser Gin Val Met Arg Glu Trp Glu Glu Ala 405 410 415

GAA CGT CAA GCA AAG AAC TTG CCT AAA GCT GAT AAG AAG GCA GTT ATC 1296

Glu Arg Gin Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val He 420 425 430

CAG CAT TTC CAG GAG AAA GTG GAA TCT TTG GAA CAG GAA GCA GCC AAC 1344

Gin His Phe Gin Glu Lys Val Glu Ser Leu Glu Gin Glu Ala Ala Asn

435 440 445

GAG AGA CAG CAG CTG GTG GAG ACA CAC ATG GCC AGA GTG GAA GCC ATG 1392

Glu Arg Gin Gin Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460

CTC AAT GAC CGC CGC CGC CTG GCC CTG GAG AAC TAC ATC ACC GCT CTG 1440

Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr He Thr Ala Leu 465 470 475 480

CAG GCT GTT CCT CCT CGG CCT CGT CAC GTG TTC AAT ATG CTA AAG AAG 1488

Gin Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495

TAT GTC CGC GCA GAA CAG AAG GAC AGA CAG CAC ACC CTA AAG CAT TTC 1536

Tyr Val Arg Ala Glu Gin Lys Asp Arg Gin His Thr Leu Lys His Phe 500 505 510

GAG CAT GTG CGC ATG GTG GAT CCC AAG AAA GCC GCT CAG ATC CGG TCC 1584

Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gin He Arg Ser

515 520 525

CAG GTT ATG ACA CAC CTC CGT GTG ATT TAT GAG CGC ATG AAT CAG TCT 1632

Gin Val Met Thr His Leu Arg Val He Tyr Glu Arg Met Asn Gin Ser 530 535 540

CTC TCC CTG CTC TAC AAC GTG CCT GCA GTG GCC GAG GAG ATT CAG GAT 1680

Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu He Gin Asp 545 550 555 560

GAA GTT GAT GAG CTG CTT CAG AAA GAG CAA AAC TAT TCA GAT GAC GTC 1728

Glu Val Asp Glu Leu Leu Gin Lys Glu Gin Asn Tyr Ser Asp Asp Val 565 570 575 TTG GCC AAC ATG ATT AGT GAA CCA AGG ATC AGT TAC GGA AAC GAT GCT 1776 Leu Ala Asn Met He Ser Glu Pro Arg He Ser Tyr Gly Asn Asp Ala 580 585 590

CTC ATG CCA TCT TTG ACC GAA ACG AAA ACC ACC GTG GAG CTC CTT CCC 1824 Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605

GTG AAT GGA GAG TTC AGC CTG GAC GAT CTC CAG CCG TGG CAT TCT TTT 1872 Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gin Pro Trp His Ser Phe 610 615 620

GGG GCT GAC TCT GTG CCA GCC AAC ACA GAA AAC GAA GTT GAG CCT GTT 1920 Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val 625 630 635 640

GAT GCC CGC CCT GCT GCC GAC CGA GGA CTG ACC ACT CGA CCA GGT TCT 1968 Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser 645 650 655

GGG TTG ACA AAT ATC AAG ACG GAG GAG ATC TCT GAA GTG AAG ATG GAT 2016 Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Lys Met Asp 660 665 670

GCA GAA TTC CGA CAT GAC TCA GGA TAT GAA GTT CAT CAT CAA AAA TTG 2064 Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu 675 680 685

GTG TTC TTT GCA GAA GAT GTG GGT TCA AAC AAA GGT GCA ATC ATT GGA 2112 Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He Gly 690 695 700

CTC ATG GTG GGC GGT GTT GTC ATA GCG ACA GTG ATC GTC ATC ACC TTG 2160 Leu Met Val Gly Gly Val Val He Ala Thr Val He Val He Thr Leu 705 710 715 720

GTG ATG CTG AAG AAG AAA CAG TAC ACA TCC ATT CAT CAT GGT GTG GTG 2208 Val Met Leu Lys Lys Lys Gin Tyr Thr Ser He His His Gly Val Val 725 730 735

GAG GTT GAC GCC GCT GTC ACC CCA GAG GAG CGC CAC CTG TCC AAG ATG 2256 Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745 750

CAG CAG AAC GGC TAC GAA AAT CCA ACC TAC AAG TTC TTT GAG CAG ATG 2304 Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin Met

755 760 765

CAG AAC 2310

Gin Asn 770

(2) INFORMATION FOR SEQ ID NO:6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 770 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 :

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15

Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30

Gin He Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gin 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser, Gly Thr Lys Thr Cys He Asp 50 55 60

Thr Lys Glu Gly He Leu Gin Tyr Cys Gin Glu Val Tyr Pro Glu Leu 65 70 75 80

Gin He Thr Asn Val Val Glu Ala Asn Gin Pro Val Thr He Gin Asn 85 90 95

Trp Cys Lys Arg Gly Arg Lys Gin Cys Lys Thr His Pro His Phe Val 100 105 110

He Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125

Val Pro Asp Lys Cys Lys Phe Leu His Gin Glu Arg Met Asp Val Cys 130 135 140

Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160

Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly He 165 170 175

Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190

Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205

Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220

Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240

Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255

Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser He 260 265 270

Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285

Glu Val Cys Ser Glu Gin Ala Glu Thr Gly Pro Cys Arg Ala Met He 290 295 300

Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320

Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335

Cys Met Ala Val Cys Gly Ser Ala Met Ser Gin Ser Leu Leu Lys Thr 340 345 350

Thr Gin Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360 365

Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380

Glu Asn Glu His Ala His Phe Gin Lys Ala Lys Glu Arg Leu Glu Ala 385 390 395 400

Lys His Arg Glu Arg Met Ser Gin Val Met Arg Glu Trp Glu Glu Ala 405 410 415

Glu Arg Gin Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val He 420 425 430

Gin His Phe Gin Glu Lys Val Glu Ser Leu Glu Gin Glu Ala Ala Asn 435 440 445

Glu Arg Gin Gin Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460

Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr He Thr Ala Leu 465 470 475 480

Gin Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495

Tyr Val Arg Ala Glu Gin Lys Asp Arg Gin His Thr Leu Lys His Phe 500 505 510

Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gin He Arg Ser 515 520 525

Gin Val Met Thr His Leu Arg Val He Tyr Glu Arg Met Asn Gin Ser 530 535 540

Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu He Gin Asp 545 550 555 560

Glu Val Asp Glu Leu Leu Gin Lys Glu Gin Asn Tyr Ser Asp Asp Val 565 570 575

Leu Ala Asn Met He Ser Glu Pro Arg He Ser Tyr Gly Asn Asp Ala 580 585 590

Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605

Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gin Pro Trp His Ser Phe 610 615 620

Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val 625 630 635 640

Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser 645 650 655

Gly Leu Thr Asn He Lys Thr Glu Glu He Ser Glu Val Lys Met Asp 660 665 670

Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gin Lys Leu 675 680 685

Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala He He Gly 690 695 700

Leu Met Val Gly Gly Val Val He Ala Thr Val He Val He Thr Leu 705 710 715 720

Val Met Leu Lys Lys Lys Gin Tyr Thr Ser He His His Gly Val Val 725 730 735

Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745 750

Gin Gin Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gin Met 755 760 765 Gin Asn 770

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7 :

CCGATGATGA CGAGGACGAT 20

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

TGAACACGTG ACGAGGCCGA 20

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Phe Arg Val Gly Ser

5

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Asp Ala Glu Phe Arg Gly Gly Cys

5

Claims

We claim:

1. A non-human transgenic mammal wherein the transgenic mammal has a nucleic acid construct stably incorporated into the genome, wherein the construct comprises a promoter for expression of the construct in a mammalian cell and a region encoding the A_/3-containing protein, wherein the promoter is operatively linked to the region, wherein the region comprises DNA encoding the A/3-containing protein, wherein the A/3-containing protein consists of all or a contiguous portion of a protein selected from the group consisting of

APP770, APP770 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, APP751, APP751 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, APP695, and APP695 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671 , 690, 692, and 717, wherein the Aβ-containing protein includes amino acids 672 to 714 of human APP, wherein the promoter mediates expression of the construct such that Aβ„ is expressed at a level of at least 30 nanograms per gram of brain tissue of the mammal when it is two to four months old, Aβ,., is expressed at a level of at least 8.5 nanograms per gram of brain tissue of the mammal when it is two to four months old, APP and APPα combined are expressed at a level of at least 150 picomoles per gram of brain tissue of the mammal when it is two to four months old, APPβ is expressed at a level of at least 40 picomoles per gram of brain tissue of the mammal when it is two to four months old, and/or mRNA encoding the A -containing protein is expressed to a level at least twice that of mRNA encoding the endogenous APP of the transgenic mammal in brain tissue of the mammal when it is two to four months old.

2. The mammal of claim 1 wherein the A 3-containing protein is selected from the group consisting of 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, 671, 690, 692, 717; APP695; 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.

3. The mammal of claim 2 wherein the DNA encoding the A3-containing protein is 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.

4. The mammal of claim 1 wherein the promoter is the human platelet derived growth factor β chain gene promoter.

5. The mammal of claim 1 wherein the region further comprises DNA encoding a second protein, wherein the DNA encoding the A/3-containing protein and the DNA encoding the second protein are operative linked such that the region encodes an Aβ-containing fusion protein comprising a fusion of the Aβ- containing protein and the second protein.

6. The mammal of claim 5 wherein the second protein is a signal peptide.

7. The mammal of claim 1 produced by introduction of the constmct into an embryo, insertion of the embryo into a surrogate mother, and allowing the embryo to develop to term.

8. The mammal of claim 1 wherein the mammal is a rodent.

9. The mammal of claim 1 produced by mating transgenic mammals expressing different constmcts.

10. The mammal of claim 1 wherein the codon encoding amino acid 717 is mutated to encode an amino acid selected from the group consisting of He, Phe, Gly, Tyr, Leu, Ala, Pro, Trp, Met, Ser, Thr, Asn, and Gin.

11. The mammal of claim 10 wherein the codon encoding amino acid 717 is mutated to encode Phe.

12. The mammal of claim 1 wherein the codon encoding amino acid 670 is mutated to encode an amino acid selected from the group consisting of Asn and Glu, or the codon encoding amino acid 670 is deleted, and/or wherein the codon encoding amino acid 671 is mutated to encode an amino acid selected from the group consisting of He, Leu, Tyr, Lys, Glu, Val, and Ala, or the codon encoding amino acid 671 is deleted.

13. The mammal of claim 12 wherein the codon encoding amino acid 670 is mutated to encode Asn, and/or the codon encoding amino acid 671 is mutated to encode Leu or Tyr.

14. The mammal of claim 1 wherein the constmct further comprises an effective amount of at least one intron, wherein the effective amount of at least one intron is located in the region of the constmct encoding a human amyloid precursor protein.

15. The mammal of claim 14 wherein the intron is an APP gene intron.

16. The mammal of claim 1 wherein the promoter mediates expression of the constmct such that A3„ is expressed at a level of at least 30 nanograms per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, A/ is expressed at a level of at least 8.5 nanograms per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, APP and APPα: combined are expressed at a level of at least 150 picomoles per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, APP0 is expressed at a level of at least 40 picomoles per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, and/or mRNA encoding the Aβ-containing protein is expressed to a level at least twice that of mRNA encoding the endogenous APP of the transgenic mammal in hippocampal or cortical brain tissue of the mammal when it is two to four months old.

17. The mammal of claim 1 wherein amyloid plaques that can be stained with Congo Red are present in brain tissue of the mammal.

18. A method for testing compounds for an effect on expression or processing of an A_/3-containing protein comprising a) administering the compound to be tested to a non-human transgenic mammal, or mammalian cells derived from the transgenic mammal, wherein the transgenic mammal has a nucleic acid constmct stably incorporated into the genome, wherein the constmct comprises a promoter for expression of the constmct in a mammalian cell and a region encoding the A/3-containing protein, wherein the promoter is operatively linked to the region, wherein the region comprises DNA encoding the Aβ-containing protein, wherein the A/3-containing protein consists of all or a contiguous portion of a protein selected from the group consisting of

APP770, APP770 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, APP751, APP751 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, APP695, and APP695 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, wherein the Aβ-containing protein includes amino acids 672 to 714 of human APP, wherein the promoter mediates expression of the constmct such that A/ is expressed at a level of at least 30 nanograms per gram of brain tissue of the mammal when it is two to four months old, Aβ,., is expressed at a level of at least 8.5 nanograms per gram of brain tissue of the mammal when it is two to four months old, APP and APPα combined are expressed at a level of at least 150 picomoles per gram of brain tissue of the mammal when it is two to four months old, APPjS is expressed at a level of at least 40 picomoles per gram of brain tissue of the mammal when it is two to four months old, and/or mRNA encoding the A/3-containing protein is expressed to a level at least twice that of mRNA encoding the endogenous APP of the transgenic mammal in brain tissue of the mammal when it is two to four months old; and either bl) measuring the amount of an APP marker or a Aβ marker produced by the transgenic animal, or by mammalian cells derived from the transgenic mammal, and comparing the amount measured to the amount of the APP marker or the Aβ marker produced by a transgenic mammal, or by mammalian cells derived therefrom, to which the compound has not been administered, wherein a change in the amount of the APP marker or the Aβ marker indicates that expression or processing of APP has been altered; or b2) determining the histopathology of an APP marker or a Aβ marker in the transgenic animal, and comparing the histopathology to the histopathology of the APP marker or the Aβ marker in a transgenic mammal to which the compound has not been administered, wherein a change in the histopathology of the APP marker or the Aβ marker indicates that expression or processing of APP has been altered.

19. The method of claim 18 wherein the AjS-containing protein is selected from the group consisting of 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, 671, 690, 692, 717; APP695; 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.

20. The method of claim 19 wherein the DNA encoding the AjS- containing protein is 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.

21. The method of claim 18 wherein the promoter is the human platelet derived growth factor jS chain gene promoter.

22. The method of claim 18 wherein the region further comprises DNA encoding a second protein, wherein the DNA encoding the Aβ-containing protein and the DNA encoding the second protein are operative linked such that the region encodes an AjS-containing fusion protein comprising a fusion of the Aβ- containing protein and the second protein.

23. The method of claim 22 wherein the second protein is a signal peptide.

24. The method of claim 18 wherein the Aβ marker is selected from the group consisting of Aβ., A , Aβ,„, A/5„(pE), AjS,-,, Aβ , A/ ~, Aβ_^, and the APP marker is selected from the group consisting of full length APP, APPα, APP/3, FLAPP + APPα, the last 100 amino acids of APP, and the last 57 to 60 amino acids of APP.

25. The method of claim 18 wherein the APP marker is selected from the group consisting of APP695, APP751, and APP770, and wherein the change in histopathology is a reduction in the amount of APP marker localized in plaques and neuritic tissue.

26. The method of claim 18 wherein the mammals are rodents.

27. The method of claim 18 wherein the codon encoding amino acid 717 is mutated to encode an amino acid selected from the group consisting of He, Phe, Gly, Tyr, Leu, Ala, Pro, Trp, Met, Ser, Thr, Asn, and Gin.

28. The method of claim 27 wherein the codon encoding amino acid 717 is mutated to encode Phe.

29. The method of claim 18 wherein the codon encoding amino acid 670 is mutated to encode an amino acid selected from the group consisting of Asn and Glu, or the codon encoding amino acid 670 is deleted, and/or wherein the codon encoding amino acid 671 is mutated to encode an amino acid selected from the group consisting of He, Leu, Tyr, Lys, Glu, Val, and Ala, or the codon encoding amino acid 671 is deleted.

30. The method of claim 29 wherein the codon encoding amino acid 670 is mutated to encode Asn, and/ or the codon encoding amino acid 671 is mutated to encode Leu or Tyr.

31. The method of claim 18 wherein the constmct further comprises an effective amount of at least one intron, wherein the effective amount of at least one intron is located in the region of the constmct encoding the Aβ-containing protein.

32. The method of claim 31 wherein the intron is an APP gene intron.

33. The method of claim 18 wherein the promoter mediates expression of the constmct such that Aβ. is expressed at a level of at least 30 nanograms per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, AjS,. is expressed at a level of at least 8.5 nanograms per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, APP and APPa combined are expressed at a level of at least 150 picomoles per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, APPjS is expressed at a level of at least 40 picomoles per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, and/or mRNA encoding the A/5-containing protein is expressed to a level at least twice that of mRNA encoding the endogenous APP of the transgenic mammal in hippocampal or cortical brain tissue of the mammal when it is two to four months old.

34. The method of claim 18 wherein amyloid plaques that can be stained with Congo Red are present in brain tissue of the mammal.