JPH11507821A - Methods for identifying therapeutic agents for Alzheimer's disease using transgenic animal models - Google Patents

Abstract

(57) SUMMARY This article describes the construction of a transgenic animal model of human Alzheimer's disease and a method for screening potential Alzheimer's disease therapeutics using this model. This model includes all three forms of the beta amyloid precursor protein (APP) (APP695, APP751, and APP770), as well as naturally occurring mutations (eg, London and Indiana familial Alzheimer's disease (FAD) at amino acid 717). Mutations, putative mutations in the APP gene, and the expression of truncated forms of APP containing the Aβ region, characterized by pathologies similar to those observed in Alzheimer's disease. Animal cells can be isolated from transgenic animals or prepared using the same constructs using standard techniques such as lipofectin or electroporation. The transgenic animal or animal cell has an effect on the amount of APP, β-amyloid peptide, and a number of other markers of Alzheimer's disease in the animal, neuropathology of the animal, as well as changes in Alzheimer's disease as measured by changes in the behavior of the animal. Used for screening for compounds that alter the pathological course.

Description

DETAILED DESCRIPTION OF THE INVENTION Methods for identifying therapeutic agents for Alzheimer's disease using transgenic animal models                                Background of the Invention   Transgenic animal models of Alzheimer's disease Screening for therapeutic agents useful for treating Alzheimer's disease using product models Along with a method for.   Alzheimer's disease (AD) is a degenerative disorder of the brain that affects Alios Alzheimer Thus, for the first time in 1907, a patient who showed a marked decline in cognitive ability and general dementia It was listed after examining one of the persons. (The early story of Alzheimer's Disea se, editor Bick et al. (Raven Press, New York 1987)). This is a problem for the elderly It is the major etiology of baldness. AD patients have difficulty with memory loss and intellectual function Have and become unable to function as normal individuals as they progress. With the lack of intellectual skills, Patients exhibit personality changes, socially ineligible behavior, and schizophrenia (A Guid e to the Understanding of Alzheimer's Disease and Related Disorders, Ed. Jorm (New York University Press, New York 1987). AD is inevitable Lack effective palliative or preventive measures for neurodegeneration in victims and families Fatigue both.   The impact of AD on society and the national economy is profound. By 2000, to the United States The elderly population with dementia in is expected to increase by 41%. For medical systems Estimated $ 40 billion annually in institutional and assisted care for AD patients What you have to offer is expensive (Jorm (1987); Fisher, "Alzheimer 's Disease ", New York Times, August 23, 1989, page D1, edited by Reisberg (The Free Press, New York & London 1983)). These factors affect the effect on AD Means that action must be taken to create a strategic action.   At the macroscopic level, the brain of AD patients is usually small, sometimes less than 1000 g . At the microscopic level, neurofibrillary tangles (N FT), neuritic plaque, and degeneration of neurons . AD patients have neurons in the frontal and temporal regions of the cerebral cortex, pyramidal neurons in the hippocampus Norepinephrine activation in the inner, inner and middle parts of the amygdala, cortex and locus Shows degeneration of sex neurons and neurons of the basal forebrain cholinergic system (Fisher (1983); Alzheimer's Disease and Related Disorders, Research an d Development Editor Kelly (Charles C. Thomas, Springfield, IL. 1984)). Thing In fact, AD is defined by neuropathology of the brain.   AD is 200 μm in diameter in cortex, hippocampus, hippocampus, hippocampal gyrus, and amygdala Up to neurites associated with plaque. One of the main components of neurite plaque Is an amyloid, which is stained with Congo Red (Fisher (1983); Kelly (1 984)). Amyloid plaque stained with Congo Red is extracellular and Pink or rust, and birefringent in polarized light. Plaque is made up of polypeptide fibers. Many are present around blood vessels, reducing blood supply to various neurons in the brain Let it.   Various factors such as genetic predisposition, infectious agents, toxins, metals, and head trauma Deviation has also been suggested as a possible mechanism of AD neuropathy. However, available evidence The evidence strongly indicates that there is a distinct type of genetic predisposition for AD. First, molecular analysis Shows that amyloid precursor protein (APP) Provided evidence of mutations in offspring (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)). What is the predominant form of early AD onset? Additional genes are present on chromosome 14 and chromosome 1 (Rogaev et al., N. ature 376: 775-778 (1995); Levy-Lahad et al., Science 269: 973-977 (1995); Sherr ington et al., Nature 375: 754-760 (1995)). Another locus associated with AD is chromosome 1. 9 and encodes a mutant form of apolipoprotein E (Corder, Science 26 1: 921-923 (1993)).   Amyloid plaques are present in AD patients and individuals with Down's syndrome who have survived to age 40. And abundant. Overexpression of APP in Down's syndrome is Has been recognized as a possible cause of the development of AD in patients with New England J .; Med. 320: 1446-1452 (1989); Mann et al., Neurobiol. Aging 1 0: 397-399 (1989)). Plaques are also present in small, but normal, elderly brains. Also exists. These plaques consist mainly of the amyloid β peptide (A β; 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)) It is also a major component in cerebrovascular amyloid deposition. Amiro Ids are fibrous materials arranged in beta pleated sheets. Aβ is 43 amino acids It is a hydrophobic peptide containing up to the acid. The determination of the amino acid sequence is based on APPcD NA (Kang et al., Nature 325: 733-735 (1987); Goldgaber et al., Science 235: 877-8 80 (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)). Led the awning. Numerous types of APP cDNA have been identified, in which three Also include abundant forms, APP695, APP751, and APP770. This These types are generated by alternate splicing from a single precursor RNA. You. This gene spans over 175 kb with 18 exons (Yoshikai et al. ( 1990)). APP binds the extracellular, transmembrane and cytoplasmic domains Including. Aβ has up to 28 amino acids just outside the hydrophobic transmembrane domain, and It consists of up to 15 residues of this transmembrane domain. Thus, Aβ can be used in the brain as well as in the heart. Cleavage products from APP normally found in other tissues such as kidney, spleen It is. However, Aβ is usually found abundantly in the brain only.   The larger forms of APP (APP751, APP770) are APP695 and APP695. And one or two additional domains. APP751 is APP695 Of all 695 amino acids and an additional 56 amino acids, Has homology to the Kunitz family of protease inhibitors (KPI) ( Tanzi et al. (1988); Weidemann et al., Cell 57: 115-126 (1989); Kitaguchi et al., Natur e 331: 530-532 (1988); Tanzi et al., Nature 329: 156 (1987)). APP770 All 751 amino acids of APP751 plus the neuronal cell surface antigen OX-2 Consisting of an additional 19 amino acid domain homologous to (Weidemann et al. (1989); Kitagu chi et al. (1988)). Unless otherwise noted, the options specified herein The amino acid site is the site as found in APP770. APP695 And the number of amino acids at the equivalent site in APP751 may be OX- 2 and the absence of the KPI domain. By convention, all of APP Amino acid sites of the type are dictated by equivalent sites of the APP770 type. Other notes Unless otherwise noted, the APPs and APP fragments referred to herein All forms (including all forms of Aβ) are based on the human APP amino acid sequence. AP P is post-translationally modified by removal of the leader sequence and addition of sulfate and sugar groups. It is.   Van Broeckhaven et al., Science 248: 1120-1122 (1990) describe two Dutch homes. In a system, the APP gene is used for inherited cerebral hemorrhage with amyloidosis (HCHWA- D) (Levy et al., Science 248: 1124-1128 (1990)). . This is the discovery of a point mutation in the APP coding region in two Dutch patients Confirmed by This mutation occurs at site 22 of Aβ (site 618 of APP695). Or replacing glutamic acid with glutamine at site 693) of APP770 did. In addition, certain families are genetically predisposed to Alzheimer's disease (this disease Is referred to as familial Alzheimer's disease (FAD)), Due to a mutation resulting from an amino acid substitution at site 717 (Goate et al. (1991); Murrel l et al. (1991); Chartier-Harlin et al. (1991)). These mutations share disease with family members Separate, not present in families with late-onset AD. The mutation at amino acid 717 is Increases the production of Aβ1-42 Aβ from PP (Suzuki et al., Science 264: 1 336-1340 (1994)). Other variants are at sites 670 and 671 of the full length protein. (Mullan et al. (1992)). To amino acids 670 and 671 This mutation increases the production of total Aβ from APP (Citron et al., Nature 36 0: 622-674 (1992)).   Although there are no robust animal models for AD studies, older non-human primates In parenchyma and in the walls of certain interstitial and cortical tracts, Aβ It seems to produce lloyd plaque. Elderly primates and dogs are Can be useful, but are expensive to maintain, require long research periods, and The range of affected pathologies can vary greatly.   Spontaneous with enough similarity to AD to be useful as an experimental model There are no mutations in healthy animals. Various models have been proposed, and these are some of the AD-like Symptoms include electrolysis, transplantation of AD brain samples, aluminum chloride, kainate or Phosphorus analogs (Kisner et al., Neurobiol. Aging 7: 287-292 (1 986); Mistry et al., J Med Chem 29: 337-343 (1986)). Flood et al., Proc. Natl. Ac ad. Sci. 88: 3363-3366 (1986) describes four synthetic plasmids homologous to Aβ in mice. We reported the memory loss effect of the peptide. These are all common signs with AD, Because they do not share biochemistry or pathogenesis, etiology or It is unlikely to give much useful information about the treatment.   Several transgenic rodent systems have been created, and these are diverse promoters. Expressing human APP gene or DNA complementary to human APP regulated by You. E. having a human APP promoter linked to E. coli β-galactosidase Transgenic mice (Wirak et al., The EMBO J 10: 289-296 (1991)), and Transgenic mice expressing human APP751 cDNA (Quon et al., N 352: 239-241 (1991)) or a subfragment of Aβ-containing cDNA. Transgenic mice (Wirak et al., Science 253: 323-325 (1991); dhu et al. Biol. Chem. 266: 21331-21334 (1991); Kawabata et al., Nature 354: 47. 6-478 (1991)). The results obtained in different studies were It appears to be dependent on the source of the promoter and the protein coding sequence. For example, Wirak et al., Science 253: 323-325 (1991) describe a form of Aβ that expresses Aβ. In transgenic mice, “A” reactive with antibodies prepared against Aβ Intracellular accumulation of “myloid-like” substances was observed, but other histopathology No significant disease signs were found. That the antibody-reactive substance is intracellular and other The absence of any signs indicates that this particular transgenic animal is not associated with Alzheimer's disease. It suggests that it is not a real model system. Subsequent studies showed that similar staining was This was shown in transgenic control mice, Wirak et al., Science 25. 3: 323-325 (1991) was partially commented in a comment on Science 255: 143-145 (1992). Was withdrawn. Thus, the staining seen by Wirak et al. Appears to be an artifact. You.   Kawabata et al. (1991) reported amyloid plaques in transgenic animals. , Neurofibrillary tangles, and neuronal cell death. Each of these studies , Aβ or an Aβ-containing fragment was expressed. Wirak et al. (1991) Kawabata et al. (1991) used the human thy- One promoter was used. However, Kawabata et al. (1991) later developed Kawabata et al. Withdrawn by Nature 356: 23 (1992) and Kawabata et al., Nature 356: 265 (1992) Was done. APP from the neuron-specific enolase promoter of Quon et al. (1991). In transgenic mice expressing the 751 cDNA, Rare small molecule extracellular deposits reactive with the prepared antibody were observed. This Considering a report describing early transgenic mice in Japan, It does not appear to have a Tuheimer's condition (Marx, Science 255: 1200-1202 ( 1992)).   Expression of APP751 from neuron-specific enolase (NSE) promoter Transgenic mice have recently been described as: McConlogue Neurobiol. Aging 15: S12 (1994), Higgins et al., Ann Neurol. 35: 598-607 (19 95), Mucke et al., Brain Res. 666: 151-167 (1994); Higgins et al., Proc. Natl. Ac ad. Sci. USA 92: 4402-4406 (1995), and U. S. Patent 5,387,742 to Cordell . Higgins et al., Ann Neurol. 35: 598-607 (1995) was described in Quon et al. (1991). The results from the same mice are described. Such mice have very early AD and Have very little Aβ deposition, which is more typical of cases of juvenile Down's syndrome . The deposition seen in this transgenic mouse, albeit at a lower level, was It was also found in transgenic control animals. More advanced obstacles For example, high frequency of concentrated plaque, neurite dystrophy, and marked gliosis No disease is seen in these mice (Higgins et al., Ann Neurol. 35: 598-607 (1 995)). McConlogue et al. (1994) found no Aβ deposits in these mice. Reported.   APP is a neuron-specific synaptophysin promoter The transgenic mouse expressed from the NSEAPP mouse was It expresses APP at low levels equivalent to that in brain tissue. These mice also It was reported that it did not show any brain damage (Higgins et al.).   Transgenic mice containing the yeast artificial chromosome (YAC) APP construct are also (Pearson and Choi, Proc. Natl. Acad. Sci. USA 90: 105 78-10582 (1993); Lamb et al., Nature Genetics 5: 22-30 (1993); Buxbaum et al., Bioc. hem. Biophys. Res. Comm. 197: 639-645 (1993)). These mice are human APP It contains the entire genomic gene and converts human APP protein to a level similar to endogenous APP. Higher than those obtained in mice using the NSE promoter. Bell expression. However, none of these mice showed evidence of a condition similar to AD. No proof.   Alzheimer's disease animal models, including transgenic models, have recently been Lannfelt et al., Behavioral Brain Res. 57: 207-213 (1993), and fukuchi et al., A nn. N. Y. Acad. Sci. 695: 217-223 (1993). Lannfelt et al. , Any of the conventional transgenic animals showing apparent plaque Point out that it does not show the characteristic neuropathological changes. Lannfelt and colleagues also We discuss possible reasons for “failure” in upcoming transgenic animal models. Similarly, Fukuchi et al. Reported that traditional transgenic animal models were associated with AD. Does not exhibit most of the known features. For example, Quon et al. Transgenic mice are reported to produce Aβ immunoreactive deposits, It is very rarely stained with Thioflavin S, never stained with Congo Red, In contrast to the staining pattern of ADAβ deposition.   Alzheimer's disease is caused by many changes in the expression levels of various proteins, Characterized by biochemical activity and histopathology, and recognizable changes in the affected individual Be charged. Such characteristic changes associated with AD are well described in the literature. ing. As noted above, the most striking change is the precipitation of Aβ into amyloid plaques. (Haass and Selkoe, Cell 75: 1039-1042 (1993)). Plaque , Apolipoprotein E, laminin, amyloid P component, and collagen IV There are a variety of other molecules such as types (Kalaria and Perry, Brain Research 6 31: 151-155 (1993); Ueda et al., Proc. Natl. Aca. Sci. USA 90: 11282-11286 (199 3)). Microtubule-associated protein such as tau, MAP-2 or neurofilament, Alterations in cytoskeletal markers are also associated with AD (Kosik et al., Science 256: 78). 0-783 (1992); Lovestone and Anderton, Current Opinion in Neurology & N eurosurgery 5: 883-888 (1992); Brandan and Inestrosa, General Pharmacol ogy 24: 1063-1068 (1993); Trojanowski et al., Brain Pathology 3: 45-54 (1993); M asliah et al., American Journal of Pathology 142: 871-882 (1993)). Alzhai Myr's disease also stimulates the immune inflammatory response, causing collagen fibrillary acidic protein (GFAP) , Α2-macroglobulin, and interleukins 1 and 6 (IL-1 and And to increase inflammatory markers such as IL-6) (Frederickson  And Brunden, Alzheimer Disease and Associated Disorders 8: 159-165 (19 94); McGeer et al., Canadian Journal of Neurological Sciences 18: 376-379 (199 1); Wood et al., Brain Research 629: 245-252 (1993)). Finally, cholinergic, Muscarinic, serotonergic, adrenergic, and adenosine Neurons and neurotransmitters such as the adensosine receptor system Changes are associated with AD (Rylett et al., Brain Res 289: 169-175 (1983); Sims et al., Lancet 1: 333-336 (1980); Nitsch et al., Science 258: 304-307 (1992); M asliah and Terry, Clinical Neuroscience 1: 192-198 (1993); Greenamyre And Maragos, Cerebrovascular and Brain Metabolism Reviews 5: 61-94 (199 3); McDonald and Nemeroff, Psychiatric Clinics of North America 14:42 1-422 (1991); Mohr et al., Journal of Psychiatry & Neuroscience 19: 17-23 (1994 )).   Accordingly, an object of the present invention is to provide an algorithm constructed using transgenic technology. It is to provide an animal model of Zheimer's disease.   It is a further object of the present invention to provide a genetic It is to provide a transgenic animal characterized by an abnormality.   A further object of the present invention is to provide one or more sets similar to Alzheimer's disease An object of the present invention is to provide a transgenic animal exhibiting tissue pathology.   It is a further object of the present invention that high levels of one or more A To provide a transgenic animal expressing a β-containing protein .   A further object of the present invention is to use a transgenic animal model, Providing a method of screening for potential drugs for the treatment of Immer's disease It is in.                                Summary of the Invention   Transgenic animals to test potential treatments for Alzheimer's disease The construction of the model is described. This model exists in natural Alzheimer's disease. Β-amyloid precursor characterized by a high similarity to the existing state Three major types of body protein (APP), APP695, APP751, and And the expression of one or more of APP770, or a subfragment thereof, And various point mutations based on natural mutations, such as amino acid 717 Based on the FAD mutation in and the expected mutation in the APP gene. APP The genetic construct harbors the native APP promoter from human, mouse or rat. It is prepared using An effective promoter is the human platelet-derived growth factor β chain (P DGF-B) gene promoter, and water or food to animals such as zinc Mouse metallothionein (metallot) which can be controlled by adding heavy metals hionine) promoters. Construct neurons Specific expression using rat neuron-specific enolase promoter Can be achieved by:   Constructs are prepared using standard techniques, such as microinjection, in animal embryos, Or into embryonic stem cells. Model based on cell culture by two methods Can also be prepared. The cell is a single cell selected from the group consisting of a transgenic animal. Can be isolated or constructed using standard cell transfection techniques Can be prepared from established cell cultures.   The constructs described herein generally comprise three types of APP: APP69. 5, all or a contiguous part of one of APP751 or APP770 And preferably encodes an Aβ-containing protein as described herein. . Examples of Aβ-containing proteins include those containing all or a contiguous portion of: Proteins: APP770, amino acids 669, 670, 671, 69 0, APP770, APP751 having a mutation at 692 and / or 717 , Amino acids 669, 670, 671, 690, 692 and / or 717 APP751, APP695, amino acids 669, 670, 67 APP695 having a mutation at 1,690,692 and / or 717; Here, these Aβ-containing proteins correspond to amino acids 672 of human APP, respectively. To 714. Some of the specific constructs described are Using the nucleotide sequence: APP770 cDNA; amino acids 669, 670, 671, A having the mutation at 690, 692, 717, or a combination of these mutations PP770 cDNA; no OX-2 domain in the construct, KPI protein APP751 cDNA containing zein inhibitor domain; amino acids 669, 670 , 671, 690, 692, 717, or a combination of these mutations APP751 cDNA having: APP695 cDNA; amino acids 669, 67 Mutations at 0, 671, 690, 692, 717, or a combination of these mutations APP695 cDNA having a bias; β-secretase or α-secretase, respectively. Truncated at amino acid 671 or 685, which is the cleavage site for the enzyme ( truncated) APP695, APP751, or APP770 cDNA; AP APPcDN truncated to encode amino acids 646 to 770 of P A: Shortened to encode amino acids 646 to 770 of APP. APP cDNA containing at least one intron; Aβ region (APP amino acid 672-714) and the remaining carboxy terminal 56 amino acids of APP. APP leader sequence; Aβ region and remaining cal with mutation at amino acid 717 APP leader sequence with boxy terminal 56 amino acids; APP with Aβ region Leader sequence; Aβ region (amino acids 672 to 714 of APP) and APP The remaining carboxy terminal 56 amino acids; The remaining carboxy terminal 56 amino acids of APP obtained; combination cDNA / Genomic APP gene construct; amino acids 669, 670, 671, 690, 692 , 717, or a combination of these mutations CDNA / genomic APP gene construct; at amino acid 671 or 685 Truncated combination cDNA / genomic APP gene construct; and Few An APP cDNA construct comprising at least amino acids 672 to 722 of APP.   These protein coding sequences provide for the transport of the encoded Aβ-related protein and And operably linked to a leader sequence that specifies secretion. Preferred leader arrangement Columns are APP leader sequences. These protein coding sequence combinations Promoter for high expression of Aβ in transgenic animal brain tissue Operably linked. A preferred promoter is human platelet-derived growth factor β chain (PDGF-B) gene promoter. Further constructs include: M: human yeast artificial chromosome construct controlled by the PDGF-B promoter; Mutations at amino acids 669, 670, 671, 690, 692, 717; Is controlled by the PDGF-B promoter plus a combination of these mutations. Human yeast artificial chromosome construct; endogenous mouse modified by homologous recombination process Or the rat APP gene, wherein homologous recombination occurs in the mouse or rat embryonic stem ( ES) Cell APP gene and amino acids 669, 670, 671, 690, 69 Human APPcDN with mutations at 2,717, or a combination of these mutations A between the vector carrying A and the resident rodent chromosome APP Recombination point in the gene (preferred site of recombination is within APP exon 9) Sequence beyond amino acids 669, 670, 671, 690, 692, 717 With a similar human sequence that has a mutation in it, or a combination of these mutations Is done. These constructs have been introduced into transgenic animals and have different constructs. Combined by copulation of animals expressing the premises.   The transgenic animal or animal cells are responsible for the pathological processes of Alzheimer's disease Is used to screen for compounds that alter Effects of the compound on the amount of Alzheimer's disease marker and / or histopathology And behavioral changes. These markers are: Including: APP and APP cleavage products; Aβ; apolipoprotein E, laminin And plaque-associated molecules such as collagen type IV; spectrin, tau, nerve Filaments and cytoskeletal markers such as MAP-2; GFAP, α2-ma Inflammatory markers such as cloglobulin, IL-1, and IL-6; and GA Neuronal and synaptic neurotransmitters such as P43 and synaptophysin quality Related markers and cholinergic, muscarinic, serotonergic, Involvement in drainergic and adenososine receptor systems A series of markers.                             BRIEF DESCRIPTION OF THE FIGURES   The boxed portion of the drawing indicates the amino acid coding portion of the construct. Drawing line (fil The led) parts indicate the various domains as shown in the footnotes of the drawing. The line is 5 ' Or a 3 'untranslated sequence, flanking genomic sequence, or intron, The sequence in the drawing is shown. The interruption of the left line of the construct in FIGS. Indicates the presence of the A sequence.   FIG. 1a is a schematic of the APP770 cDNA coding sequence.   FIG. 1b shows the APP770 cDNA coding sequence with a mutation at position 717. It is a schematic diagram.   FIG. 2a is a schematic of the APP751 cDNA coding sequence.   FIG. 2b shows the APP751 cDNA coding sequence with a mutation at position 717. It is a schematic diagram.   FIG. 3a is a schematic of the APP695 coding sequence.   FIG. 3b shows the APP695 cDNA coding sequence with a mutation at position 717. It is a schematic diagram.   FIG. 4a is a schematic of the coding sequence of the carboxy-terminal portion of APP.   FIG. 4b shows a copy of the carboxy-terminal portion of APP with a mutation at position 717. FIG.   FIG. 5 is a schematic diagram of the coding sequence of the Aβ portion of APP.   FIG. 6a shows alternative splicing of KPI and OX-2 exons. FIG. 1 is a schematic representation of a combination cDNA / genomic coding sequence, which enables it.   FIG. 6b shows KPI and OX-2 exons with a mutation at position 717. Combination cDNA / geno to enable alternative splicing FIG. 4 is a schematic diagram of a code sequence.   FIG. 7a is a schematic of the YAC coding sequence of human APP.   FIG. 7b shows a schematic of the YAC coding sequence of human APP with a mutation at position 717. It is a schematic diagram.   Figures 8a and 8b show a schematic of the genetic modification of the mouse APP gene by homologous recombination. In a schematic diagram, homologous recombination is performed using mouse APP gene of mouse ES cells and human AP. Vectors carrying PCDNA (wild type (FIG. 8a) or FAD mutant type (FIG. 8b)) And is directed to exon 9 of the gene. The consequences of this recombination event As a result, APP exon 9 in the resident mouse chromosomal APP gene Sequences beyond a certain recombination point are replaced by similar human sequences.   FIG. 9 shows a combination cDNA / genomic APP construct, PDAPP. It is a schematic map of a vector.   FIG. 10 shows a vector of the genomic region of APP present in the PDAPP construct. It is a chart. The diagram shows the size of the original introns 6, 7 and 8, and the final intron. Indicates the size of thetron. Intron 6 and in the PDAPP construct The site of the deletion in 8 is also shown.   FIG. 11 shows the construction of the APP splicing cassette and PDAPP vector. 3 is a chart showing the intermediate constructs used.   FIG. 12 shows the production of PDAPP-wt vector and PDAPP-wt vector. 4 is a table showing plasmids used for synthesis.   FIG. 13 shows PDAPP-Sw / Ha vector and PDAPP-Sw / Ha FIG. 2 is a table showing plasmids and intermediate constructs used for preparing vectors.   FIG. 14 shows PDAPP695V-F vector and PDAPP695V-F vector. 2 is a table showing plasmids and intermediate constructs used for the construction of the plasmid.   FIG. 15 shows PDAPP751V-F vector and PDAPP751V-F vector. 2 is a table showing plasmids and intermediate constructs used for the construction of the plasmid.                             Detailed description of the invention   The constructs and transgenic animals and animal cells are prepared according to the methods and materials described below. It was prepared using the ingredients. Source of material   Restriction endonucleases are available from New England Biolabs (Beverly, MA. ), Promega Biological Research Products (Madison, WI. ), And Stratagene (La Joll , CA. ) From common commercial sources. Radioactive materials are Dupont / NEN Or from common commercial sources such as Amersham. Site-specific mutation Custom designed oligonucleotides are available from Bio-Synthesis Inc. , Lewisville, TX, etc. Is available from any of several commercial providers of such materials. Part special Kits for making mutations are available from Promega Biological Research Products and Available from commercial suppliers such as Stratagene. APP mRNA APP6 CDNA clones containing 95, APP751, and APP770 types were obtained from Dr. D Obtained directly from mitry Goldgaber, NIH. DNA libraries are available from Stratagene, La  Jolla, CA. And Clontech, Palo Alto, CA. Available from commercial suppliers such as It is. PC12 and 3T3 cells were obtained from the ATCC (#CRL, respectively. 1721 and # CCL92). An additional PC12 cell line is available from Harvard Medical School Dr. Charles Marotta, Massachusetts General Hospital, and McLean  Obtained from Hospital. Standard cell media suitable for this cell line include Gibco / BRL From common commercial sources. Mouse stem cell, D3 strain is Dr. Rolf Kemler (Doetschman et al., J. et al. Embryol. Exp. Morphol. 87:27 (1985)). DNA Selection of lipofectin and stable transformants for transfection G418 is available from Gibco / BRL. Definition of APP cDNA clone   cDNA clone APP695 was described by Kang et al. (Nature 325: 733-735 (1987)). And the most predominant form of APP in the brain. You. The cDNA clone APP751 was obtained from Ponte et al. (Nature 331: 525-527 (1988)). Is the type described by. This form is 168 to APP695 cDNA. Includes nucleotide inserts. The 168 nucleotide insert is in the KPI domain Code. The cDNA clone APP770 is described in Kitaguchi et al. (Nature 331: 53). 0-532 (1988)). This form is included in the APP695 cDNA. In contrast, it contains an insert of 225 nucleotides. This insert is an APP751cD 168 nucleotides present at the insert of NA and APP751 cDNA Contains an additional 57 nucleotide region not found. Insertion of 225 nucleotides The section encodes a KPI domain and an OX-2 domain. All three types , Generated by alternative splicing from the same precursor RNA. 1 The 68 nucleotide insert contains APP751 cDNA and APP770cDN. A is present in both.   The sequence encoding APP695 is shown in SEQ ID NO: 1. This array contains the start code Starting from the first base of AUG, it encodes a 695 amino acid protein. Arrangement The region from nucleotides 1789 to 1917 of SEQ ID NO: 1 encodes Aβ. A The amino acid sequence of PP695 is shown in SEQ ID NO: 2. Amino acid 59 of SEQ ID NO: 2 7 to 639 form Aβ. The amino acid composition of APP695 is A57, C1 2, D47, E85, F17, G31, H25, I23, K38, L52, M2 1, N28, P31, Q33, R33, S30, T45, V62, W8, Y17 And the calculated molecular weight is 78,644. It becomes 45. These arrays are described in Kang et al. (1988).   The sequence encoding APP751 is shown in SEQ ID NO: 3. This array contains the start code Starting from the first base of AUG, it encodes a 751 amino acid protein. Arrangement Nucleotides 866 to 1033 of SEQ ID NO: 3 are found in the APP695 cDNA. I can't. The region from nucleotides 1957 to 2085 encodes Aβ. AP The amino acid sequence of P751 is shown in SEQ ID NO: 4. Amino acid 289 of SEQ ID NO: 4 To 345 are not found in APP695. This 57 amino acid region is the KPI domain. Including in. Amino acids 653 to 695 of SEQ ID NO: 4 form Aβ. this These sequences were taken from Ponte et al. (1988).   The sequence encoding APP770 is shown in SEQ ID NO: 5. This array contains the start code Starting from the first base of AUG, it encodes a 770 amino acid protein. Arrangement Nucleotides 866 to 1090 of SEQ ID NO: 5 are found in the APP695 cDNA. I can't. Nucleotides 1034 to 1090 of SEQ ID NO: 5 correspond to APP751 Not found in cDNA. The region from nucleotides 2014 to 2142 encodes Aβ. To load. The amino acid sequence of APP770 is shown in SEQ ID NO: 6. SEQ ID NO: 6 Amino acids 289 to 364 are not found in APP695. This 76 amino acid region The region includes the KPI and OX-2 domains. From amino acid 345 of SEQ ID NO: 6 364 is not found in APP751. This 20 amino acid region is the OX-2 domain. Including Amino acids 672 to 714 form Aβ. APP's putative transmembrane The transmembrane region occurs at amino acids 700 to 723. Unless stated otherwise Reference to a nucleotide position herein refers to the numbering of SEQ ID NO: 5. You. This is a numbering derived from the APP770 cDNA. Otherwise Unless otherwise indicated, references to amino acid positions herein are numbered SEQ ID NO: 6. Indicates numbering. This is a numbering derived from APP770. This numbering According to the rules, for example, amino acid 717 is amino acid 717 of APP770, A Refers to amino acid 698 of PP751 and amino acid 642 of APP695. Up The sequence shown was taken from Kang et al. (1988) and Kitaguchi et al. (1988).   Unless noted otherwise, the APPs and APs referred to herein All types of fragments of P (including all types of Aβ) are human APP amino acids Based on the array. For example, Aβ is human Aβ, APP is human APP, APP77 0 refers to human APP770. As used herein, the term cDNA refers to the mRNA Not only the DNA molecule actually prepared by reverse transcription, but also the coding region is interrupted. Any DNA molecule that encodes a protein without DNA molecules having a continuous open reading frame. Follow As used herein, the term cDNA means that the protein coding region has an intron sequence. (Or any other sequence that does not encode a protein) It provides a convenient means of referring to a DNA molecule that encodes a protein. Definition of APP genomic locus   Phage and cosmids of human genomic DNA clones listed in Table 1 below The characterization of the loan is at least 100 kb for the Alzheimer gene. Small size was established for the first time. There are a total of 18 exons in the APP gene ( Lemaire et al., Nocl. Acid Res 17: 517-522 (1989); Yoshikai et al. (1990); Yoshikai et al. Nucleic Acids Res 102: 291-292 (1991)). Yoshikai et al. (1990) reported APP genetics. The sequence of the exon-intron boundary of the offspring is described. Taken together these results, The minimum size of the Alzheimer gene is shown to be 175 kb.   Table 2 shows where 17 introns interrupt the APP coding sequence. Numbered Indicates the nucleotide position of the APP770 cDNA shown in SEQ ID NO: 5 . The starting nucleotide of exon 1 represents the first transcribed nucleotide. This It is negative because the +1 nucleotide is customarily the first nucleotide of the AUG start codon. This is because it is otide (Kang et al. (1988)). The terminal nucleotide of exon 18 is m Represents the last nucleotide present before the poly (A) tail in RNA (Yosh ikai et al. (1990)). Yoshikai et al. (1990) and Yoshikai et al. (1991) position exon 8 Was found to be erroneous. Figure 1 of Yoshikai et al. (1991) The EcoRI fragment between the EcoRI fragment containing exon 7 and exon 8. Including In fact, this intermediate EcoRI fragment is located immediately after exon 8. Due to the location, an EcoRI fragment containing exon 7 and exon 8 The EcoRI fragments are adjacent to each other. APP gene mutation   Certain families are genetically susceptible to Alzheimer's disease and this condition is familial. It is called Alzheimer's disease (FAD), which is at position 717 of the full length protein. Mutations resulting in amino acid substitutions (Goate et al. (1991); Murrell et al. (1991); Chartier -Harlin et al. (1991)). These mutations are co-segregated with disease in the family (co-segregate ). For example, Murrell et al. (1991) described exon 17 (Murrell et al. No. 5), the mutation at position 717 was identified in this mutation. Phosphorus is replaced by phenylalanine.   Other FAD variants have amino acids at positions 670 and 671 of the full-length protein. (Mullan et al. (1992)). In one type of this mutation, lysine at position 670 Is replaced by asparagine and methionine at position 671 is replaced by leucine. Is replaced. The effect of this mutation increases Aβ production in cultured cells by about 7-fold (Citron et al., Nature 360: 672-674 (1992); Lai et al., Science 259: 514-516 (1993)) . Substitution of methionine at position 671 with leucine itself could increase Aβ production. Was shown. Further mutations at amino acids 669, 670 and 671 are It has been shown to reduce the amount of Aβ processed from PP (Citron et al.). , Neuron 14: 661-670 (1995)). The APP construct with Val at amino acid 690 is Produce increased amounts of truncated forms of Aβ.   Amino acids 669, 670, 671, 690, 692 of the full-length protein, or An APP expression clone having the mutation at 717 can be constructed. Amino acids 670 and Mutations from Lys to Asn and Met to Leu at Each is sometimes referred to as a Swedish mutation. Further mutations Acid 669, 670, or 671 which are processed from APP Increase or decrease the amount of Aβ that is shinged. Any APP clone or Mutations at these amino acids in transgenes are created by site-specific mutations. Can be issued (Vincent et al., Genes & Devel. 3: 334-347 (1989)) Once done, it can be introduced into other constructs using standard genetic engineering techniques. amino Some mutations at acid 717 are sometimes referred to as Hardy mutations. That Such mutations include the wild-type Val717 codon at Ile, Phe, Gly, Ty. r, Leu, Ala, Pro, Trp, Met, Ser, Thr, Asn, and May include the conversion of Gln to codons. A preferred substitution for Val717 is Phe It is. These mutations can cause individuals expressing the mutant protein to have Alzheimer's disease. Make it easier to develop. The mutation may affect APP expression and / or processing. It is thought that the balance may be tilted toward Alzheimer's pathology. Ami The mutation at acid 669 is a change from the wild-type Val669 codon to the Trp codon. Or may include codon deletions. The mutation at amino acid 670 is the wild-type Ly Conversion of s670 codon to Asn or Glu codon, or codon deletion May be included. The mutation at amino acid 671 is due to the modification of the wild-type Met671 codon, Le. conversion of u, Val, Lys, Tyr, Glu, or Ile to codons, or It may include deletion of codons. A preferred substitution for Lys670 is Asn, A preferred substitution for t671 is Leu. These mutations result in mutant proteins Make the appearing individual more susceptible to developing Alzheimer's disease. Amino acids 669, 67 Other mutations listed to 0 and 671 are Aβ processed from APP. (Citron et al. (1995)). These mutations are Is thought to affect the processing of Aβ, resulting in altered Aβ production.   Truncated forms of APP can also be expressed from the transgene construct. For example, A APPcD truncated to encode amino acids 646-770 of PP NA. It is truncated to encode amino acids 646-770 of APP. APP cDNA construct, which is operably linked to the PDGF-B promoter. The concatenation is called PDAPPc125. Nucleic acid constructs encoding Aβ-containing proteins   Constructs for use in transgenic animals can be found in mammalian cells. Promoters for expression of the constructs, and three types of APP: APP69 5, all or a contiguous part of one of APP751 or APP770 (Including the specific amino acids described above as described herein) With or without mutation). The encoded protein is Aβ -It preferably encodes the containing protein. As used herein, Aβ-containing The proteins are in three forms of APP: APP695, APP751, or APP7. A protein comprising all or a contiguous portion of one of the 70 (as described herein) With or without the specific amino acid mutations described above) The protein contains all or part of amino acids 672 to 714 of human APP . Preferred Aβ-containing proteins include amino acids 672 to 714 of human APP. Including. Preferred forms of such Aβ-containing proteins include all of the following: Or contiguous parts: APP770, amino acids 669, 670, 671, 6 APP770 having a mutation at 90, 692 and / or 717, APP 751, amino acids 669, 670, 671, 690, 692 and / or 71 APP751, APP695, amino acids 669, 670 with mutations at 7 , 671, 690, 692 and / or 717 95, wherein these Aβ-containing proteins are each amino acids of human APP 672-714.   A preferred form of the Aβ-containing protein described above is: APP77 0; from the group consisting of amino acids 669, 670, 671, 690, 692, 717 Has a mutation in the codon encoding one or more selected amino acids APP770; APP751; amino acids 669, 670, 671, 690; One or more amino acids selected from the group consisting of APP751 having a mutation at the codon to be inserted; APP695; amino acid 66 9, 670, 671, 690, 692, 717 APP695 having mutations in codons encoding amino acids or more A protein consisting of amino acids 646 to 770 of APP; amino acid 6 of APP 70-770; a sequence consisting of amino acids 672-770 of APP. And a protein consisting of amino acids 672 to 714 of APP.   In the constructs disclosed herein, D encoding Aβ-containing protein The NA can be a cDNA or a cDNA / genomic DNA hybrid, where The cDNA / genomic DNA hybrid has at least one APP intron sequence And the intron sequence is sufficient for splicing.   Preferred constructs include: DNA encoding APP770; Codons encoding amino acids 669, 670, 671, 690, 692, 717 Encoding APP770 having the mutations in, or a combination of, these mutations NA; encodes an amino acid sequence containing amino acids 672 to 714 of APP770 A fragment of DNA encoding APP770; encoding APP751 DNA; codes for amino acids 669, 670, 671, 690, 692, 717 APP751 with mutations at different codons, or a combination of these mutations DNA containing amino acids 672 to 714 of APP770 A fragment of DNA encoding APP751 encoding APP695 Encoding DNA; amino acids 669, 670, 671, 690, 692, 717 Having mutations in the codons encoding, or a combination of these mutations A DNA containing amino acids 672 to 714 of APP770. Fragment of DNA encoding APP695 encoding amino acid sequence; AP APPcDN truncated to encode amino acids 646 to 770 of P A: Combination cDNA / genomic DNA hybrid APP gene construct A code encoding amino acids 669, 670, 671, 690, 692, 717; Combination cD having mutations in the mutation, or a combination of these mutations NA / genomic DNA hybrid APP gene construct; or amino acid 67 Combination cDNA / genomic DNA Hybrid APP gene construct.   A preferred type of such a construct is the following: APP770 cDNA A code encoding amino acids 669, 670, 671, 690, 692, 717; APP770 cDNA having a mutation in the mutation, or a combination of these mutations; Encodes an APP amino acid sequence comprising amino acids 672 to 714 of APP770 A fragment of APP770 cDNA; APP751 cDNA; amino acid 66 Mutations at codons encoding 9,670, 671, 690, 692, 717 Or an APP751 cDNA having a combination of these mutations; APP751cDN encoding an amino acid sequence comprising amino acids 672 to 714 Fragment of A; APP695 cDNA; amino acids 669, 670, 671, Mutations in the codons encoding 690, 692, 717, or these mutations APP695 cDNA with the combination of: amino acids 672 to 7 of APP770 A fragment of an APP695 cDNA encoding an amino acid sequence comprising APPcD truncated to encode amino acids 646-770 of PP NA; Combination cDNA / genomic DNA hybrid APP gene construction A product encoding amino acids 669, 670, 671, 690, 692, 717 Combination c with mutations in the don and combinations of these mutations A DNA / genomic DNA hybrid APP gene construct; and amino acid 67 Combination cDNA / genomic DNA Hybrid APP gene construct. Building a transgene   Construction of the various APP transgenes can be performed by any suitable genetic engineering technique, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N. Y. , 1989). Manipulation or mutated Region of the isolated APP clone is a convenient restriction present in the APP cDNA clone It can be exchanged using an enzyme site. The NruI site is located at the first nucleus of the AUG start codon. Begins at the -5 position (relative to nucleotide). The KpnI and Asp718 sites were The shift also starts at position 57 (these are iso-restriction enzymes, leaving different cohesive ends) . The XcmI site starts at position 836 and truncates at position 843. The ScaI site is 1004 Starts with a digit. The XhoI site starts at position 1135. BamHI site at position 1554 Starts with The BglII site starts at position 1994. EcoRI site at position 2020 Starts with The SpeI site starts at position 2583. Another EcoRI site is 3076 Starts with a digit.   Clones having various portions of the human APP gene sequence shown in FIGS. It can be constructed in a general manner using standard genetic engineering techniques. For example, these First, the polyA addition signal from the SV40 virus was The base pair BclI to BamHI fragment (Reddy et al., Science 00: 494-502 (1978) )) By cloning into a modified vector from the pUC series. Can be constructed. Next, the cDNA coding sequence (APP770, APP751, or Or one of APP695). Orientation of inserted fragment And that the content is correct, restriction endonuclease mapping and limited Can be determined by sequencing. Of the human APP gene sequence shown in FIGS. Clones with various carboxy-terminal moieties are a few additional steps above. Can be built with additional steps. For example, an APP cDNA clone (SEQ ID NO: 5) It can be digested with Asp718, which cuts after nucleotide position 57. Arise The 5 'end was filled in using the Klenow enzyme (Sambrook et al., (1989)) and BglII It is linked to the following sequence which is a recognition site: AGATCT hexanucleotide. B After cleavage at glII, which also cuts after position 1994 and religation The translation reading frame is preserved. The shortened tongue thus coded The protein contains a leader sequence, about 6 amino acids preceding Aβ, followed by Aβ, And the 56 terminal amino acids of APP. The clone of FIG. 5 is the clone of FIG. Converts the nucleotide at position 2138 to T by site-directed mutation in Created by creating a stop codon immediately after the last amino acid codon of Aβ It is. The APP cDNA clone naturally contains a NruI site, which Cut two nucleotides upstream from the onin codon. This site has a different promotion Can be used to attach and complete individual constructs.   APP transgenes can also be constructed using PCR cloning techniques. . Such techniques provide for accurate coupling of DNA fragments in the transgene. Enable Compilation cDNA / genomic DNA clone   As a result of endogenous APP expression, alternative mRNAs are transcribed following precursor mRNA transcription. Pricing occurs, producing three major types of APP. This alternative Splicing generates a pattern of APP expression involved in Alzheimer's disease Can be important in doing so. Also, the presence of introns in the expression construct For example, target precursor mRNA to mRNA processing and transport pathways (Huang et al., Nucleic Acids Res. 18: 937-947 (1990)), level of expression And properties can be affected. Therefore, the combination of cDNA and genomic DNA Transgenes containing an intron sequence are a preferred type of construct.   The mechanism of RNA splicing is based on a small number of specific and well-known consensus sequences. Request. Such a sequence is described in APP genomic DNA by Yoshikai et al. 90). The disclosed transgene comprises one or more transgenes. It can be constructed using complete and intact intron sequences. But the transgene A truncated plasmid containing an effective amount of intron sequences to allow splicing It is preferable to construct using an intron sequence. Generally, a splicing donor Sites, splicing acceptor sites, and splicing branch point sequences. The truncated intron sequence it carries constitutes an effective amount of the intron. Any short To determine whether the truncated intron sequence is sufficient, use the following method Test for the presence of correctly spliced mRNA in And can be determined by   Other intron sequences and splicing signals not derived from the APP gene sequence Nulls can also be used in the transgene construct. Such an intron arrangement The columns enhance the expression of the transgene construct. Preferred heterologous introns are Denovirus major late region exon 1 and intron junctions, and IgG It is a hybrid of the variable region splice acceptor. This hybrid in Tron can be prepared as described in Bothwell et al., Cell 24: 625-637 (1981). 162 bp PvuII to HindIII fragment of the major late region of influenza virus (Including 8 bp of the first exon and 145 bp of the first intron); 99 bp HindIII-P of G variable region splice acceptor clone 6 It can be constructed by ligating the stI fragment. Manley et al., Nucleic Acid Res. 18: 937-947, a similar splice signal is It has been shown to enhance expression of the attached construct. Heterogeneous introns are promoted It is preferable to arrange between the promoter and the region encoding APP.   Preferred APP combination cDNA / genomic expression clones are shown in FIG. As described, contains an effective amount of introns 6, 7 and 8. Such a tran The gene can be constructed as follows. A preferred construction method is described in Example 5. It is. The plasmid containing the cDNA portion of the clone was first cloned into APP770cDN The TaqI site at position 860 in the A clone was ligated to the XhoI site by site-directed mutagenesis. It can be constructed by converting to places. Cleavage of the resulting plasmid with XhoI Occurs at a new XhoI site and an already existing XhoI site at position 1135, Releases KPI and OX-2 coding sequences. The plasmid thus created is , KPI and OX-2 alternative splicing cassettes Served as a putter.   Alternative splicing cassettes are a series of clips that involve genomic DNA. It can be created by a roning process. First, exon 6 and the adjacent downstream The TaqI site at position 860 in the genomic clone containing the Can convert to an XhoI site. Cleavage of the resulting plasmid with XhoI Occurs at the new XhoI site and the XhoI site within intron 6 or 7. D This fragment comprising exon 6 and at least a portion of the adjacent intron 6 Can then be cloned into the XhoI site in a plasmid vector. next, A genomic clone containing exon 9 and the adjacent upstream genomic sequence was cloned into XhoI And cloned into the XhoI site at position 1135 (Kang et al. (1987) numbering). At position 910) and intron 7 or 8 at the XhoI site. . This comprises a portion of exon 9 and at least a portion of the adjacent intron 8. The fragment is then cloned into the XhoI site in another plasmid vector I can do it. These two exon / intron junction fragments are then Yes From these plasmid vectors with XhoI and BamHI or BglI And cloned together into an XhoI site in another plasmid vector. I can do it. This exon / intron junction fragment was prepared by BamHI. Is preferably cut out. Before cutting out the BamHI site, Introducing the intron / intron junction fragment into the intron Is also preferred. This is due to the long extra intron sequence from the cDNA / genomic clone. Enables column elimination.   Obtained by cloning two exon / intron junction fragments together The XhoI fragment obtained was used with either enzyme in the above excision step. Depending on whether it is possible to cut with BamHI or BglII, and 5. The genome, which contains the OX-2 and OX-2 coding regions together with its flanking intron sequences. 8 kb A BamHI segment may be inserted. This fragment was prepared by Kitaguchi et al. (1988) By the TaqI-AvaI fragment of 212 bp of APP770 cDNA. (Nucleotides 862 to 1073) as hybridization probes BamHI digested phosphorus from one normal individual and eight Alzheimer's disease patients Identified by Southern blot analysis of papilla DNA. 225 bp insertion area A genomic DNA clone containing, for example, a 212 bp TaqI-AvaI Fragment as a probe can be isolated from a human leukocyte DNA library. Get In nome DNA, the 225 bp sequence is a 168 bp exon (exon 7). And about 57 bp exon (exon 8), about 2. 6 kb intron (int 7) are separated and located. Both exons are intron-exon Flanked by consensus sequences. Exon 7 is nucleotide 8 of APP770 From 66 to 1033, exon 8 corresponds to nucleotides 1034 to 1090 You. Exon 7 is the high domain of the Kunitz-type protease inhibitor family domain. Code the saved area each time.   After cleavage with XhoI, this alternative contains exon and intron sequences. The active splicing cassette is then cut out by cutting with XhoI, The modified APP770 cDNA plasmid constructed above (acceptor plasmid C) into the XhoI site. These cloning steps are Resulting in a transgenic cDNA / genomic expression clone, In the cells in the product, the KPI and the natural alternative splicing mechanism And OX-2 domain inclusion. Amino acids 669, 6 70, 671, 690, 692, 717, or a set of these mutations Similar genes with matching can be constructed directly by in vitro mutation. Ami The mutation in acid 717 can also be performed using the mutant form of APP770 cDNA described above. It can be made by constructing an acceptor plasmid. promoter   Using different promoter sequences, a nucleic acid encoding an Aβ-containing protein It can control the expression of the otide sequence. In transgenic animals, Aβ-containing The ability to regulate the expression of the gene encoding the protein can be attributed to the different A It is useful in assessing the role of the PP gene product. Aβ in cultured cells -The ability to regulate the expression of the gene encoding the containing protein is different for different Aβ- Useful in assessing expression and processing of contained gene products, It can provide the basis for cell culture drug screening. Preferred promoters are Platelet-derived growth factor β (PDGF-B) chain gene promoter (Sasaha ra et al., Cell 4: 217-227 (1991)).   Preferred promoters for the disclosed APP constructs are protein-encoded When operably linked to a sequence, for one of the disclosed APP constructs, One or more of the following expression products, from 2 to 4 month old transgenic animals: It mediates at least a specific level of expression in brain tissue. Product And its expression level, Aβtot is at least 30 ng / g (6 . 8 pmoles / g), and preferably at least 40 ng / g (9 . Levels of Aβ 1-42 are at least 8. 5 ng (1. 82 pmoles / g), and preferably at least 1 / g of brain tissue 1. 5 ng (2. 5 pmoles / g), full length APP (FLAPP) and A The sum of PPα (FLAPP + APPα) is at least 150 / g of brain tissue pmoles levels, APPβ, at a level of at least 42 pmoles / g brain tissue , And the mRNA encoding the Aβ-containing protein are transgenic Thing At least twice the level of mRNA encoding endogenous APP. A βtot is the sum of all forms of Aβ. Aβ 1-42 is amino acids 1 to 4 of Aβ 2 (corresponding to amino acids 672 to 714 of APP). FL APP + APPα is the first 12 amino acids of the Aβ region (amino acids 672 of APP). To 684). Therefore, FLAPP + APPα is Represents a mixture of the full-length form of APP and APP cleaved at the α-secretase site (Esch et al., Science 248: 1122-1124 (1990)). APPβ is a β-secretase moiety APP truncated at the position (Seubert et al., Nature 361: 260-263 (1993)).   The above expression level refers to the amount of expression product present, and It is not limited to a unit of measurement. Thus, expression levels are, for example, In grams per tissue gram, in moles per tissue volume, and And grams per tissue volume. For these units of measurement, Can be determined by a known conversion method.   The above expression levels need not occur in all brain tissues. Therefore, there is The promoter is responsible for the above expression levels in at least one type of brain tissue. If at least one occurs, it is preferred. Where expression is tissue specific If the expression level is sufficient in the specific brain tissue, its promoter Is preferred, even if the expression level in the whole brain tissue does not reach the threshold Well, and need not be. This expression level is dependent on the hippocampus and / or Or it is preferably observed in the cortex. The promoter is the expression product described above. Expression of the product at the above levels can be mediated constitutively or by induction. Induction For example, by administration of an activator molecule, by heat, or by Aβ-containing For a promoter operably linked to a gene encoding a protein This can be achieved by expression of a protein activator of transcription. Suitable for this purpose Many inducible expression systems are known to those skilled in the art.   In the above measurement, it is preferable to prepare brain tissue by the following method. G The brain is excised from the transgenic animal and the tissue is removed unless otherwise specified. And keep on ice throughout the homogenization procedure. 10x brain tissue Volume (w / v) of 5M guanidine-HCl, 50 mM Tris-HCl, pH8. Homogenize in 5. This sample is then gently mixed for 2-4 hours at room temperature I do. The homogenate was then placed in cold casein buffer # 1 (0. 25% casein / phosphorus Acid buffered saline (PBS) 05% sodium azide, pH7. 4, 1X Pro 1:10 dilution with a T.ase inhibitor cocktail) to give a final pH of 0.1. 5M guanidi And keep on ice. 100X Protease Inhibitor Cocktail 2 mg / ml aprotinin, 0. 5M EDTA, pH8. 0, 1mg / ml It is composed of ipeptin. The diluted homogenate is then transferred to an Eppendorf Micro Centrifuge in a fuge at 14000 rpm for 20 minutes at 4 ° C. Further dilution If required, use cold guanidine buffer # 2 (part of guanidine buffer # 1 in case In buffer # 1 to 9 parts).   Preferred promoters have their ability to mediate Aβ expression at the above levels. Therefore, identification using the following assay is preferred. Antibody 266 (Seuber et al., Nature 359: 325-327 (1992)) in buffer (0. 23g / L NaH2PO4-H 2O, 26. 2 g / L NaHPO4-7H2O, 1 g / L sodium azide, p H7. Dissolve at 10 μg / ml in 100 μl / well in 96-well Coat on a well immunoassay plate (Costar) and allow to bind overnight. This The rate is then aspirated to a 0. 25% human serum albumin solution (25 g / L Claus, 10. 8g / L Na2HPO4-7H2O, 1. 0g / L NaH2PO 4-H2O, 0. 5 g / L sodium azide, pH7. At least 4) Block for 1 hour. This 266 coated plate is then used for Skatron play Using a washer, wash buffer (PBS / 0. 05% Tween20) Wash (IX). 100 μl / well of Aβ1-40 standard and brain tissue sample The plate is added to the plate in triplicate and incubated overnight at 4 ° C. Aβ1-40 target Standards were stored at -40 C in DMSO. 0156, 0. 0312, 0. 06 25, 0. 125, 0. 250, 0. 500, and 1. 000μg / ml From DMSO-only controls for stock and background determination Made. Aβ standards were guanidine buffer # 3 (1 in BSA buffer) for each standard. 1: 100 dilution in guanidine buffer # 1 to 9 parts) followed by casein buffer. Consisting of a 1:10 dilution into Implant # 1 (Note: Final Aβ concentration range is 15. From 6 1000 pg / ml, final guanidine concentration is 0. 5M). BSA buffer , 1% bovine serum albumin (BSA, immunoglobulin-free) / PBS / 0. Consists of 05% sodium azide. Plate and casein buffer # 2 (0. 2 5% casein / PBS / 0. 05% Tween 20 / pH7. 4) Next to room temperature (RT). The plate is then washed (3X) with wash buffer. Next, 3D6 -Biotin (0.5 in casein buffer # 2) 5 μg / ml) 100 μl / well Is added to each well and incubated for 1 hour at RT.   Monoclonal antibody 3D6 is a cysteine antibody to sheep anti-mouse immunoglobulin. To the synthetic peptide DAEFRGGC (SEQ ID NO: 10) linked via Generated. This antibody did not recognize the secreted APP, and did not recognize the Aβ1 position (Asp) Recognize species that begin with For biotinylation of 3D6, for labeling of IgG Pierce's NHS-biotin protocol (cat. # 20217X), except that 100 mM sodium bicarbonate, pH8. 5, and 24 mg NHS-biotin ( Per ml of DMSO).   The plate is then washed again (3X) with wash buffer. Next, casein buffer Horseradish peroxidase (HRP) -avidin diluted 1: 4000 in # 2 (Vector abs, cat. # A-2004), add to each well Incubate at RT for a while. Wash plate (4X) with wash buffer, then TMB substrate (Slow TMB-ELISA (Pierce cat. # 34024)) 100 μl / Wells are added to each well and incubated for 15 minutes at RT. Finally, 2 The enzymatic reaction was stopped by adding 25 μl / well of NH 2 SO 4 to each well, Rates are measured from 450 nm to 650 nm using a Molecular Devices Vmax reader. Read in.   MRNA encoding human Aβ-containing protein and transgenic dynamics The relative levels of mRNA encoding endogenous APP of a product can be determined by the method of Bordonaro et al., Biotech. niques 16: 428-430 (1994) and Rockenstein et al. Biol. Chem. 270: 28257-28267 (1995). Aβ1-42, FLAP Preferred methods for measuring the expression levels of P + APPα and APPβ are described in Examples 8 is described. Yeast artificial chromosome   The construct shown in FIG. 7 can be constructed as follows. Large sections of human genomic DNA Fragment is a self-replicating unit in yeast when cloned into certain vectors. Can grow as The segment on such a vector is called a yeast artificial chromosome. (YAC; Burke et al., Science 236: 806 (1987)). Average insert size of 250,000 base pairs (from 180,000 to 500,000 base pairs) (Clontech, Palo Alto, CA). Alzheimer's Disease library was screened with human APP770 cDNA for this library. It can be isolated directly by leaning. Essential gene regions in clones Inclusion of all regions can be confirmed by PCR analysis.   The YAC-APP clone shown in FIG. 7a is encoded by the YAC vector. In embryonic stem (ES) cells by selecting for neomycin resistance Can stand. Using ES cells having the YAC-APP clone, Established under the headings “Sgenic mice” and “Embryonic stem cell method” Transgenic mice can be made by the methods described. At amino acid 717 The YAC-APP gene having the mutation of By creating a YAC library using genomic DNA from affected individuals, Can be produced. Such clones were identified as described above and confirmed in ES cells. Can stand. Genetic modification of mouse APP gene   Nucleotide sequence between human and mouse Alzheimer's protein genes The homology is about 85%. Within the Aβ-coding region, there is 3 between the two sequences. There are two amino acid differences. Amino acids that can mutate to alter APP processing The acids Lys670, Met671 and Val717 are present in mice, rats and humans. Are saved between Wild-type rodents do not develop Alzheimer's disease, Similar to that found in human Alzheimer's patients in the central nervous system (CNS) No deposits or plaques occur. Thus, human (as opposed to rodent) Aβ There is a possibility that the disease may occur. Homologous recombination (Capecchi, Science 244: 1288-1292 ( 1989)), the mouse Alzheimer gene was It can be converted to a gene encoding human Aβ in Ju. This recombination A natural alternative splicing mechanism suitable for all cells in nick animals Downstream from the KPI and OX-2 domains so that they can be used for expression of the final gene product. It is directed to the site of the flow, for example, exon 9.   Both wild-type (FIG. 8a) and mutant (FIG. 8b) forms of human cDNA It can be used to create a transgenic model that expresses a wild type or mutant type. set The replacement vector is a human APP cDNA (APP695, APP751 or AP P770 type) wild type, amino acids 669, 670, 671, 690, 692, 7 17 or a combination of these mutations. Recombinant vector Cleavage of the promoter (eg, at the XhoI site in exon 9) To promote homologous recombination (Capecchi (1989)). Endogenous A resulting from this event The PP gene is normal up to the point of recombination (in this example within exon 9). , And thereafter consist of human cDNA. Preparation of constructs for transfection and microinjection   DNA clones for microinjection are bacterial plasmid sequences With appropriate enzymes such as SalI and NotI to remove The fragments are electrophoresed on a 1% agarose gel in TBE buffer (Sambro ok et al. (1989)). DNA bands were visualized by ethidium bromide staining, and APP Cut out the band containing the current sequence. Next, the cut band is set to 0. 3M sodium acetate PH7. Place in dialysis bag containing 0. The DNA is electroeluted into the dialysis bag , Extracted with phenol-chloroform (1: 1) and precipitated with 2 volumes of ethanol. Let The DNA was mixed with 1 ml of low salt buffer (0. 2M NaCl, 20mM Tris, pH7. 4, and 1 mM EDTA) and Elutip-D^TMKara Purify with a system. The column was first loaded with 3 ml of high salt buffer (1 M NaCl, 20 mM T ris, pH 7.4, and 1 mM EDTA). Then wash with 5 ml of low salt buffer. Pass the DMA solution through the column three times, DNA is bound to the matrix. After washing with 3 ml of low salt buffer, DN A is eluted with 0.4 ml of high salt buffer and precipitated with 2 volumes of ethanol. D N A concentration is measured by absorption at 260 nm in a UV absorption spectrophotometer. Microin For injection, the DNA concentration was 5 mM Tris, pH 7.4, and Adjust to 3 μg / ml in 0.1 mM EDTA. Micro injection Other methods for purification of DNA for use are also described in: Hogan et al., Man. ipulating the Mouse Embryo (Cold Spring Harbor Laboratory, Cold Spring Ha rbor, NY, 1986); Palmiter et al., Nature 300: 611 (1982); The Qiagenologist, Ap replication Protocols, 3rd edition, published by Qiagen, Inc., Chatsworth And Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Sp. ring Harbor Laboratory, Cold Spring Harbor, NY, 1989). Construction of transgenic animals A. Animal sources   Animals suitable for transgenic experiments are available from standard commercial sources, such as Char les River (Wilmington, MA), Taconic (Germantown, NY), and Harlan Sprague  Available from Dawley (Indianapolis, IN). Many strains are appropriate, but Swiss W ebster (Taconic) female mice are preferred for embryo collection and transfer. B6D2F1 ( Taconic) Males can be used for mating and vasectomized Swiss Webster breeding individuals Can be used to stimulate pseudopregnancy. Vasectomized mice and rats are supplied Available from the public. B. Microinjection procedure   Procedures for manipulation of rodent embryos and microinjection of DNA are described in Ho gan et al., Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory, Col. d Spring Harbor, NY, 1986). Its teaching is generally known And incorporated herein by reference. C. Transgenic mouse   In a 6-week-old female mouse, 5 IU of pregnant horse serum gonadotropin (PMSG; Sigma) Injection (0.1 cc, ip) was performed and human chorionic gonadotropin (h) 48 hours later CG; Sigma) induced superovulation by 5 IU injection (0.1 cc, ip) . Immediately after hCG injection, the females coexist with the males. Copulated 21 hours after hCG injection Females were sacrificed by CO2 asphyxiation or cervical dislocation and embryos were collected from excised fallopian tubes. , Dulbecco's phosphate buffer containing 0.5% bovine serum albumin (BSA; Sigma) Place in saline. The surrounding cumulus is removed with hyaluronidase (1 mg / ml). The pronuclear embryos are then washed and moistened with 5% CO2, 95% air until injection. In a 37.5 ° C. incubator under an atmosphere, a mixture containing 0.5% BSA (Ea rle) in adjusted salt solution (EBSS). Embryos can be transferred at the two-cell stage.   Randomly cycled adult female mice are paired with vasectomized males. Swiss Webster Or other comparable strains can be used for this purpose. The recipient female is mated at the same time as the donor female. Let At the time of embryo transfer, the recipient female was 0.015 ml of 2.5% Anesthetize with intraperitoneal injection of avertin. Cut the fallopian tube into a single midline back Expose by opening. An incision is then made through the body wall and directly over the fallopian tubes. Ovary The wrapper is then incised with watchmaker tweezers. The embryo to be transferred is In a pet chip (about 10-12 embryos), DPBS (Dulbecco's phosphate buffered saline) Water). The embryo is transferred by inserting the tip of a pipette into the fallopian funnel. Transplant Thereafter, the incision is closed with two sutures. D. Transgenic rat   The procedure for creating transgenic rats is the same as that for mice (Hammer Cell 63: 1099-112 (1990)). 30-day-old female rats were given PMSG (0.1 cc). ) Was injected subcutaneously and 48 hours later each female was provided with a male Let it exist. At the same time, a 40-80 day old female rat was paired with a vasectomized male in a cage. To This provides a foster mother for embryo transfer. The next morning, check the female vaginal plug You. Females mated to vasectomized males are retained until transplantation. Mating donor females Kill (CO2 asphyxiation), remove fallopian tubes, DPBS containing 0.5% BSA (Dulbeth (Phosphate buffered saline) and the embryos were harvested. The cumulus cells around the embryo , Hyaluronidase (1 mg / ml). The embryos are then washed and injected EBS containing 0.5% BSA in a 37.5 ° C. incubator until the Place in S (Earl's adjusted salt solution).   After the embryos are injected, the live embryos are transferred to DPBS for transfer to foster mothers. Nourishment Mothers received ketamine (40 mg / kg, ip) and xylazine (5 mg / kg, i Anesthetize in p). A midline dorsal incision is made through the skin and the ovaries and fallopian tubes are removed Exposure is by incision of the muscle layer just above the nest. Split the ovarian sac and transfer the embryo And insert the tip of the transfer pipette into the fallopian tube funnel. About 10-12 Embryos are implanted into the oviduct of each rat through the oviduct funnel. The incision is then sutured and closed , House the foster mother alone. E. FIG. Embryonic stem (ES) cell method 1. Introduction of cDNA into ES cells   Method for culturing ES cells and subsequent method for producing transgenic animals, Cloporation, calcium phosphate / DNA precipitation, and direct injection Transfection of DNA into ES cells by various methods, such as nd Embryonic Stem Cells, A Practical Approach, edited by E.J.Robertson, (IRL P ress 1987). The teachings are generally known and are described herein. Used in Selection of the desired transgene-containing ES cell clone is This can be achieved in one of several ways. APP for random gene integration Clones are co-precipitated with the gene encoding neomycin resistance. Transfer Action: Teratocarcinomas and Embryonic Stem Cells, A Practical Appro ach, editor E.J.Robertson, Lovell-Badge in (IRL Press 1987), or Potter et al. Proc. Natl. Acad. Sci. USA 81: 7161 (1984). obtain. Lipofection is performed using a commercially available kit, for example, DOTAP (Boehringer-Man). nheim) or lipofectin (BRL). Rin Calcium acid / DNA precipitation, lipofection, direct injection, and Electroporation is a preferred method. In these procedures, 0.5 X10⁶ES cells are plated in tissue culture dishes and transfected with the following mixture: The linearized APP clone and 1 mg of pSV2neo DNA (Southe rn and Berg, J. Mol. Appl. Gen. 1: 327-341 (1982)) and 50 mglipof Precipitated in the presence of ectin (BRL) to a final volume of 100 μl. Cells are selective culture Ground, supplemented with G418 (between 200 and 500 μg / ml) 10% fetal bovine serum in DMEM. A colony of cells resistant to G418 , Isolated in a cloning ring and expanded. Drug resistant clones From the DNA and Southern blot using APP770 cDNA probe. for Thus, a clone having the APP sequence can be identified. Using the PCR detection method, Elephant clones can be identified.   DNA molecules introduced into ES cells are also homologous to those described in Capecchi (1989). It can be integrated into the chromosome by a recombination step. Direct injection is highly effective Incorporation of rate results. The desired clone is a pool of the injected ES cells. Can be identified by PCR of DNA prepared from DNA. Positive cells in the pool Can be identified by PCR following cell cloning (Zimmer and Gruss, Natur e 338: 150-153 (1989)). DNA transfer by electroporation has high efficiency Lower and requires a selection step. Positive selection of recombination events (eg neo resistance), And double positive-negative selection (eg, neo resistance and ganciclovir resistance), and And subsequent methods for identifying desired clones by PCR are described in Joyner et al., Nature 3 38: 153-156 (1989) and Capecchi (1989). The teaching is general And are incorporated herein by reference. 2. Embryo recovery and ES cell injection   ES cells were used in naturally cycling or superovulated female mice mated with males. Embryos can be harvested for transfer of the cells. When using a mouse, C57BL is used for this purpose. / 6 strains are preferred. Embryos of appropriate age are collected approximately 3.5 days after successful mating You. Mated females were sacrificed by CO2 asphyxiation or cervical dislocation, and excised uterine horns Of the embryos from the culture (Dulbecco's modified essential medium containing 10% calf serum) Put it in the ground. About 10 to 20 ES cells were obtained using a glass microneedle having an inner diameter of about 20 μm. And inject into the blastocyst. 3. Embryo transfer to pseudopregnant females   Randomly cycled adult female mice are paired with vasectomized males. Swiss Webster, ICR or other mouse strains can be used for this purpose. Recipient females are embryos containing ES cells Copulate as required for blastocyst transplantation, 2.5-3.5 days after copulation You. At the time of embryo transfer, the recipient female was 0.015 ml of 2.5% Abel / g body weight. Anesthetize with intraperitoneal injection of tin. By incising the body wall just above the fallopian tube, To expose the ovaries and uterus (externalized). 25 gauge in the uterine horn The needle is pierced and the blastocyst is implanted therethrough. After transplantation, the ovaries and uterus And the incision is closed with two sutures. Use this procedure if you need to do more transplants Repeat on the other side. Identification, characterization, and utilization of transgenic mice and rats   Transgenic rodents can be identified by analyzing their DNA . For this purpose, tail samples (1-2 cm) can be removed from 3 week old animals. DNA from these or other samples is then prepared, Southern blot, PCR Or transgenic founder (founder ) (F0) animals and their progeny (F1 and F2) can be detected. A. Pathological research   Various F0, F1 and F2 animals with the transgene were subjected to Aβ deposition, APP Or expression of APP cleavage products, neuronal or neurite abnormalities, and in the brain Evidence of an inflammatory response can be analyzed by immunohistology. Each transgenic Mouse and rat brains from strains are fixed and sectioned. APP section And / or stained with antibodies reactive with Aβ. Fluorescein, loader Binds to min, horseradish peroxidase, or alkaline phosphatase The primary antibody is detected by using the secondary antibody. These methods work in specific areas of the brain Allows the identification of amyloid plaques and other pathological lesions in A. 9? Plaques ranging in size> 50 μm are found in the cerebral cortex in the brain of AD patients. Characteristically, but also observed in dense gray matter including the amygdala, striatum and diencephalon Can be done. Sections are also stained with other antibodies that can diagnose Alzheimer's plaque These antibodies can be used to identify APP, Alz-50, tau, A2B5, neurofilament. , Synaptophysin, MAP-2, ubiquitin, complement, neuron-specific Recognizes antigens such as nolase and other antigens 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); Ar USA et al., Proc. Natl. Acad. Sci. USA 87: 2249-2253 (1990); Majocha et al., Alner. Assoc. Neuropathology Abs 99:22 (1988); Masters et al., Proc. Natl. Acad. Sci. 82: 4245-4 249 (1985); Majocha et al., Can J Biochem Cell Biol 63: 577-584 (1985)). Chioff Staining with Rabin S and Congo Red was also observed in neurite plaques and NFTs. of It can be done to analyze the presence of amyloid and co-localization of Aβ deposition. B. Analysis of APP and Aβ expression 1. mRNA   Messenger RNA is derived from cell lines and tissues of transgenic animals. Guanidium thiocyanate-phenol: isolated by chloroform extraction (Chomaczynski and Sacchi, Anal Biochem 162: 156-159 (1987)). Determined by Northern blot, RNAse and nuclease protection assays. Can be determined. 2. protein   APP, Aβ, and other fragments of APP are derived from the APP extracytoplasmic domain. Aβ region, Aβ1-42, Aβ1-40, APPβ, FLAPP + APPα, and A Detection using polyclonal and monoclonal antibodies specific for the C-terminus of PP It can and has been served. Various antibodies specific to human sequences, such as 10D5 And 6C6 are very useful for this purpose (Games et al. (1995)). 3. Western blot analysis   Protein fractions are isolated from tissue homogenates and cell lysates, e.g., Western blot analysis can be performed as described below: Harlow et al., Antibo dies: A Laboratory Manual, (Cold Spring Harbor, NY, 1988); Brown et al., J. Ne. urochem 40: 299-308 (1983); and Tate-Ostroff et al., Proc Natl Acad Sci 86: 745. -749 (1989).   Briefly, the protein fraction was denatured in Laemmli sample buffer and Run on a DS-polyacrylamide gel. The protein is then nitro Transfer to a cellulose membrane by electroblotting. Filter Block, incubate with primary antibody, and finally react with enzyme-conjugated secondary antibody Respond. Subsequent incubation with the appropriate chromogenic substrate is performed by Clarify the location of parkin. C. Pathological and behavioral studies 1. Pathological research   Immunohistological and Thioflavin S staining are described elsewhere herein. It is done as follows.   In situ hybridization: radioactively or enzymatically labeled nucleic acid Using the probe, mRNA can be detected in situ. The probe is For better penetration, digest or prepare to about 100 nucleotides in length. You. For fixed or paraffin-embedded samples, see Chou et al., J. Psych. Res. Two The hybridization procedure of 4: 27-50 (1990) is outlined below, but a similar procedure is used. Can also be used on sectioned samples as frozen material. In situ hybridization Paraffin slides for stabilization should be dewaxed in xylene and Rehydrate with a series of ethanol and finally rinse with phosphate buffered saline (PBS) . Sections are post-fixed in fresh 4% paraformaldehyde. Slide the PBS And wash twice for 5 minutes to remove paraformaldehyde. Then, cut the slices into 20 μg Permeabilized by treatment with / ml protein kinase K solution . Sections were refixed in 4% paraformaldehyde and background The base molecule that generates the thiol bond is 0.1 M triethanolamine and 0.3 M acetic acid-free. Acetylation with aqueous solution for 10 minutes. Wash slides with PBS, then step-by-step Dehydrate with a series of ethanol and air dry. Control sections and sense probes And hybridized with an antisense probe. After proper cleaning, The combined radioactive probe was detected by autoradiography or enzyme-labeled. The probe is detected by reaction with an appropriate chromogenic substrate. 2. Behavioral research   Behavioral tests set up to evaluate learning and memory deficits are used. This Examples of such tests include the Morris water maze (Morris, Learn Motivat. 12: 239-260). (1981)). In this procedure, the animal is filled with water and sheltered under water. Placed in a circulating pool with a platform. The animal faces the adjacent visible display Visible markers on the platform so you can find them Is placed. Alternatively, animals may not have a position indicator indicating the position of the platform. And are subjected to more complex types of testing. In this version, the animal is associated with a distant visible display. You need to learn the position of the platform in the staff, reference and working memory (working) memory). Learning deficiencies in the water maze are Genetic mice. Transgenic expression of Aβ-containing proteins An example of behavior analysis for evaluating the effect is described in Example 9.   Operant Behavioral Study of Memory Function: Disclosed Transgenic Animal Memory Machine Performance can be assessed by testing for memory-related dietary behavior (Dunnett, "O. Perrant delay matching and non-matching position "Behavioral Neuroscience, Vo lume I: A Practical Approach (editor Sagal, IRL Press, N.Y., 1993) 123-136;  Zornetzen, Behav.Neur.Biol. 36: 49-60 (1982)). Transgenic and Non-transgenic mice get dietary reward in two-component operant procedure Train to do so. One component is a delayed spatially variable schedule. This schedule The mouse is rewarded by pressing another lever in the current trial Over the course of a different delay time, You have to remember if you pushed the lever. This is the immediate or "effect" of the animal. Give an indication of memory. The second component is a discriminating spatially variable schedule. Under this schedule, the mouse presses one of the illuminated levers. Earn rewards. This discriminating action is an example of reference storage. Transgenic And non-transgenic groups of mice over time, eg, from 3 months of age. It has been studied to the end of its useful lifespan, which indicates the sensitivity to cholinergic antagonists. Contributes to assessing fertility and the development of behavioral disorders with respect to these memory tasks . The disclosed transgenic mice have a memory of cholinergic antagonists. Sensitivity to perturbation effects is increased, and With disability, it is expected to be a model for cognitive deficits in Alzheimer's disease. Administration-challenges with cholinergic antagonists were tested at various ages. Can do. These memory behavioral tests were also performed on the disclosed transgenic animals. It can also be used to compare the effects of compounds on behavioral disorders. In this case, two groups of mice The transgenic mice treated with the test compound and the test compound administered And not transgenic mice.   Emotional responsiveness and object recognition: various features of the disclosed transgenic animals Noh is an ambulatory activity, emotional responsiveness to a new environment or new subject, and It can be assessed by testing the recognition of the subject. The first set of assessments is for different ages Done on the same animal (the individual animal is its own control) Work is a type of memory that shows severe disability in AD patients. Test the emotional responsiveness to a normal environment or new subject, and the perception of the subject. Serve the purpose of testing. Day 1, transgenic and non-transgenic Control mice are individually square open with a central platform Placed in the field. Horizontal and vertical activity for each animal for 30 minutes , And platform crossings are recorded in 5 minute blocks. On the second day, Each animal is subjected to two trials at one hour intervals. First trial Two identical objects are placed in the open field and animals are allowed 3 minutes of exploration. Is done. In a second trial, one of the subjects is replaced with a new subject and the animal is already The time spent searching for existing and new objects is recorded in the next 3 minutes ( Ennaceur and Delacour, Behav. Brain Res. 31: 47-59 (1988)). Animals then Test for neophobic behavior, which is considered a guide to anxiety . Here, animals are given the opportunity to move freely between existing and new environments. Can be   After that, the same animals are given various learning tasks and their learning and memory abilities are investigated. You. First, for spatial recognition memory, in the T-maze delay variable task, 6 hours And a delay time of 24 hours. This type of memory is It has always been shown to be sensitive. Half of the animals in each group are then Train on just reinforced lever pushing tasks. This is the training of animal work To measure later improvement, which has been shown to be involved in hippocampal activation Can be used. Another half of the animals are used for spatial discrimination in an 8-arm radical maze. (Oltons and Samuelson, J. Exp. Psychol. [Animal Behav,] 2: 9 7-116 (1976)), measures that evaluate action and reference, and provide good guidelines for memory ability (Angle preference) for analysis purposes. Bar-lever pushing task 2-3 months old animals that were trained and tested were 9 to 10 months old in the radical maze It can be trained and tested, and vice versa. Working memory defect, radical arm stray Tracts were shown in PDAPP transgenic mice.   Two additional groups, 9 to 10 months of age, may be subjected to the same behavioral tests as above, Suggests that behavioral screening performed at the age of two to three months can increase learning and memory skills Serve the purpose of deciding whether to affect.   These memory behavioral tests also demonstrate the behavioral disorders of the disclosed transgenic animals. Can also be used to compare the effects of compounds on In this case, two groups of mice Transgenic mice receiving the test compound and whether the test compound was not administered And transgenic mice.   The procedure applicable for transgenic mice is The same is true for the case. D. Preferred features   The phenotypic characteristics of the disclosed transgenic animals are Can be used to identify preferred types of transgenic animals as animal models. You. Among the disclosed transgenic animals, the preferred type as an animal model is the same. Measure additional phenotypic features and these features, which can also be used to determine The assay to perform this is described in Example 6. These features are preferably It is similar to the phenotypic features observed in Alzheimer's disease. Disclosure Of transgenic animals selected for use as animal models APP and Aβ markers, also useful for the following, are described below. These Or any or all of the phenotypic features alone or in combination Can be used to identify preferred types of the disclosed transgenic animals. For example, the presence of plaques that can be stained with Congo Red in brain tissue has been disclosed. Phenotypic features that can identify transgenic animals that are preferred. Good Certain APP-related tags present in transgenic animals (described above) Protein expression levels are used to identify preferred transgenic animals. It is intended to be a standing feature. Therefore, the most preferred transgenic The animal has an expression level disclosed for one or more of the APP-related proteins. And indicates one or more of the above phenotypic features. Especially preferred Phenotypic characteristics (the presence of which identifies the animal as a preferred transgenic animal) ) Is: the presence of amyloid plaques that can be stained with Congo Red (K elly (1984)), extracellular amylo identified by electron microscopy by 12 months of age Presence of id fibrils and identified by electron microscopy by 12 months of age Presence of type I dystrophic neurites (including synaptic proteins and APP) , Composed of spherical neurites; Dickson et al., Am J Pathol 132: 86-101 (1988);  Dickson et al., Acta Nuropath. 79: 486-1493 (1990); Masliah, J Neuropathol Ex p Neurol 52: 135-142 (1993); Masliah et al., Acta Nuropath. 87: 135-142 (1994); W ang and Munoz, J Neuropathol Exp Neurol 54: 548-556 (1995)). These features An example of signature detection is provided in Example 6. Transgenic animals are 14 months old At the time, it is most preferred to have amyloid plaques that can be stained with Congo Red. New Screening compounds for the treatment of Alzheimer's disease   Transgenic animals or animal cells derived from transgenic animals Uses standard methodologies to assess potential effects in treating Alzheimer's disease Can be used to screen compounds. AD screen like this In a tuning assay, compounds are administered over a period of time and at various dosages. Administered to animals or introduced into the culture medium of cells derived from these animals The animal or animal cells may then be processed as described above and in the Examples below. Changes in the expression or processing of APP, the development of other AD markers, Current level or localization, histopathology and / or behavior in the case of animals To be tested. In general, any improvement in behavioral testing (changes in AD association markers, Reduced severity of AD-related histopathology, reduced expression of Aβ or APP cleavage products, and And / or the presence of other compounds that are correlated with the AD observed in treated and / or treated animals. Presence or change in level (compared to untreated animals) is due to Alzheimer's disease Is an indicator of compounds useful for treating. Specific protein, code transcription Substance, enzyme activity or biochemical activity, and / or tissue of these proteins Pathology is related to and characteristic of AD and is referred to herein as a marker . Both the expression or localization of these markers characteristic of AD have been detected Or is present in the disclosed transgenic animals. This These markers can be measured or detected, and these measurements Comparison and testing between transgenic animals and untreated transgenics Compound The effect of the object can be determined. A useful marker for AD screening assays is AD Selection is based on the detectable change in these markers in relation. Many of this Such markers have been identified in AD and disclosed transgenes. Has been detected in nick animals or is expected to be present in these animals. Is either measured. These markers indicate their nature, location, Are divided into several categories based on function. AD screening ass Preferred examples of markers useful for (a) are the Aβ-related markers described below, Plaque-related markers, cytoskeletal and neural markers, inflammatory markers Group of neurons and neurotransmitter-related markers . A. Aβ-related marker.   Expression of various forms of APP and Aβ are described above and in the Examples below. Can be measured directly by both immunohistochemistry and quantitative ELISA. And to treated and untreated transgenic animals. Can be compared. Currently, two forms of APP products (APP and Aβ) have been discovered (Haass and Selkoe, Cell 75: 1039-1042 (1993)). This They have been shown to be endogenously associated with AD pathogenesis in a time-dependent manner. Therefore, the preferred assays are APP and A in transgenic mice. Compare age-related changes in β expression. As described in Example 6, The increase is shown during the aging of PDAPP mice.   A preferred target for assay measurements is in individuals with Alzheimer's disease. Aβ markers that are known to increase in total Aβ (Aβ_tot) , Aβ1-42 (Aβ1_1-42Aβ with amino acids 1-42), Aβ_1-40(With amino acids 1-40 Aβ), AβN3 (pE) (Aβ_N3(PE)); AβX-42 (Aβ_X-42Ends with amino acid 42 Aβ form); AβX-40 (Aβ_X-40; Aβ form ending with amino acid 40); insoluble Aβ (Aβ_{ins oluble} ); And soluble Aβ (Aβ_solubleKuo et al. Biol. Chem. 271 (8): 407 7-4081 (1996)). Aβ_N3(PE) has pyroglutamic acid at position 3 (Saido, N euron 14: 457-466 (1995))._X-42Is Aβ_13-42Any C-terminal form of Aβ such as Say. Aβ_insolubleIs Gravina, J .; Biol. Chem. 270: 7013-7016 (1995) As mentioned This is the form of Aβ recovered. APPβ also assesses the amount of β-secretase activity (Seubert et al., Nature 361: 260-263 (1993)). this Some of their Aβ forms and their association with Alzheimer's disease are described by Haass and And Selkoe (1993). Aβ_tot, Aβ1_1-42, And Aβ_X-42of Detection and assays are described in Example 6. Generally, the specific form of Aβ is specific Antibodies can be assayed either quantitatively or qualitatively as described below. When referring to the position of an amino acid in the form of Aβ, this position is relative to the Aβ region of APP. Respond. Amino acid 1 of Aβ corresponds to amino acid 672 of APP, and amino acid 1 of Aβ 42 corresponds to amino acid 714 of APP.   Preferred as targets for assay measurements are also APP markers. An example For example, the different forms of secreted APP (called APPα and APPβ) are also measured. (Seubert et al., Nature 361: 260-263 (1993)). Other forms of APP also Assays to assess potential for compounds affecting Ruzheimer's disease Can act as a target for These include FLAPP + APPα, full-length APP, APP C-terminal fragments (particularly C100 (the last 100 amino acids of APP) and C57-C 60 (the last 57-60 amino acids of APP)), and Aβ_1-40Including the area corresponding to Any form of APP.   APP form also has potential for compounds affecting Alzheimer's disease Is a preferred target for assays to assess Absolute APP and APP transcripts Levels, different APP forms and the relative levels of their cleavage products, and the expression of APP Current or processing localization is all a marker associated with Alzheimer's disease And can be used to determine the effect of treatment with potential therapeutic compounds . Localization of APP to plaque and neural tissue is particularly important for these assays. It is a preferred target.   Quantitative measurements can be achieved using a number of standard assays. For example, transcription Product levels are determined by RT-PCR and hybridization methods including PNase protection, It can be measured using Zan analysis, as well as Rdot analysis. APP and Aβ levels Of tissue sections stained by ELISA, ELISA, Western analysis and immunohistologically Can be assayed by comparison. Immunohistological staining can also be performed on specific tissues and cell types. What Can be used to assay the localization of APP and Aβ. Such an ass A is described above, and specific examples are provided below. B. Plaque association marker.   A variety of other molecules are also present in the plaques of individuals with AD and the disclosed trans. Is present in transgenic animals and is absent in plaque and neural tissue Can be detected. The amount of these markers present on plaques and nervous tissue has not yet been determined. It is expected to increase with the age of the transgenic animal of the treatment. Like A new plaque-related marker is apolipoprotein E, a final glycosylation product Products, amyloid components, advanced glucosylation end products (Smith et al., Pr oc. Natl. Acad. Sci. USA 91: 5710 (1994)), growth inhibitory factor, laminin, type IV Collagen (Kalaria and Perry (1993); Ueda et al. (1993)), progressive glycosylation Product receptor (RAGE), and ubiquitin.   The markers described above are used to detect specific components of plaque and neural tissue. Although plaque location and extent may also be determined by well-known histochemical staining (eg, For example, by using Congo Red and Thioflavin S), And may be determined as described in the following several examples. C. Cytoskeletal and neural markers.   Many changes in cytoskeletal markers associated with AD are also due to transgenic Detected in PDAPP mice. These markers are AD screening And the effect of the compound on AD can be achieved. cell Many changes in skeletal markers are associated with plaque-associated neurofibrillary tangles or Occurs in any of the dystrophic neurites (Kosik et al. (1992); Loves tone and Anderton (1992); Brandan and Inestrosa (1993); Trojanowski et al. (1993); Masliah et al. (1993)).   The following is a preferred cytoskeleton showing changes in and / or associated with AD Markers and neural markers. These markers can be detected and altered. Can be determined and the effect of the compound on the disclosed transgenic animals measured. Can be determined. Spectrin shows increased degradation in AD. Tau and nerve fibers Lament shows an increase in hyperphosphorylation in AD, And ubiquitin levels are increased in AD. Tau, ubiquitin, MAP-2, God Transfilaments, heparin sulfate, and chondroitin sulfate are Localization to neurons and nervous tissue and generally varies from normal localization. GAP4 3 levels are reduced in the hippocampus and abnormally phosphorylated tau and nerve Filament is present in PDAPP transgenic mice. D. Inflammatory markers.   Alzheimer's disease is also associated with immunoinflammatory, with a corresponding increase in inflammatory markers. It is known to stimulate responses (Frederickson and Brunden (1994); McG eer et al. (1991); Wood et al. (1993)). The following are in and / or related to AD Are preferred inflammatory markers that show changes that occur. Inspection at these manufacturers Changes in expression are useful in AD screening assays. Acute phase (acute ph ase) protein and glue markers (eg, α1 antitrypsin, C-reactive protein, α2-macroglobulin (Tooyama et al., Molecular & Chemical  Neuropathology 18: 153-60 (1993)), collagen fibrillary acidic protein (GFAP), Mac I, F4 / 80, and cytokines (eg, IL-1α and β, TNFα, IL-8, MIP-1 α (Kim et al., J. Neuroimmunology 56: 127-134 (1995)), MCP-1 (Kim et al., J. Neuro logical Sciences 128: 28-35 (1995); Kim et al., J. Neuroimmunology 56: 127-134 (1 995); Wang et al., Stroke 26: 661-665 (1995)), and IL-6), all of which Increases in AD and increases in the disclosed transgenic animals It is expected that Complement markers (eg, C3d, C1q, C5, C4d, C4bp, and And C5a-C9) localize to plaque and neural tissue. Major histocompatibility complex ( MHC) Glycoproteins (eg, HLA-DR and LA-A, D, C) are increased in AD I do. Microglial markers (eg, CR3 receptor, MHC1, MHCII, LCD31, CD11a, CD 11b, CD11c, CD68, CD45RO, CD45RD, CD18, CD59, CR4, CD45, CD64, and CD4 4 (Akiyama et al., Brain Research 632: 249-259 (1993)) increases in AD You. Additional inflammatory markers useful for AD screening assays include: But Α2 macroglobulin receptor, fibroblast growth factor (Tooyama et al., Neuro oscience Letters 121: 155-158 (1991)), ICAM-1 (Akiyama et al., Acta Neuropath). ologica 85: 628-634 (1993)), lactotransferrin (Kawamata et al., America) n Journal of Pathology 142: 1574-85 (1993)), C1q, C3d, C4d, C5b-9, Fcγ RI, FcγRII, CD8 (McGeer et al., Can J Neurol Sci 16: 516-527 (1989)), LCA (C D45) (McGeer et al. (1989); Akiyama et al., Journal of Neuroimmunology 50: 195-201. (1994)), CD18 (β2 integrin) (Akiyama and McGeer, Journal of Neur oimmunology 30: 81-93 (1990)), CD59 (McGeer et al., Brain Research 544: 315-31). 9 (1991)), vitronectin (McGeer et al., Canadian Journal of Neurological S ciences 18: 376-379 (1991); Akiyama et al., Journal of Neuroimmunology 32: 19-28. (1991)) vitronectin receptor, β3 integrin (Akiyama et al. (1991)) , Apo J, clusterin (McGeer et al., Brain Research 579: 337-341). (1992)) Type 2 plasminogen activator inhibitor (Akiyama et al., Neuro oscience Letters 164: 233-235 (1993)), CD44 (Akiyama et al., Brain Research 6) 32: 249-259 (1993)), Midkine (Yasuhara et al., Biochemical & Biophysical Research Communications 192: 246-251 (1993)), Macrophor Dicolony stimulating factor receptor (Akiyama et al., Brain Research 639: 171-174 (1994) )), MRP14, 27E10, and interferon alpha (Akiyama et al., Journal of Neurology). immunology 50: 195-201 (1994)). Further associated with inflammation or oxidative stress Markers include 4-hydroxynonenal protein binding. Combination (Uchida et al., Biochem. Biophys. Res. Comm. 212: 1068-1073 (1995); And Stadtman, Methods in Enzymology 233: 371-380 (1994); Yoritaka et al., Proc. N atl.Acad.Sci.USA 93: 2696-2701 (1996)), IκB, NFκB (Kaltschmidt et al., Mole cular Aspects of Medicine 14: 171-190 (1993)), cPLA_Two(Stephenson et al., Neuro obiology Dis. 3: 51-63 (1996)), COX-2 (Chen et al., Neuroreport 6: 245-248 (199) 5)), matrix metalloproteinases (Backstrom et al., J. Neurochemistry 58: 9 83-992 (1992); Bignami et al., Acta Neuropathologica 87: 308-312 (1994); And Gottschall, J. Neurochemistry 66: 1641-1647 (1995); Peress et al., J. Neuropat. hology & Experimental Neurology 54: 16-22 (1995)), membrane lipid peroxide , Protein oxidation (Hensley et al., J. Neurochemistry 65: 2146-2156 (1995); Smith et al., Pr. oc. Natl. Acad. Sci. USA 88: 10540-10543 (1991)) and reduced ATPase activity. (Mark et al., J. Neuroscience 15: 6239 (1995)). These markers can be detected And a compound against the disclosed transgenic animal, wherein the change can be detected Is measured. E. FIG. Neuron and neurotransmitter related markers.   Changes in neuronal and neurotransmitter biochemistry are associated with AD and Present in indicated PDAPP animals. Cortical and hippocampal cholinergic action in AD Sexual innervation is greatly reduced. This is the synthase choline acetyl transferer Very large deficiency of zeo, acetylcholinesterase, synaptosome choline uptake Incorporation (measured by hemicolinium inding), As demonstrated by the synthesis and release of acetylcholine (Rylett et al. (1983); Sim Coyle et al., Science 219: 1184-1190 (1983); Davies and Maloney, La. ncet 2: 1403 (1976); Perry et al., Lancet 1: 189 (1977); Sims et al., J. Neurochem. 40: 503-509 (1983)). All of these are useful markers. These markers Can be used in an AD screening assay to determine the effect of a compound on AD. Can be determined. Basal forebrain neuron deficiencies are also present, and gas The galanin system becomes hypertrophic in AD.   In addition to the above marker changes in AD, pre-basal protruding into the cortex and hippocampus Atrophy and deficit of brain cholinergic neurons (Whitehouse et al., Science 215: 1237- 1239 (1982)), as well as changes in entorhinal cortical neurons (V An Hoesan et al., Hippocampus 1: 1-8 (1991) also exists. Based on these observations Measurements of these enzyme activities, neuron size, and neuron count. Are expected to decrease in the disclosed transgenic animals. Therefore, it is a useful target for detection in AD screening assays. You. Basal forebrain neurons are dependent on nerve growth factor (NGF). Brain-derived nerve Nutrition molecules (BDNF) are also reduced in the hippocampus of the disclosed transgenic animals. A little and therefore a useful marker for detection in AD screening assays It is a target.   Release of APP and Aβ in both in vitro tissue culture and brain sections. Outbreaks have also been shown to be affected by muscarinic receptor stimulation. A similar finding is that phosphoinosital turnover (Nitsch et al. (1992) Hung et al., J. Biol. Chem. 268: 22959-22962 (1993); Nitsch et al., Proceedings of t. he Eighth Meeting of the International Study Group on the Pharmacology o f Memory Disorders Associated with Aging 497-503 (1995); Masliah and Te rry (1993); Greenamyre and Maragos (1993); McDonald and Nemeroff (1991) Mohr et al. (1994); Perry, British Medical Bulletin 42: 63-69 (1986); Maslia h et al., Brian Research 574: 312-316 (1992); Schwagerl et al., Journal of Neurochemi. stry 64: 443-446 (1995)). Based on these observations, the neurotransmitter Agonis Can reduce Aβ production in the disclosed transgenic animals It is. Based on this theory, the effects of compounds on neurotransmitter receptors Measuring screening assays identify compounds useful for treating AD Or can be used.   In addition to well-documented changes in the cholinergic system, Other receptor systems (eg, serotonergic receptor systems, adrenergic Sexual receptor system, adenosine receptor system, and nicotine receptor system) Dysfunction has also been shown. Markers characteristic of these changes, and Other neuronal markers that show both metabolic and structural changes in AD include: It is described below. Changes in the level and / or localization of these markers Using similar techniques described to measure and detect early markers, Can be measured.   The following is a preferred cytoskeleton showing changes in and / or associated with AD Markers and neural markers. Cathepsin (cat) D, B and neurons Neuronal Thread Protein Levels and Elongation Factor 2 Phosphorus Oxidation increases in AD. Cat D, B, protein kinase C, and NADPH Localizes in plaque and neurite tissue in AD. Nicotine receptor , 5-HT_TwoReceptor, NMDA receptor, α2-adrenergic receptor, China Putophycin, p65, glutamine synthetase, glucose transporter, P PI kinase, drebrin, GAP43, cytochrome oxidase, heme Oxygenase, calbindin, adenosine A1 receptor, monoamine metabolite , Choline acetyltransferase, acetylcholinesterase, The activity and / or level of naptosome choline uptake are all Decrease.   Additional markers associated with AD or after treatment of cells with Aβ Examples include: (1) cPLA_Two(Stephenson et al., Neurobiology of Di seases 3: 51-63 (1996)), which is up-regulated in AD, (2 ) Heme oxygenase-1 (Premkumar et al., J. Neurochemistry 65: 1399-1402 (1995) Schipper et al., Annals of Neurology 37: 758-768 (1995); Smith et al., American Jo urnal of Pathology 145: 42-47 (1994); Smith et al., Molecular & Chemical Neurop. athology 24: 227-230 (1995)), c-jun (Anderson et al., Experimental Neurology) 125: 286-295 (1994); Anderson et al., J. Neurochemistry 65: 1487-1498 (1995)), c-fos (Anderson et al. (1994); Zhang et al., Neuroscience 46: 9-21 (1992)), HSP27 (Renkawek et al., Acta Neuropathologica 87: 511-519 (1994); Renkawek et al., Neurore port 5: 14-16 (1993)), HSP70 (Cisse et al., Acta Neuropathologica 85: 233-240) (1993)), and MAP5 (Geddes et al., J. Neuroscience Research 30: 183-191 (199) 1); Takahashi et al., Acta Neuropathologica 81: 626-631 (1991)), these are AD, And induced in cortical cells after Aβ treatment, and (3) junB, junD, f osB, fral (Estus et al., J. Cell Biology 127: 1717-1727 (1994)), cyclin D1 (Freeman et al., Neuron 12: 343-355 (1994); Kranenburg et al., EMBO Journal 15: 46-54. (1996)), p53 (Chopp, Current Opinion in Neurology & Neurosurgery 6: 6-1) Sakhi et al., Proc. Natl. Acad. Sci. USA 91: 7525-7529 (1994); Wood and Youle, J. Neuroscience 15: 5851-5857 (1995)); NGFI-A (Vaccarino et al., Molecul) ar Brain Research 12: 233-241 (1992)), as well as NGFI-B, which are treated with Aβ Induced in later cortical cells. F. Determination of the amount and localization of AD markers.   Quantitative measurements can be achieved using a number of standard assays. For example, transcription Product levels are determined by RT-PCR and hybridization methods (RNase protection, Northern Analysis, including R-dot analysis). Protein marker Levels were stained by ELISA, Western analysis and immunohistochemically It can be assayed by comparing tissue sections. Immunohistochemical staining is also Used to assay the localization of protein markers to tissues and cell types obtain. The localization and histochemical binding of AD markers are determined by histochemical detection methods (antibody Determined by staining, laser scanning confocal imaging, and immunoelectron microscopy Can be done. Examples of such techniques are described in Masliah et al. (1993) and Example 6 below. Are listed.   For receptor and enzyme markers, the activity of the receptor or enzyme is measured. Can be For example, neurotransmitter metabolizing enzymes (eg, choline acetyltransfer Acetylcholinesterase) activity is measured using standard radiometric enzymes. Can be measured using an activity assay.   The activity of certain neurotransmitter receptors is determined by phosphoinositol (PI) turnover. It can be determined by measuring the bar. This is because of the receptor Includes measuring inositol accumulation after stimulation. Useful agonists include Carbochol and glutaminergic receptors As for ter, there is norepinephrine. Of receptors present in brain tissue The number is assessed by quantitatively measuring the binding of the ligand to the receptor. obtain.   Receptor ligand and neurotransmitter levels and turnover are dependent on species It can be determined by quantitative assays performed at various times. Dopamine turnover Can be measured using DOPAC and HVA. MOPEG sulfate is norepinephrine Can be used to measure turnover, and 5-HIAA is a serotonin turnover Can be used to measure over. For example, norepinephrine levels In the hippocampus of 12-13 month old PDAPP transgenic mice compared to controls At 20%. In general, the above-described assay should read the following statement For example, Rylett et al. (1983); Sims et al. (1980); Coyle et al., Science 219: 1184-119. 0 (1983); Davies and Maloney, Lancet 2: 1403 (1976); Perry et al., Lancet 1: 189. (1977); Sims et al., J. Neurochem. 40: 503-509 (1983) obtain. These markers are also described by Bymaster et al., J. Pharm. Exp. Ther. 269: 282-289 ( 1994). G. FIG. Screening assay using cultured cells.   Screening assays to determine the therapeutic potential of compounds are also open Cells from a transgenic animal for the indicated APP construct, and It can be performed using cell cultures stably transfected with the disclosed constructs. You. For example, such an assay can be performed on cultured cells in the following manner. . Cell cultures are described in International Patent Application No. 94/10569 and in Citron et al. (1995). In general, they can be transfected. Then, the resulting transgenic Check cells or transfected cell cultures can be plated in Corning 96-well plates. Dulbecco's minimum essential medium plus 10% fetal bovine serum, 1.5 to 2. 5 × 10^FourCells can be plated.   Overnight ink at 37 ° C in an incubator equilibrated with 10% carbon dioxide After incubation, the medium is removed and incubated for 2 hours with medium containing the compound to be tested. The period of pre-treatment was replaced and the cells were incubated as described above. The final concentration of the compound used in the treatment, when the concentration of dimethyl sulfoxide exceeds 0.5% The stock containing the compound to be tested should not be more than about 0.1%. , First prepared in 100% dimethyl sulfoxide.   At the end of the pretreatment period, the medium is again removed and tested as described above. Replace with fresh medium containing compound and incubate cells for an additional 2-16 hours. To After processing, plates are centrifuged at 1200 rpm for 5 minutes at room temperature in a Beckman GPR. Centrifuge and pellet cell debris from conditioned media. 100 μL from each well Conditioned medium or a suitable dilution thereof is described in International Patent Application No. 94/10569. Pre-coated with antibody 266 (antibody directed against amino acids 13-28 of Aβ) And stored at 4 ° C. overnight. Labeled antibody 6C6 (Aβ ELISA assays (directed against amino acids 1 to 16) can be performed The amount of Aβ can be measured. Different capture and detection antibodies can also be used.   The cytotoxic effects of the compounds are described in Hansen et al. Immun. Method. 119: 203-210 (198 It is measured by modification of the method of 9). 25 μl of cells remaining in the tissue culture plate 3, (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) Add stock solution (5 mg / mL) to a final concentration of 1 mg / mL. 1 cell Incubate for hours at 37 ° C. and allow cell activity to equal volume of MTT lysis buffer (50% For the addition of 20% w / v sodium dodecyl sulfate in dimethylformamide, pH 4.7) Stop more. Complete extraction is achieved by overnight shaking at room temperature. OD_{562n m} And OD_650nmDifference between UV and Molecular Device_maxMicroplate reader Or its equivalent as an indicator of cell survival.   Aβ ELISA results are fitted to a standard curve and expressed as ng / mL Aβ . These results are divided by the MTT results to standardize cytotoxicity, and Percentage of results from control assays performed in the absence of compound It is expressed as   All publications cited herein are hereby incorporated by reference. . The information contained in these publications (in which respect they are cited) Generally known. Example 1: pMTAPP-1 expression in NIH3T3 and PC12 cells.   Clone pMTAPP-1 is one example of an APP770 expression construct shown in FIG. 1a. Use here The promoter used is the metallothionine promoter It is. Stable cell lines were used for the NIH3T3 and PC12 cell lines (ATCC # CCL92 and CRL1721 ) Obtained by transfection. 500,000 NIH3T3 or PC12 cells Was plated in a 100 mm dish and 50 mg in a final volume of 100 μl. 5 mg SalI fragment precipitated in the presence of lipofectin (Gibco, BRL) With a mixture of 1 mg of pSV2neo DNA (Southern and Berg (1982)). Sfected. For PC12 cells, use a plate coated with polylysine. Using. These cells usually do not adhere well to tissue culture dishes. Cells, DMEM Or grown on selective medium containing 10% fetal calf serum in RPMI and supplemented with G418 I let it. Using 500 mg / ml (biological weight) and 250 mg / ml of G418, Colonies were selected from NIH3T3 and PC12 cells. 15 days after transfection , A colony of cells resistant to G418 were isolated by cloning ring and Expanded in flask. The presence of APP cDNA in cells was determined by Mullis and Faloona, M. ethods Enzymol. 155: 335-350 (1987) (this teaching is generally known, and (Incorporated herein).   Expression of APP in 25 colonies from each cell line was determined by immunostaining (Majocha et al. (1988) ). Cells are grown to sub-confluence and 4% para Formaldehyde, 0.12M NaCl, and 20mm Na_ThreePO_FourIn a solution containing (pH 7.0) Fixed. These were synthesized Aβ sequences (Masters et al. (1985); Glenner and Wong). Primary Monoclonal Antibodies to Dr. Ronald Majocha, Massachusetts Genera l, provided by Hospital, Boston, MA) Anti-mouse antibody conjugated to otine (Jackson ImmunoResearch Labs, PA) Overnight. Then, avidin-horseradish peroxidase (H RP) (Vector Labs, Burlingame, CA) and diaminobenzide as a chromogen Immunostaining was performed by adding gin (Majocha et al. (1985)). Result is PMTAPP-1 vector expresses APP in both NIH3T3 and PC12 cells It was shown. Example 2: pEAPP-1 expression in PC12 cells.   pEAPP-1 is one example of an APP770 expression construct shown in FIG. 1a. The programs used here The promoter is a 25 kb human APP gene promoter. DNA from this construct Was transfected into PC12 cells as described above. Transfer with pEAPP-1 Certain clones of isolated cells were isolated from PC12 cells treated with nerve growth factor (NGF). Thus, it showed a differentiated phenotype morphologically similar to the differentiated phenotype indicated. PC12 cells Are usually fairly round and flat cells. Transfected with pEAPP-1 These cells have a cytoplasmic extension that resembles a neurite. Processed by NGF The treated PC12 cells extended very long neurite outgrowths. Transform with pEAPP-1 Thirteen infected PC12 cell clones were selected and propagated. These details Eight of the vesicle clones showed a spontaneous differentiation phenotype, clones 1-8, 1 -1 and 1-4 showed the strongest phenotypes. Against Aβ as described in Example 1 Staining of PC12 cells transfected with pEAPP-1 using the These cells exhibiting metabolism were also shown to express APP. pMTAPP-1 PC12 cells transfected with a loan, even if APP770 cDNA is expressed Did not show this phenotype, so these results Suggest that the expression of APP770 has novel properties with respect to cell physiology. Example 3: pMTA4 expression in PC12 cells.   pMTA4 is one example of the type of construct shown in FIG. 4a. Where used The promoter is a metallothionein promoter. This construct is the code The different proteins are slightly different from the proteins shown in FIG. 4a. APP770 cDN The A clone was digested with Asp718. This cleaves after position 57 (Kang et al. (1987 A) number system). The obtained 5 'extension site was filled in using Klenow enzyme. (Sambrook et al. (1989)). The same DNA preparation was also cut with EcoRI. This too Also cut after 2020. The obtained 5 'extension site was filtered using Klenow enzyme. (Sambrook et al. (1989)). The self-association of this molecule is thus coded Truncated protein is the leader sequence, followed by the sequence of Aβ Phe-Arg-Val-Gly-Se truncated version of Aβ beginning with r-, followed by 56 terminal amino acids of APP , Resulting in an expression clone. DNA from this construct was transferred to PC12 cells as described above. Was transfected. Example 4: Transgenic mouse expressing APP under control of MT-1 promoter Generation.   Microinjection of pMTAPP-1 vector DNA into pronuclear embryos Thus, transgenic mice were produced. pMTAPP-1 is a member of the construct shown in FIG. It is an example of a type. Where the APP770 coding sequence is the metallothionein promoter Operably connected to the For microinjection into mouse embryo Procedure is described in Manipulating the Mouse Embryo by Hogan et al. (1986). . Only a brief description of this procedure is provided below.   Mice were obtained from Taconic Laboratories (German Town, New York). Sw iss Webster female mice were used for embryo retrieval and transfer. B6D2F₁Swiss webster slaughter using male mice for mating and vasectomy Was used to stimulate pseudopregnancy.   A. Embryo recovery.   Female mice (4-8 weeks old) were given 5 IU of pregnant horse serum gonadotropin (PMSG; Sigma). ), Followed 48 hours later with 5 IU of human chorionic gonadotropin (hCG; Sigma) Superovulation was induced. Immediately after hCG injection, females were placed with males. 21:00 hCG Shortly afterwards, embryos are harvested from the resected oviducts of the mated females and 0.5% bovine serum albumin (BSA; Sigma) in Dulbecco's phosphate buffered saline. Surrounding Cumulus cells were removed using hyaluronidase (1 mg / ml). Then the pronucleus Wash embryos and add 7% CO_Two, 5% O_Two, And 88% N_TwoContains a humid atmosphere at 37. In Earle Balanced Salt Solution (EBSS) containing 0.4% BSA in a 5 ° C incubator At the time of injection.   B. Microinjection.   Elutip-D^TMSalI DNA purified in 5 mM Tris (pH 7.4) and 0.1 mM EDTA Was dissolved at a concentration of 3 μg / ml for microinjection. Microneedles and Holding pipette, Fisher coagulation tube (Fisher) of DKI model 720pipette puller Pulled from. The holding pipette was then folded at about 70 μm (0.D.) and Nar Mouth baked to about 30 μm I.D. with ishige microforge (model MF-83). Pipette , Equipped with a Narishige micromanipulator attached to a Nikon Diaphot microscope. Fold the tip against the holding pipette into the injection pipette filled with air. After that, the chip was filled with a DNA solution. Embryos (30-40 groups), micromanifold Placed in a 100 μl drop of EBSS under paraffin oil for purification. Embryo Oriented and held with a holding pipette. Next, the injection pipe Pets were inserted into male pronuclei (usually larger). Pipette does not pass through membrane If so, the stage was tapped immediately to assist penetration. Then inject the nucleus And the injection was monitored by nuclear swelling. Recipe embryo group after injection They were placed in EBSS until transfer to ent females.   C. Population.   Adult female mice with random cycles are mated with male vaccinated Swiss Webster males. I let you. Recipient females were mated simultaneously with donor females. At the time of transfer, Anesthetized with rutin. The fallopian tubes were exposed by a single midline dorsal incision. Then eggs An incision was made through the body wall just above the tube. Then, the ovarian sac is removed by tweezers for clock And teared. Embryos to be transferred were placed in DPBS and at the tip of a transfer pipette (About 10-12 embryos). Pipette tip was inserted into fallopian tube funnel and embryos transferred . After transfer, the incision was sutured with two sutures.   D. Analysis of mice for transgene integration.   At approximately 3 weeks of age, approximately 1 cm long tail samples were excised for DNA analysis. Tail Samples were prepared with 0.7 ml of 5 mM ris (pH 8.0), 100 mM DTA, 0.5% SDS, and 350 μg Incubation at 55 ° C with shaking in the presence of proteinase K Digested by doing. Extract the digested material once with an equal volume of phenol And extracted once with an equal volume of phenol: chloroform (1: 1 mixture). The supernatant was mixed with 70 μl of 3 M sodium acetate (pH 6.0) and the DNA was added to an equal volume of Precipitated by adding 100% ethanol. Spin DNA by microcentrifugation Down, wash once with 70% ethanol, dry, and 100 μl TE buffer ( Dissolved in 10 mM Tris (pH 8.0) and 1 mM EDTA).   10-20 μl of DNA from each sample is digested with BamHI and run on a 1% agarose gel. Electrophoresis, blotting on nitrocellulose paper, and³²P sign Was hybridized with the APP cDNA fragment. Transgenic animals For autoradiography of hybridized nitrocellulose filters Therefore, it was identified. This DNA is also synthesized to produce an 800 bp fragment of APP DNA. Analysis was performed by PCR using primers.   A total of 671 pronuclear embryos were microinjected, 73 of which survived A child, and six dead children, were born. 9 trans Transgenic mice (5 females and 4 males) were identified and₁and F_TwoMating was done to give rise to transgenics. These animals are As such, APP mRNA and protein expression in various tissues, and behavior It can be analyzed for analysis of sexual and pathological abnormalities. Toe with this construct Transgenic mice express transgenic RNA. Example 5: Construction of an APP construct containing a cDNA / genomic coding sequence combination.   The cDNA / genomic APP construct containing introns 6, 7, and 8 was cloned into exons 1-6 And cDNA encoding introns 6, 7, 8, and exo Prepared by combining with genomic APP sequences encoding (See FIG. 6). Small enough for convenient insertion in pUC vector Two deletions in the intron sequence to create a simple splicing cassette. Produced. In one, exon from intron 6 (position 143 of intron 6) To the BamHI site located upstream of the start of 7 (1658 bp before the start of exon 7) Was made. In the other, in intron 8 (the first in intron 8 A deletion was made from the BamHI site) to a site at 263 bp before the beginning of exon 9. . To avoid confusion, these truncated APP introns 6 and 8 are referred to herein as Referred to as intron Δ6 and intron Δ8. The BamHI site has these deletions Site, so that they are marked by the presence of the BamHI site. You. In this construct, called PDAPP, exons 7 and 8 and Ron 7 is identical to that of Intron 7, except that only one XhoI site in Intron 7 has been destroyed. It is a contact genome sequence.   DNA fragments containing truncated introns are generated as follows: Bam The HI site was engineered at 143 bp in the nucleotides of intron 6 by PCR mutagenesis. (“Mutation by PCR” PCR technology: Current Innovations (Griffith and And Griffith, CRC Press, 1994) 69-83), and another BamHI site by PCR mutation The induction manipulated 263 bp before the beginning of exon 9. These parts are Exon 6 and Intron 6, and Intron 8 and Exon, respectively Engineered and modified APP genomic DNA into another APP genomic DNA clone containing 9 junctions A clone was generated.   All cassettes were assembled into APP cDNA clones as follows (FIG. 11). Eku 889 bp BamHI-XcmI flag of APP cDNA containing sons 1 to 5 and part of exon 6 (Including nucleotides 1 to 843 of SEQ ID NO: 5) from Bam downstream of the insertion site. APP770x-oligo-x was created by cloning into a vector containing HI and XhoI sites. Was. The APP770x-oligo-x was then cut with XcmI and BamHI. Then two Fragment containing the junction of exon 6 and intron 6 described above. From the obtained APP genomic DNA clone by cutting with XcmI and BamHI. Obtained 34 bp fragment from XcmI in exon 6 to XcmI in intron 6 And artificially prepared from XcmI in intron 6 at position 143 bp of intron 6. In the three-way ligation step, 131 bp to the BamHI site Ligation into P770x-oligo-x created APP770x-E6 oligo-x. Fragment The orientation was confirmed by sequencing. Next, APP770x-E6 oligo-x was transferred to BamHI and And XhoI. Then, a modification including the junction between intron 8 and exon 9 was performed. 313 bp BamHI and XhoI fragments from the APP genomic DNA clone Ligation into x-E6 oligo-x created APP770xE6E9x.   APP770xE6E9x is then cut with BamHI and the KPI and OX-2 domains (E 6.8 kb BamHI fragment of APP genomic DNA encoding exons 7 and 8) Was inserted into the site. This fragment contains a 1658 bp Ba upstream of the beginning of exon 7. Starting at the mHI site and extending to the first BamHI site in intron 8. this The BamHI fragment is used to replace the lambda phage genome encoding this part of the APP gene. Obtained from clone. This clone is a Lambda FIXII vector obtained from Stratagene. Obtained from a human placental genomic library in the United States. This BamHI fragment Originally contained XhoI. This XhoI site cuts, fills in, and religates Destroyed by The location of the deletion is illustrated in FIG. Exons 1-8 and 9 And clones containing introns 6, 7, and 8 "Pricing cassette". Insert the APP splicing cassette into NruI and NruI-XhoI cDNA flag of APP cDNA with XyI and XhoI and having Hardy mutation Used to replace the statement. This mutant form of APP cDNA is It was made by converting G at nucleotide position 2145 to T by mutagenesis. This changes the encoded amino acid from Val to Phe. The resulting construct is , The combination of the cDNA genome APP is a “mini gene”.   APP exon 7 from the APP genomic clone used to generate this construct Sequencing of a 6.8 kb BamHI fragment containing b length and the first BamHI site in intron 8 (APP minigene The site upstream of the deletion in intron 8 engineered into the construct) is the end of exon 8. It was shown to be 2329 bp downstream. This has been demonstrated by Yoshikai et al. (1990) and Does not match the APP gene restriction map published by Yoshikai et al. (1991). By comparing their maps with our sequences, Yoshikai et al. It suggests that the order of the two EcoRI fragments was exchanged in the ping. The 1.60 kb EcoRI fragment containing exon 8 is actually a 1.48 kb EcoRI fragment. 1.48 kb which is mapped upstream in the intron 7 by Yoshikai et al. The EcoRI fragment is actually in intron 8. We have determined from human DNA By measuring the size of the fragment generated by PCR, the EcoRI plasmid containing exon 8 was determined. This position was confirmed with regard to   This APP minigene is operably linked to the PDGF-B promoter and Provides expression of the APP cDNA / genomic construct in a cell. 5 'flanking sequence of PDGF β chain Was inserted upstream of the NruI site at the beginning of the APP minigene. This fragment 1.3 kb upstream of the transcription start site (where the PDGF-B promoter is present), and Containing approximately 70 bp of the 5 'untranslated region, ending at the AurII site (Higgins et al. (1994)). 240 Late SV40 polyadenylation signal with a bp BamHI to BclI fragment Was added downstream of the APP minigene. PDGF-B promoter, APP splicein Cassette, the Hardy mutation, and the SV40 polyadenylation signal. Is referred to as PDAPP (FIG. 9). Example 6: Transgenic mouse containing PDAPP construct.   Generating transgenic mice using the PDAPP construct described in Example 5 did. Microinjection using standard techniques described above Sgenic mice were generated. PDAPP DNA is microinjected into embryos at the 2-cell stage. Injection. Before microinjection, plasmid sequence (pUC) Was removed by digestion of SacI and NotI. 7 founder mice Generated and 109 lines were used for extensive analysis. Using only heterozygous animals Was. Southern analysis of 104 animals from 4 generations showed that about 40 copies of the transzyme Was inserted at a single site and was shown to have been inherited in a stable manner. Human APP messenger RNA was produced in several tissues of transgenic mice Was produced at particularly high levels in the brain. RNA protection assays show that Quon et al. (1 991), Mucker et al., Brain Res. 666: 151-167 (1994), McConlogue et al., Neurobiol. A ging 15: S12 (1994), and Higgins et al., Ann Neurol. 35: 598-607 (1994) Driven by the previously described neuron-specific enolase (NSE) promoter Of 109 animal strains than in a mouse strain expressing the APP transgene Showed at least a 20-fold higher expression of APP.   A. Expression of APP transcripts and proteins.   RNA was purified from Chomaczynski and Sacchi, Analyt. Biochem. 162: 156-159 (1987) As described, isolated from brain tissue and human-specific APP primers (5'-CC GATGATGACGAGGACGAT-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 And Wang et al., Proc. Natl. Acad. Sci. U.S.A. 86: 9717-9721 (1989) Was subjected to RT-PCR as described above. RT-PCR analysis is performed in the brain of transgenic animals. Demonstrated the presence of transcripts encoding the 695, 751, and 770 isoforms of human APP , Did not demonstrate its presence in brain from non-transgenic littermates. G The identity of the human APPRT-PCR band from transgenic mouse RNA was Confirmed by sequencing and sequencing.   PDAPP transgenic mouse, NSE-APP transgenic mouse, non-tiger Transgenic mice and humans with and without AD Relative levels of APP transcripts in the brain and alternative splicing In a Nase protection assay (Rockenstein et al., J. Biol. Chem. 270: 2825). 7-28267 (1995)). PDAPP mice are about less than non-transgenic controls 5-fold higher total APP mRNA levels, and most NSE-APP transgenic mice Expressed at least 20-fold higher human APP mRNA levels. Driven by NSE Human APP expression does not affect mouse APP mRNA levels, but PDAPP trans The transgenic mice showed a significant 30% decrease in mouse APP transcript. Non Relative expression of mouse APP770: 751: 695 mRNA in the brain of transgenic mice Although the production was about 1: 1: 35, the brain of PDAPP transgenic mice The corresponding human APP mRNA level in was 5: 5: 1.   Analysis of holo-APP was performed using 0.5 mM EDTA, 10 μg ml^-1Leupeptin and 1 mM PMSF Was performed by homogenization of the brain in 10 volumes of PBS containing Sun The pull is spun at 12,000 g for 10 minutes and the pellet is washed with RIPA (150 mM NaCl, 50 mM  Tris (pH 8.0), 20 mM EDTA, 1.0% deoxycholate, 1.0% Triton X-100, 0.1% SDS, 1 mM PMSF, and 10 μg ml^-1(Leupeptin). Sun Pulls (each containing 30 μg of total protein) were analyzed by SDS-PAGE and obilon membrane and transfer the holo-APP antibody anti-6 (anti-Bx6) (Oltersdorf et al. . Biol. Chem. 285: 4492-4497 (1990)), or 8E5 monoclonal Reacted with any of the antibodies. 8E5 is a bacterial fusion containing residues 444-692 of human APP Prepared for proteins (Oltersdorf et al. (1990)) and human-specific. And show essentially no cross-reactivity to mouse APP. Transgenic Total APP expression in human 109 littermate and control litter brain tissue (human and And mouse) by immunoblot analysis using the C-terminal APP antibody anti-6. The transgenic mice showed much higher expression levels. Holo -APP polyclonal antibody, anti-6 or human specific APP monoclonal antibody Immunoblot analysis of brain homogenates using any of the 8E5 Transgenic mouse APP levels or APP levels in the brain of AD Overexpression of human APP in sgenic mice resulted in at least 3 Showed at twice the level.   For immunoblot analysis of Aβ, the brains of 9 month old mice were Homogenized in Niidine HCl, 50 mM Tris (pH 7.5). Homogenate, 100,0 Centrifuge at 00g for 15 minutes and wash the supernatant with H_TwoO overnight, dialysate 1 mM PMSF and And 25μg ml^-1Adjusted to PBS containing leupeptin. Using this material with antibody 266 resin And immunoprecipitated, and described by Seubert et al., Nature 359: 325-327 (1992). Was performed using the human-specific Aβ antibody 6C6. This 4kD β-amyloid immunoreactive peptide using human specific Aβ antibody (6C6) Was isolated from the brains of transgenic animals. This is due to the relative molecular weight of Aβ Matches. Aβ levels in the brain were measured using the previously described human APP transgenic matrix. At least 10 times higher in 109 strains of animals than in mice. Trance Early 16-day cortical cell cultures from transgenic animals are available from Seubert et al. (1992) and And as described by McConlogue et al. (1994) and in Example 8. As detected in the medium by a human-specific Aβ enzyme-linked immunosorbent assay A substantial fraction of Aβ 1-42 (5 ng ml^-1Of total Aβ; 0.7 ng ml^-1Aβ1-42) Was secreted constitutively. Thus, 109 strains of animal Highly overexpressed human APP mRNA, holo-APP and Aβ.   B. Histopathology of PDAPP transgenic mice.   180 transgenic and 160 months old, corresponding to 5 generations of 109 lines Comparable to non-transgenic moons of comparable control (4 to 20 months of age) Brains from the sources were extensively examined histopathologically. Take out some mouse brain, And placed in an alcohol fixative for 48 hours, then embedded in paraffin (Arai et al., Proc. Natl. Acad. Sci. U.S.A. 87: 2249-2253 (1990)). Menstrual diet of other mice Perfused with saline followed by 4% paraformaldehyde in 0.1 M sodium phosphate. Transgenic and non-transgenic brains embedded in paraffin A 6 μm coronal or parasaggital section from a synthetic mouse was subjected to poly-L- Placed close to each other on lysine coated slides. Remove the section Raffinized, rehydrated, and 0.03% H_TwoO_TwoFor 30 minutes, then 1: Aβ antibody at a dilution of 1,000, R1280 (Tamaoka et al., Proc. Natl. Acad. Sci. U.S.A. 89: 1345-1349 (1992)) at 4 ° C. overnight. For adsorption experiments For example, synthetic human Aβ1-40 peptide in 10% aqueous dimethyl sulfoxide (Games Et al., Neurobiol. Aging 13: 569-576 (1992)) to the diluted antibody 7.0 μM and incubated at 37 ° C. for 2 hours. Dilutions are sectioned Applied and processed under the same conditions as the standard antibody solution. Next, Peru Use the oxidase rabbit IgG kit (Vector Labs) as a chromogen, as recommended. Used with 3,3'-diaminobenzidine (DAB). Similarly fixed human AD brain Were processed simultaneously under the same conditions.   Prior to 4 months of age, no apparent Aβ deposition was detected. But until about 4 months old In addition, the transgenic animals have human Aβ in the hippocampus, callosum and cerebral cortex. Began to show deposition. These Aβ plaques increase with age and 8 By age, numerous deposits of 30-200 μm were observed. If the animal is older than 9 months Plaque density increases until the Aβ staining pattern resembles that of AD. Added. Vascular amyloid, another characteristic of AD pathology, occurs in older mice Was. Strong pathology is also due to another transgenic line produced from the PDAPP vector. Were also observed in the Tohoku line (35 lines).   Various forms of Aβ deposition can be attributed to various Aβ antibodies (well-characterized human specific Specific for Aβ antibodies and the free amino and carboxy termini of Aβ 1-42 Was clearly evident as a result of the use of Antibody 9204 (Saido et al. J. Biol. Chem. 289: 15253-15257 (1994)) is specific for Aβ1-5 And 7.0 μg ml of this^-1Used at a concentration of Specific for Aβ 1-42 Antibody 277-2 was prepared using standard immunization protocol (500 μg per injection) Cysteine-amino linked to activated BSA ("Super Carriers"; Pierce) Prepared by immunizing New Zealand white rabbits with heptanoic acid-Aβ33-42 did. Specific antibodies were raised against the immunogen immobilized on agarose beads. Was subjected to affinity purification. Before incubation with antibody 277-2, section Was treated with 80% formic acid for 1-2 minutes. For detection, the antibody is The reaction was carried out using a rabbit IgG kit (Vector Labs). The product is then dyed Visualized using DAB as source. Then, several sections were taken at a 1: 500 dilution. Incubated overnight at 4 ° C. with riclonal anti-GFAP (Sigma). GFA P antibody was purified using alkaline phosphatase anti-rabbit IgG kit and alkaline phosphatase. Using Vatase Substrate Kit 1 (Vector Labs; used according to manufacturer's recommendations) Reacted. Additional sections were prepared with the F480 antibody (Serotec) used at a 1:40 dilution. Both were incubated overnight to visualize microglia. Next, The luoxidase kit (Vector Labs) was used according to the manufacturer's recommendations. How many The sections were prepared according to standard procedures (Dickson et al., Acta Neuropath. 79: 486-493 (1990)). Stained with Thioflavin S, using a FITC filter with a maximum wavelength of 440 nm And examined using ultraviolet light passed through.   Serial sections show numerous plaques positive for both 9204 and 277-2 antibodies It was proved that it was stained. Is the form of Aβ deposition diffused and irregular? From plaques to concentrated plaques with cores. Almost spherical and nebula-like Random deposits were labeled with an antibody 9204 specific for the free amino terminus of Aβ. Astrocyte gliosis associated with Aβ deposition involves collagen fibrillary acidic protein (GFAP) And after double immunolabeling with an antibody against human Aβ. concentrated Aβ core and “halo” were apparent in some plaques . Non-transgenic littermates did not show these neuropathological changes . Immunostaining is sufficiently adsorbable with the relevant synthetic peptides and Processing conditions (including fixation with paraformaldehyde and Trojanowski method) )). Many plaques were stained with Thioflavin S and Some are also stained using the Bielschowsky silver method, and with Congo Red Birefringent, indicating the true amyloid nature of these deposits.   Most of the plaques, like the gliosis found in AD plaques, Individually surrounded by GFAP-positive reactive astrocytes. Transgenic The mouse neocortex has an amoebic appearance, shortened protrusions, and It contained widely activated microglia, as defined by staining. Rin Staining with antibodies recognizing oxidized neurofilament and phosphorylated tau Colors indicate that abnormal phosphorylation occurred in the brain of PDAPP similar to AD . These phosphorylations are observed in AD and the formation of neurofibrillary tangles It is believed to hinder. Paired Helical Filament (PHF) is still a PDA Abnormally phosphorylated neurofilament not detected in PP mice And tau detection is associated with the formation of PHFs in AD, and It is believed that there is.   Clear evidence of neurite pathology was obtained using both conventional and confocal immunomicroscopy. it is obvious. Sections of a 40 µm thick vibratome are Null anti-synaptophysin (1: 150; Dako) or 8E5 (7.0 μg)^-Combine with 1) And incubated at 4 ° C. overnight with R1280 (1: 1000). A few Sections from anti-synaptophysin or monoclonal anti-MAP2 (1:20, Boehrin ger-Mannheim) and then goat anti-rabbit biotin Antibody (1: 100) followed by FITC-conjugated horse anti-mouse IgG (1:75) and And Avidin D Texas Red (1: 100) (Vector Labs) . Double immunolabeled sections were analyzed using the laser confocal scanning system MRC600 (Bio-Ra Examined on a Zeiss Axiovert 35 microscope fitted with d). Texas Red Channel Collects R1280 or synaptophysin labeled images, and FITC channel Collected synaptophysin, 8E5, or MAP2 labels. 0.5μm thick optical z -Sections were obtained from Masliah et al. Neuropath. Exp Neurol. 52: 619-632 (1993) Collected from each area as well as image processing and storage.   Many Aβ plaques have been identified with human APP-specific and anti-synaptophysin antibodies. Closely related to the distorted neurites that can be detected, this means that these neurites Suggests that it is partially derived from axonal buds, as observed in AD brains Was. Plaque also compresses and distorts the surrounding neural network, like the brain in AD. Was. Synaptic and dendritic densities are also important in hippocampal teeth of transgenic mice. Decreased in the molecular layer of gyroid This is a presynaptic marker in the AD brain For synaptophysin, a steroid, and MAP-2, a dendritic marker (Masliah et al., Am. J. Path. 138: 235-246 (19 91)).   Confirmation of the presence of extracellular Aβ was obtained using immunoelectron microscopy. Immunoelectron microscope For the method, mice were perfused with saline, followed by 2.0 ml in cacodylate buffer. Perfused with 1.0% paraformaldehyde and 1.0% glutaraldehyde. 40μm thickness Vibratome sections were incubated with the R1280 antibody and The reaction was carried out using a luoxidase rabbit IgG kit (Vector Labs). Then Immunolabeled sections with Aβ deposition were fixed in 1.0% ammonium tetraoxide. And embed in Epon / Araldite, ultra-fine on a Jeol CX100 electron microscope Thin sections were examined (Masliah et al., Acta Neuropath. 81: 428-433 (1991)).   Tables 3 and 4 provide a summary of the above results and the cytology between AD and PDAPP mice. Shows similarity and pathological similarity. Paired helical for all features examined Except for the filaments, PDAPP mice showed pathology characteristic of AD. these The findings indicate that human APP production in transgenic (TG) mice Causes not only deposition but also many of the complex subcellular degenerative changes associated with AD To indicate that it is enough.   The most prominent feature of these transgenic mice is their Alzheimer's -Like neuropathology. This involves local similarities similar to those of AD , Extracellular Aβ deposition, dystrophic neurite component, gliosis, and Includes loss of pass density. The plaque density of these transgenic mice As in humans (Selkoe, Rev. Neurosi. 17: 489-5). 17 (1994)), increased with age, which has also been proposed for AD ( Maggio et al., Proc. Natl. Acad. Sci. U.S.A. 89: 5462-5466 (1992)) Mean progressive Aβ deposition over clearance. PDAPP transgenic Mice are the first potent new candidate for APP expression and Aβ deposition in AD neuropathology Provide evidence. Such mice can also have A Compounds that reduce beta production and / or reduce their neurotoxicity in vitro To test whether an object can produce a beneficial effect in this animal model, Provide a sufficiently robust AD model. Example 7: Construction of an APP transgene expressing APP from the PDGF-B promoter.   PDAPP transgenic mice express all three major human APP isoforms. Includes a splicing cassette that allows for realization. The expression here is the PDGF-B promoter. And this is amino acid 71, the site of familial AD mutation 7 contains mutations. These properties, and the other properties listed above, are used independently to Producing transgenic mice useful as a model for Zheimer's disease Is expected. Some specific examples of such constructs are described below.   A. Construction of PDAPP-wt.   CDNA / genomic clone PDAPP in which the mutation for amino acid 717 has been replaced with the wild type A wild-type version of was constructed. This is from the 1448 bp XhoI to SpeI of PDAPP. Fragment (this is the APP cD encoding Hardy mutation in which Val717 has been replaced by Phe NA fragment) containing the 1448 bp XhoI to SpeI fragment of the wild-type APP clone. Achieved by replacing with ragments. This fragment is SEQ ID NO: 5 Corresponds to the region from position 1135 to position 2588. All intron sequences of PDAPP Not replaced or removed by substitution. This construct is called PDAPP-wt. PDAP A schematic diagram of p-wt and its construction is shown in FIG.   B. Build PDAPP-SwHa.   CDNA / genomic clone PDAP with Swedish mutations introduced at amino acids 670 and 671 Built another version of P. Plasmid pNSE751._delta3'spl.sw contains amino acids Swedish mutations from Lys to Asn and Met to Leu at 670 and 671, respectively Includes human APP751 cDNA. From 563 bp EcoRI to SpeI derived from this plasmid Fragment containing the same portion of the APP cDNA sequence except for the Hardy mutation Phe717. Replace with the corresponding 563 bp EcoRI to SpeI fragment of PDAPP did. This fragment corresponds to the region from position 2020 to position 2588 of SEQ ID NO: 5. You. This is pNSE._deltaBring 3'spl.sw / ha. It consists of amino acids 670 and 67 Includes both the Swedish mutation at 1 as well as the Hardy mutation at amino acid 717.   The 1448 bp XhoI to SpeI fragment was then replaced with the Swedish mutation and Har. pNSE752 containing both dy mutations._delta3'spl.sw / ha 1448 bp XhoI to SpeI The fragment was replaced to form PDAPP-Sw / Ha. Overview of PDAPP-Sw / Ha and its construction A schematic is shown in FIG.   C. PDAPP695_VFBuilding.   Encodes APP695 only, but has the Hardy mutation, PDGF-B promoter, and PDAPP Constructs can be made that retain the vector sequence. This is the C-terminal part of the APP sequence, And PDAPP-derived containing polyadenylation, pUC, and PDGF-B promoter sequences A 6.6 kb fragment from XhoI to NruI was hybridized to a hybrid splice signal. And the remaining N-terminal part of the APP sequence (from 911 bp XhoI to NruI of APP695 cDNA) To the 1.2kb XhoI to BclI fragment of pCK695 This can be achieved by linking. The hybrid splice signal is And the vector pohCK751 (this is described in Dugan et al., J. Biological  Chem. 270: 10982-10989 (1995)). pCK695 is Herpes simplex virus replication and packaging sequences of pohCK751 have been removed That this plasmid encodes APP695 instead of APP751 Except for, it is the same as pohCK751.   In this vector, the PDGF-B promoter contains an APP695 containing a Val717 to Phe mutation. Drives the expression of Hybrid splice signal potentially enhances expression To be included. Further vectors derived from this (any splice signal Or the addition of another splice signal to perform the same function. Get) can be built.   D. PDAPP751_VFBuilding.   Encodes only APP751 but has the Hardy mutation, PDGF-B promoter, and PDAPP Constructs carrying the vector sequence can be made. This is part of the APP sequence, poly 6.65 kb XhoI containing adenylation signal, pUc, and PDGF-B promoter sequence Fragments from PDAPP to KpnI were replaced with the remainder of the human APP751 cDNA sequence (SEQ ID NO: 1.0 kb KpnI to XhoI fragment containing the 3 nucleotides 57-1084) Ligation, the intermediate plasmid PDAPPδsp751_VFCan be achieved by making You. The 1.0 kb KpnI to XhoI fragment encoding the portion of human APP751 is: Plasmid poCK751 (this means that the herpes simplex virus sequence has been removed Except that is identical to pohCK751).   Then, to introduce the splicing sequence, the first intron of PDAPP ( This is intron Δ6), PDAPPδsp751_VFInsert it inside PDAPP751_VF Is prepared. To achieve this, exons 2 through 6, intron Δ6, and 2,758 bp Asp718 to ScaI fragment of PDAPP containing exon 7 and part of exon 7 The PDAPPδsp751_VFObtained by complete digestion with Asp718 and partial digestion with ScaI Ligated to a 6,736 bp fragment. This 6,736 bp fragment Additional APP sequences (part of exon 1, rest of exon 7, and exon 9 18), providing a polyadenylation signal, pUC, and a DGF-B promoter sequence I do. The resulting construct is called PDAPP751_VFThat.   In this vector, the PDGF-B promoter has an A containing a mutation of Val717 to Phe. Drives expression of PP751. One splicing signal (from intron 6) Is included to potentially enhance expression. Further veins derived from this (Any splice signal deleted or other splice signal To obtain the same function).   E. FIG. PDAPP770_VFBuilding.   Encodes APP770 only, but has the Hardy mutation, PDGF-B promoter, and PDAPP Constructs carrying the vector sequence can be made. This is exon 2-7 of APP And PDAPP751 containing part of exon 9_VFFragment from KpnI to XhoI Was converted from KpnI to XhoI of APP770 cDNA containing exon 2 to 8 and part of exon 9. Can be achieved by substituting This fragment , SEQ ID NO: 5, corresponding to nucleotides 57-1140. The resulting construct is PDAPP770_{V- F} That.   In this vector, the PDGF-B promoter has an A containing a mutation of Val717 to Phe. Drives expression of PP770. PDAPP770_VFIs PDAPP751_VFIntrons inside Contains the same intron sequence as the sequence. Further vectors derived from this (splice A signal is added to gain enhanced expression). Example 8: Expression level of APP expression product in PDAPP mouse brain tissue.   The strains of PDAPP mice described in Example 6 were used for the hippocampus, cortex, And the levels of several derivatives of APP in cerebellar regions. β section APP cleaved at the retase site (APPβ) and at least 12 amino acids of Aβ The level of APP (FLAPP + APPα; mixture of APPα and full length APP (FLAPP)) was evaluated At all ages, it was found to be nearly constant within a given brain region. Sea Horses expressed the highest levels of all APP forms. In contrast, Aβ guanine Gin extractable levels were determined by immunohistochemically observed amyloid plaque precipitation. A marked age-dependent increase was seen in a manner that reflected dressing. Specifically, A in the hippocampus The level of β is up to 8 months of age compared to the level found in 4 month old animals. 17-fold, and 106-fold by age 1. At one age, Aβ is the total tan in the hippocampus It makes up about 1% of the protein. The cerebral cortex also has a significant increase in Aβ with age. Indicated. In contrast, the mean level of Aβ in the cerebellum was across all age groups. Relatively low, and did not change.   Aβ_1-42Analysis of Aβ in these brains using ELISA specific for This longer version provides 19 pmol / g of Aβ present in the brain of young animals. Of 690 pmol in 12-month-old animals / g increased to 97%. Aβ_1-42And the spatial distribution of Aβ deposition are: Ongoing pathological processes in PDAPP transgenic mice There is further evidence that it resembles a Ruzheimer's disease state.   Aβ, Aβ_1-42Cleaves at the β-secretase site APP (Seubert et al. (1993)) and an APP containing the first 12 amino acids of Aβ (FLA PP + APPα; specific for a mixture of full-length APP and α-secretase-cleaved APP (Esch et al.) Measured by use of an ELISA formed with the antibody. Local changes and Aβ deposition Significant similarities in both morphology are noted between mouse models and human AD status It is. This result also indicates that the magnitude and temporal prediction of Aβ deposition is possible. , PDAPP mice inhibit the processing of APP to Aβ or Demonstrates a practical model for testing drugs that delay idosis You.   A. Materials and methods.     1. Preparation of brain tissue.   Heterozygous transgenic (109 strains, Games et al .; Rockenstein et al.) And non-transgenic animals with Nembutol (1: 5 solution in 0.9% saline) ) And perfused intracardially with ice-cold 0.9% saline. Take out the brain And one hemisphere was prepared for immunohistochemical analysis. On the other hand, four brains The regions (cerebellum, hippocampus, thalamus, and cortex) are dissected from the other hemisphere and Aβ And used for APP measurements.   Use a motorized pestle (Kontes) to prepare each brain area to prepare tissue for ELISA And 10 volumes of ice-cold guanidine buffer (5.0 M guanidine HCl, 50 mM Tris-Cl (PH 8.0)). The homogenate is incubated at room temperature for 3-4 hours or gently mix on a plate and then assay directly or Stored at -20 ° C before quantification. Preliminary experiments indicate that analytes are safe under these storage conditions. It was shown to be constant.     2. Aβ measurement.   The brain homogenate was treated with ice-cold casein buffer (0.25% casein, phosphate buffered saline). Physiological saline (PBS), 0.05% sodium azide, 20 μg / ml aprotinin, 5 mM Further diluted 1:10 with EDTA (pH 8.0), 10 μg / ml leupeptin, and The final concentration was reduced to 0.5 M and then centrifuged (16,000, 20 min, 16,000 Xg). Aβ standard (1-40 or 1-42 amino acids) with 0.1% bovine blood in final composition It was prepared to contain 0.5M guanidine in the presence of clear albumin (BSA).   The “total” Aβ sandwich ELISA is a two monoclonal antibody against Aβ (m Ab). The capture antibody, 266, is specific for amino acids 13-28 of Aβ (S eubert et al. (1992)) On the other hand, antibody 3D6 specific for amino acids 1 to 5 of Aβ And is acting as a reporter antibody. The 3D6 biotinylation procedure requires 100 mM Except for using a sodium carbonate (pH 8.5) buffer, the N Use the manufacturer's (Pierce) protocol for HS biotin labeling. 3D6 antibody Does not recognize secreted APP or full-length APP, but does not recognize the amino terminus of aspartic acid. Only the detected Aβ species are detected. This assay has a sensitivity of about 50 pg / ml (11.4 pM) And at concentrations up to 1 ng / ml against endogenous mouse Aβ peptide No cross-reactivity was shown.   Aβ_1-42The construction of the specific sandwich ELISA is for amino acids 33-42 of Aβ Use the generated mAb 21F12. This antibody can be used for ELISA or competitive radioimmunoassay. Aβ in any of the_1-40Shows less than 0.4% cross reactivity You. Biotinylated 3D6 is also a reporter antibody in this assay, Has a lower limit of sensitivity of about 125 pg / ml (28.4 pM).   266 and 21F12 mAbs were placed in a 96-well immunoassay plate (Costar) Coated at 10 μg / ml overnight at room temperature. The plate is then aspirated, and Block with 0.25% human serum albumin in PBS buffer for at least 1 hour at room temperature And then dried and stored at 4 ° C. until use. Plate before use Was rehydrated with wash buffer. Add samples and standards to plate, and Incubated for 1 hour at room temperature. Plates are placed between each step of the assay. Wash at least three times with wash buffer (Tris buffered saline, 0.05% Tween 20) Was.   Casein assay buffer (0.25% casein, PBS, 0.05% Tween 20 (pH 7.4)) Biotinylated 3D6 diluted to 0.5 μg / ml in the wells for 1 hour at room temperature It was cubified. Avidin-HRP diluted 1: 4000 in casein assay buffer (Vector, Burlingame, CA) is added to the wells and incubated for 1 hour at room temperature. Was an option. Slow TMB-ELISA (Pierce), a colorimetric substrate (100 μl), was added. And allowed to react for 15 minutes, after which the enzyme reaction was_TwoSO_FourStop at . Reaction products are quantified using Molecular Devices Vmax and absorbance at 450 nm and 650 nm. The difference in degrees was measured.     3. APP ELISA.   Two different APP assays were utilized. The first is APPα and full-length form Recognizes APP (FLAPP + APPα), while the second recognizes APPβ (Aβ domain Recognizes APP ending with a methionine in front of a () (Seubert et al. (1993)). FLAPP + The capture antibody for both the APPα and APPβ assays was human APP amino acids 444-59. Monochrome evoked against bacterially expressed fusion protein corresponding to 2 The null antibody is 8E5 (Games et al.). Reporter for FLAPP + APPα assay -MAb (2H3) was generated for amino acids 1-12 of Aβ. For 8E5 / 2H3 assays The lower limit of sensitivity is about 11 ng / ml (150 pM). About the APPβ assay Used polyclonal antibody 192 as the reporter. This antibody is called antibody 92. It has the same specificity (Seubert et al. (1993)). That is, β-secretase cleavage of APP Specific for the carboxy terminus of the cleavage site. β secretase 8E5 / 192 ass The lower limit of sensitivity for A is about 43 ng / ml (600 pM).   For both APP assays, apply 8E5 mAb to 96 wells as described above for 266. Coated on Costar plates. APPα secreted by the purified recombinant (APP 751 form) and APP596 used in FLAPP + APPα and APPβ assays, respectively Was the reference standard to be used. APP was purified as previously described (Esch et al.) And the concentration of APP was determined by amino acid analysis. 5M guanidine brain homogen Nate samples were prepared in a final buffer composition of 0.5M NaCl, 0.1% NP-40, 0.5M guanidine. 1:10 dilution in sample diluent. APP standards for each assay Was diluted in a buffer of the same final composition as the sample. APP standard and sample Plates were added and incubated at room temperature for 1.5 hours. Plate Washed thoroughly with wash buffer during each step of the assay. Reporter antibody 2H3 and 192 were biotinylated according to the same procedure as 3D6 and at room temperature for 1 hour, Incubated with sample. 1: 1000 dilution in sample diluent Streptavidin-alkaline phosphatase (Boehringer Mannheim) Incubated in the wells for 1 hour at warm. 4-methyl-umbe, a fluorescent substrate Add riferyl-phosphate and plate to Cytofluor^TM2350 (Millip ore) at 365 nm excitation and 450 nm emission.     4. Production of monoclonal antibodies.   Immunogens for 3D6, 21F12, and 2H3 were converted to maleimidohexanoyl-N-hydr Sheep anti-mouse immunoglobulin (Jack) using roxysuccinimide (Pierce) son Immunoresearch Labs). A / J mouse (Jackson Laboratori es), 100 μg of the appropriate lysate emulsified in Freund's complete adjuvant (Sigma) An intraperitoneal injection (IP) of the epithelium and Freund's incomplete Two subsequent IP injections of 100 μg of immunogen in Subant (Sigma) were performed. Third time Two to three weeks after the boost, the highest titers of a given immunogen are given 5% in PBS. Intravenous and intraperitoneal injections were used with 0-100 μg of immunogen. 3 days after injection , Remove the spleen, isolate the spleen cells, and combine with SP2 / 0-Ag14 mouse myeloma cells Fused. Hybridoma supernatants were described previously (Seubert et al. (1992)). Screening for high affinity monoclonal antibodies by RIA Was. Purified monoclonal antibodies were prepared from ascites fluid.     5. Immunohistochemistry.   Tissue from one brain hemisphere from each mouse was dropped into 4% paraformaldehyde Drop-fix and post-fix for 3 days. Tissue, coronal Embed coronally, and collect 40 μm sections using a vibratome did. Sections were stored in antifreeze at −20 ° C. and then stained. Behind the cortex Every 6 sections from to the hippocampus using biotinylated 3D6 at 4 ° C overnight Immunostaining was performed. The sections were then subjected to horseradish peroxidase avidin-biotin. Incubate with the complex (Vector) and add 3,3'-diaminobenzyl The color was developed using gin (DAB) as the chromogen.   B. result.     1. Aβ and APP assays.   FLAPP + APPα assay recognizes secreted APP containing the first 12 amino acids of Aβ I do. Reporter antibody (2H3_1-12) Is between amino acids 16 and 17 of Aβ Because it is not specific for the alpha clip site (Esch et al.), The assay also recognizes full-length APP. Full-length APP to deplete the mixture Preliminary experiments using immobilized APP antibodies against the cytoplasmic tail of FLA It is suggested that about 30-40% of PP + APPα is full length. APPβ assay Due to the specificity of the clonal reporter antibody 192, Recognizes only clipped APPs (Seubert et al. (1993)).   The specific nature of the immunoreactivity of Aβ was further characterized as follows. Brain (small Guanidine homogenate (excluding brain and brain stem) is subjected to size exclusion chromatography. (Superose 12) and fractions obtained were analyzed using a total Aβ assay. Was analyzed. Homogenates of the brains of 2, 4 and 12 month old transgenic mice And the homogenate of the non-transgenic mouse brain (this Aβ at levels approximately equivalent to those found in sgenic mice_1-40Add (Spike)). Transgenic brain homogene The elution profile of the plate showed that the fraction of the peak of Aβ immunoreactivity Broad symmetric peaks (this is due to the added Aβ_1-40And immunoreactivity peaks ). Next, an antibody against Aβ (Aβ_13-28 266), an antibody against the secreted form of APP (AP in the form of APP695)_P444-592To MAb 8E5), an antibody against the carboxy terminus of APP (APP695 form of APP)_676-695To MAb 13G8) or heparin agarose bound resin to An attempt was made to immunodeplete immunoreactivity. Only 266 resins have Aβ immunoreactivity Capture, full-length APP or the carboxy-terminal fragment of APP contributes to the measurement of Aβ Not demonstrated. Aβ_1-42ELISA is Aβ_1-42Aβ_1-40Recognize Use no capture antibody. As with the Aβ assay, Aβ_1-42Assays are similar As shown by immunodepletion studies, up to the full length, including the Aβ region, in the homogenate. Or unaffected by the carboxy-terminal form of APP.     2. Measurement of total Aβ and APP.   Table 5 shows the transgenic mouse hippocampus, cortex, cerebellum, and Shows the levels of total Aβ, FLAPP + APPα, and APPβ in the and thalamus. Each day Data points indicate the average value for each age group. FLAPP + in all four brain regions The relative levels of APPα and APPβ remain relatively constant over time. Hippocampus Expresses the highest levels of FLAPP + APPα and APPβ, and the thalamus, cortex, and cerebellum Follow each. In the hippocampus, FLAPP + APPα levels are higher than APPβ at all ages About 3.5 to 4.5 times higher. All for FLAPP + APPα and APPβ assays in hippocampus The mean values for all ages were 674 (± 465) pmol / gram and 175 (± 11) pmol, respectively. / G. From this, the pool of APP in the brain is about 50% APPα, 30% It can be estimated to consist of full length APP, and 20% APPβ. AD = not measured   In contrast to APP levels, Aβ levels are associated with aging in the hippocampus and cortex. Dramatically increased. However, such an increase was observed in PDAPP transgenic mice. Was not shown at all in the cerebellum and a modest increase was observed in the thalamus ( Table 5). The increase in Aβ is higher in the hippocampus compared to the cortex. This is also , Also correlate with 3D6 immunohistochemistry results (see discussion below). 4 months old Aβ levels were 10 times higher by 8 months of age, and At age 41 (660 ± 380 pmol Aβ / gram tissue at 12 months of age). In the hippocampus The corresponding increase in Aβ observed was 8 months at 15 times the age of 4 months, and 12 months. It increases up to 106-fold at the age of a month (4,040 ± 1750 pmol Aβ / gram tissue at the age of 12 months), Even more impressive. At 12 months of age, Aβ is a protein in the hippocampus of PDAPP mice About 1% of   To investigate whether a dramatic increase in brain Aβ concentration was due to amyloid deposition, The authors then used the opposite mouse hemisphere as the mouse used for Aβ measurement. Aβ deposition was visualized immunohistochemically. Significantly, Aβ plaque load (burden ) And a parallel increase in Aβ levels. These findings are based on ELISA. Increase in brain Aβ concentration determined by age-dependent amyloidosis Show.     3. Aβ in the brain of transgenic mice_1-42Measurement.   Aβ in the cortex of transgenic mice_1-42At different ages did. As shown in Table 6, Aβ in the cortex of transgenic mice_1-42so The proportion of certain Aβ also increases with age. ELISA data is Aβ_1-42But Preferential deposition in transgenic mice and mAb 3D6 The deposits detected by immunostaining of_1-42Implies that     4. Aβ immunostaining in PDAPP transgenic brain.   Transgenic animals with Aβ values representing the mean Aβ value of the aged group were Used for epidemiological staining. Progression of Aβ deposition in animals at 4, 8, 10 and 12 months of age Is observed in At 4 months of age, the transgenic brain has small sparse spots Included a globular deposit (20 μm in diameter). This includes the hippocampus, and the frontal cortex and cingulate Only rarely observed in the bundle cortex. By eight months of age, these areas Has a large number of thioflavin positives that form plaques as large as 150 μm in diameter. Aβ aggregates were included. At 10 months of age, numerous large Aβ deposits were found in the frontal cortex and It was found throughout the cortex, as well as throughout the hippocampal molecular layer. Entryal Teeth undergoing afferent perforant pathways from the cortex The outer molecular layer of the gyrus clearly outlined Aβ deposition. This common putter Was more pronounced at one age due to greater Aβ deposition. Aβ deposition Anatomical localization is strikingly similar to that observed in Alzheimer's disease doing.   C. Consideration.   Aβ amyloidosis is an established diagnostic criterion for Alzheimer's disease (Mi rra et al., Neurology 41: 479-486 (1991)), and more in affected subjects. It is consistently observed in high cortical areas as well as hippocampal formation in the brain. Aβ amylo Idosis is a relatively early event in the pathogenesis of AD, followed by a complex Leading neuronal dysfunction and dementia via cades (Mann et al., Neurodegen eration 1: 201-215 (1992); Morris et al., Neurology 46: 707-719 (1996)). APP Various pathways of processing have been described (Schenk et al., J. Med. Chem. 38: 4141-4154). (1995)), which includes the major APP cleavage that occurs with Aβ α-secretase pathway (Esch et al.), and the cleavage of APP at the N-terminus of Aβ Amyloidogenic pathway or β-secretase pathway (Seubert et al. (1993) ) Is included. Further cleavage of the APP results in the Aβ form (Aβ form ending at position 40 (Aβ form_{1 -40} ) Or the Aβ form ending at position 42 (Aβ_1-42) Leads to a constitutive production of PDAPP Ma ELISA detecting specific APP products emanating from these individual pathways in the mouse brain Differential processing of APP may lead to region specificity of amyloid formation and deposition. Or to contribute to temporal specificity.   Aβ amyloid deposits observed in PDAPP mouse brain are highly age-specific And is region specific. Amyloid deposition begins at about 7 months of age, By 12 months of age, amyloid deposits have formed in rostral areas of the hippocampus and cortex Very deep-rooted throughout. Age-dependent increase in amyloid deposition was determined by ELISA assay As measured, well correlates with the dramatic increase in Aβ levels in these brain regions Related. The increase in Aβ is measurable by 7 months of age and by 10 months Horses have 2180 pmol / g of Aβ (which is equal to the concentration of cytoskeletal proteins, and Comparable to levels found in the cortex of the human AD brain (Gravina et al., J. Biol. Ch. em. 270: 7013-7016 (1995))). Cerebellum (unaffected) Aβ levels in the (brain area) are still 4 pmole / g (levels at 4 months of age). Amyloid precipitate as determined by histological analysis) Correlates again with wearing. These results indicate that in aged PDAPP mice, Aβ Bell reflects amyloid load and therefore direct immunoassay for Aβ levels in the brain The assay can be used to test for compounds that reduce amyloid plaque burden. And   PDAPP mice, individuals with Down syndrome, and individuals with certain forms of FAD In this regard, Aβ overproduction occurs not only in Aβ deposition but also in subsequent neuropathology. Arguably an accelerating factor (Citron et al., Manm et al., Miller et al., Archives o f Biochem. Biophys. 301: 41-52 (1993)). Aβ observed in PDAPP mice Some significant comparisons between measurements and those reported for AD brain tissue Similarity is indicated. For example, in PDAPP mice, young mice (2 months old) versus The relative levels of Aβ peptide in the hippocampus from aged mice (10 months old) were approximately 100 times. Similar findings show comparison of control brain tissue to AD brain tissue In Gravina et al. Aβ levels in the brain in PDAPP mice The rise is quite pronounced at ages between 6 and 9 months. Furthermore, this time course In an accelerated manner, down syndrome brain tissue (where amyloid deposits last for about 30 years) Observed at age (Mann) starting at age and substantially increasing to about 60 years of age (Mann) Similar to what is.   In summary, the above results indicate that a reproducible increase in measurable Aβ Occurs in human brain tissue and this increase correlates with the severity of amyloid deposition And These findings demonstrate pharmacologically Aβ peptide To identify drugs or compounds that reduce or affect the production of To gain. Example 9: Behavioral differences in PDAPP transgenic mice.   Alzheimer's disease involves cognitive deficits (including memory loss and impaired memory function) More characterized. Disclosed transgenic mice show similar defects To determine whether transgenic (TG) mice and non-transgenic Transgenic (nTG) mice to test working and reference memory The three types of maze devices used (Y maze, radial arm maze (RAM), and The task performance in the water maze was evaluated. Test The transgenic mouse derived from the PDAPP mouse described in Example 6 Equivalent to generation. Using the Y maze and the radial arm maze, one of the working memories We evaluated the function, spontaneous alternation. About the Y maze task The mouse into the stem of the Y maze twice, each time selectively invading one of the arms Made it possible. Penetration of both arms requires the memory of the previously entered arm. Successful change, while failing to enter the same arm for both attempts It is. By chance the correct response rate is a 50% change, ie 50% of the mice change.   For the radial arm maze task, multiple mice extending radially from the center of the mouse Place in the center of the maze with the game. In the test below, the radial eight arm Maze was used. By allowing only eight intrusions, different Using the number of frames as the measurement of the correct response rate, the change correct response rate is measured. Different arm penetration Can be compared to the number of different arm intrusions predicted by chance. This coincidence , 5.25 (Spetch and Wilkie, "A program that stimulates random cho ices in radial arm mazes and similar choice situations '' Behavor Research  Methods & Instumentation 12: 377-378 (1980)). The coincidence of the positive response rate That is, 5.25) above requires the storage of previously invaded arms.   The water maze used for the tests described below was a pool of water on which a flooded table was placed. Consisting of This hidden platform (HP) may be accidental (first attempt) or Either by remembering the clues of the position visible from the link (subsequent trials) And can be found by swimming mice. Subject mice are subjected to standard procedures. Trained on a hidden platform task. Easily move your mouse to a small pooh First (47 cm diameter, 20 cm table), which allows mice to How to proceed, the platform is the goal, there is no other way out, and the side of the pool Find that you have to resist the intrinsic nature of mice that stay along Educated. Then a larger pool and a smaller platform (71cm pool, 9cm Mouse) to find the position of a single platform in the hidden platform task Trained.   Place visible cues inside the pool during the HP task (maze cues; Placed in three quadrants at the top of the wall, which was 38 cm above water level , Black pieces of cardboard (circles, plus, or horizontal lines)) and clues to various places Made visible outside the pool (cue outside the maze).   Mice evaluated for the cohort study of characterization were Was tested for the above behavioral task for three days during a two week period. Them The transgenic status was not reported to the tester. Non-transgenic Litters were used for comparison. Each morning, the subject mouse is placed in the Y maze and And run in RAM. They are then tested for slope (INP) testing. The general durability was tested. For this, made of ridged plastic The mouse was placed on a 10 cm wide runway with the upper part raised to 35 degrees. Then the mouse The angle was increased until it slipped off and the angle was recorded. Repeat this three times daily I returned. Average score for 3 days, Y maze (0 = repetition, 1 = change), and RAM (Number of different arms and end time, 10 minute limit), and INP (9 trials) (Average of all rows) was calculated for each mouse. General activities are also Evaluation was made on the first day of the test. Observe each mouse in the cage, and Raised and left. Mice that remained calm in their hands were scored 1 and active. As the movement and response grew, they were scored up to 4.   Following the daily test described above, mice were tested in the water maze as described above. Simple In the small pool, the mouse is the only way for the mouse to escape from the water. Trained to climb a flooded table. The mice were then given 6 out of 4 trials. The blocks were given and each learned the position of a small platform in a large pool. analysis For, all four trials within each block were averaged. Exception is the first hidden A pedestal block for which only the last three trials were averaged. First try Rows were analyzed separately. Because the position of the platform cannot be known and is therefore relevant for spatial learning Because it is the only trial that does not. Therefore, non-spatial factors (eg, motivation and Used as a control for swimming speed). Effect of positive response rate between blocks The results were analyzed as repeated measures for the hidden platform task. Variance (ANOVA) calculation Standard analysis was used to assess the significance of the results.   TG performed significantly worse than nTG over all ages, according to results in RAM (Group effect: P = 0.00006). End time is also TG mouse And nTG mice (group effect: P = 0.005). End time and The correlation between the number of arms selected was small (for each group, R = -0. 15, p = 0.245). This suggests that a consistent obstacle in RAM is due to TG mice. Suggests that the increased time to complete the performed test is not explained I do. The results in the Y maze were also significantly different for TG and nTG mice. (Group effect: p = 0.011). Performed on non-transgenic mice Confirmed studies show that the Y-maze is not a more sensitive measurement than RAM .   Endurance (INP) and activity measurements show differences between TG and nTG mice Absent. These are considered very rough measurements, where only large differences can be detected. available. However, over time, activity scores decreased for all mice (age Effect: P = 0.070). TG is 8% lighter than control, mainly in female TG mice And there was a difference in body weight (group effect: P = 0.0003). But this is T Between G and nTG mice, endurance (see above) or swimming speed (see below) As shown by the absence of any differences in , Seems to have no effect on the results.   Hidden platform task results (this is considered a reference memory test in this example) ) Shows consistent differences between TG and nTG mice. By ANOVA , Transgenic effect (group effect: P = 0.00016) and trial The effect of the block (block effect: p <0.0001) is shown to be significant. Correct response The effect of transgenic bears on rates was significant for all trial blocks and ages. Explained by the slower positive response rate by the TG mice across. By the analysis of Trial 1 The effect of the transgenic state (group effect: p = 0.018) is shown. Indicates a difference in the positive response rate before learning occurs. However, co-random variables ( Analysis of covariance with Trial 1 as a covariate still shows significant impairment in TG mice. Harm was shown (p = 0.00051).   Some physical differences between TG and nTG mice are more water than cognitive differences. It is also thought that it can play a role in some of the differences in positive response rates found in maze tasks Can be However, no significant differences in endurance or activity were observed (top Description). Another possible possibility is the effect of swimming speed on positive response rate Met. This is because slower swimming individuals (slower swi mmer) because it took longer to reach the platform. Try this Use video tracking in a hidden platform task to test Distance traveled (a measure of the amount of quest made by the mouse in relation to cognitive abilities), Swimming speed (a measure of physical performance not related to cognitive abilities), and finding the platform Measures the amount of time required (combination of both distance traveled and swim speed measurements) did. This uses three trials per block, and without pre-training, Performed in older or younger mice than reported above. Look at the table The time required for delivery is longer for TG mice, And nTG mice were significantly different (group effect: p <0.0005). But, Swimming speed was not significantly different between TG and nTG mice (group effect : P = 0.879). Therefore, the difference in the time required to find the platform It appears to be due to differences in cognition between mice and nTG mice. This means that Is confirmed by measuring the distance traveled to find out. TG mouse is nTG mouse Before reaching the platform, they moved significantly more (group effect: p <0.0005). This These results indicate that TG mice and nTG mice Suggest that the differences found between the two are due to differences in cognitive abilities. I have.   Test whether nTG mice retain better memory of platform position than TG mice To test, a probe trial was performed immediately after hidden platform training with the platform removed. Using video tracking, a crossing of a platform-less The number of crossings at the previous platform location was measured. The relative crossing between TG and nTG mice There were significant differences seen between (group effect: p = 0.006). This is nTG Evidence that mice remember their previous platform position better than TG mice .   Between the TG and nTG mice at the time needed to reach the platform Observed differences are affected by differences in perception of cues or differences in motives. It was also conceived that this could be done. To test this, TG mice and nTG Mice were subjected to a visible platform task in the water maze. For these challenges, And placed above the water. 3 different platforms (25mm black on the surface Table (most visible), gray table 25mm on surface, and black table 5mm on surface (both Also hard to see))). The results show that the most TG and nTG mice No difference in the time to find a visible platform (group effect : P = 0.403). When using a platform that is difficult to see, the positive response rate in TG mice is large. There was no significant decrease. This means that TG mice have as good vision as nTG mice Implies that From these results, differences in perception and motivation It shows that the time to reach the platform in the water maze task is not affected.   The difference in the positive response rate between TG mice and nTG mice was 4 to 8 months old. (2-12 months for water maze), RAM, Y maze, and water maze recognition tasks Was shown against. All of these differences are transgenic as a group. Shows and is coherent with cognitive impairment in mouse. Combine various issues Test working memory and reference memory, both of which are associated with Alzheimer's disease. Affected by cognitive impairment observed in Example 10: Detection and measurement of Alzheimer's disease marker   A. GFAP detection and measurement   Collagen fibrillary acidic protein (GFAP), a marker that increases in AD brain tissue, is It was measured in a manner. Tissue extracts were prepared as described in Example 6 at 14 months of age. Roll and PDAPP were prepared from hippocampus of transgenic mice. Organization, 10 Volume (v / w) of 10 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF, 10 μg / ml Leupeptin was sonicated in 5 μg / ml calpain inhibitor 1. Tampa A quality measurement is performed on the extract, and 5 minutes after adding SDS-PAGE sample buffer. The sample was boiled for minutes. SDS-PAGE loaded on 10% Tris-glycine gel (Novex) This was performed using 12.5 μg of each sample. Pro-Blot PVDF membrane Was transferred in a standard manner. 1: 2,000 dilution of GFAP immunoreactive protein Detection was performed using the anti-GFAP antibody from Sigma (G9269) used in. Generally, immune response Increased responsivity was observed and in transgenic animals, a smaller anti-GFAP It has been found that seeding is also substantially increased. In non-transgenic animals This approximately 40 kD fragment produced an average decimeter signal of 142.47. In contrast, the transgenic animals had an average decimeter signal of 591.51 Occurred. This difference was significant, with a P value of 0.0286.   B. Gliosis detection   Gliosis is one of the changes associated with the neuropathology of Alzheimer's disease. Isoquinolinecarboxamide PK 11195 is a peripheral benzoylase associated with gliosis. It has been shown to be the preferred marker for diazepine sites. These sites are Several diseases associated with activated necroglia, including nerve damage and seizures Disease and animal models (Stephenson et al., J. Neuroscience 15: 5263-5274 (1991) And Alzheimer's disease (Diorio et al., Neurobiology of Aglng 12: 255-258 (1991)) It has been shown to be increased. In particular, Diorio and colleagues adapted for age In some brain regions (eg, temporal cortex) of AD patients compared to controls Match [^ThreeH] PK 11195 showed an increase of about 200%. In this example, the PDA To correlate brain from PP mice (described in Example 6) with AD disease pathology described above , [^ThreeH] PK 11195 was examined for qualitative and quantitative changes in binding. Two A different approach was utilized; radiorecept to homogenates of different brain regions Binding and receptor autography.   1. Method   For homogenate binding studies, PDPAA mice were euthanized by cervical dislocation, The brain was then quickly dissected on ice. Cerebral cortex, hippocampus in 50 mM Tris HCl (pH 7.4) And cerebellar homogenates (10 mg / ml wet weight) were prepared. 0.3, 1.0 and 3.0 nM of[^ThreeH] PK 11195 was incubated with these brain regions at 23 ° C. for 60 minutes, Then quickly filter on a Whatman GF / B filter using a Brandell cell harvester. I have. Non-specific knots were determined using 1 μM unlabeled PK 11195. Quantitation, liquid The measurement was performed by body scintillation spectrometry.   In an autoradiographic study, PDAPP mice were euthanized using carbon dioxide. The brains were removed and snap frozen in methyl butane / dry ice. This brain An incision was made in the frontal plane through the hippocampus. Place a 20 micron thick section on a glass slide. Mounted and stored at -20 ° C. Sections were 1 nM [^Three170 containing H] PK 11195 Incubated for 1 hour at 23 ° C in mM Tris-HCl (pH 7.4). Non-specific binding, The measurement was performed using 1 μM of unlabeled PK 11195. Incubate the sections twice Stop by rinsing in ice-cold buffer for 5 minutes, then briefly with ice-cold distilled water Was washed. After a brief drying, the sections were trimmed with tritium Hyperfilm (Amersham International al) for up to 5 weeks.   2. result   Four heterozygous transgenic mice 3-4 months of age were homogenated. Evaluated in binding studies and compared to littermate controls. Transgenic No significant differences are observed between any of the brain regions of animals and their respective controls Was not done. [^ThreeH] PK 1119 autoradiography performed 12 month-old heterozygous Non-transgenic congener adapted to zygote transgenic mouse and age The binding on the trolls was compared. From autoradiograms exposed for 5 weeks Preliminary results indicate that some plaque-like structures were Be labeled in the posterior lamella cortex, a region containing Aβ deposition invariably showed that. The labeling pattern is similar to that of hippocampal and cortical parenchymal astrocytes and microglia. Microglia or stellate cells that bind to plaque rather than a more expanded pattern Corresponding to vesicle clumps. Nontransgenic mice do not show this labeling pattern Was.   No changes were observed in 3-4 month old animals,^ThreeH] PK 11195 for increased binding Some evidence for it was found in 12 month old animals.   C. Detection and measurement of cholinergic nerve endings   The population of cholinergic neurons that protrude from the forebrain is called Alzheimer's disease. It has been shown to be selectively reduced in the postmortem brain of diagnosed patients. F Micolinium-3 is a potent inhibitor of high-affinity choline uptake, It has been shown to be a good marker for cholinergic nerve endings (Pascual et al., J. Neurochem 54: 792-800 (1990)). PDAPP transgenic animals (described in Example 6) The total number of high-affinity choline uptake sites in^Three H] hemicolinium-3 ([[^ThreeH] HCh-3) was used to prepare crude whole brain preparations and selective brain regions. It was determined using both homogenates from the field.   [^ThreeH] -hemicolinium-3 binding was determined using a modification of the method described in Pascual et al. Was. Mice are euthanized by carbon dioxide asphyxiation and brains are quickly removed And cut on ice. Cortex, cerebellum, striatum and hippocampus were removed from 5 ml of 10 mM NaCl-free Homogenized in phosphate buffer. Spin the sample at 17,000 xg for 10 minutes, And pellet to 5 ml of 10 mM PO_FourWashed twice with buffer. 5 ml of final pellet Resuspend in 1 × phosphate buffered saline (PBS) to yield a protein concentration of 0.5 mg / ml I did Brain region^ThreeAddition of H] HCh-3 (final concentration of 3 nM) allows high-affinity choline Uptake sites were assayed in triplicate. After a 20 minute incubation, Rapid filtration through Whatman GF / B filters using Brandell cell harvesters The assay was terminated by washing with PBS. Scintillation filter Transfer vial and specific binding by liquid scintillation spectroscopy. Was evaluated.   D. Detection and measurement of sodium-potassium ATPase   Ouabain specifically binds to high affinity sites in the mammalian brain, and The site is shown to correspond to the neuronal form of sodium-potassium ATPase (Na / K-ATPase; Hauger et al., J. Neurochem 44: 1709-1715 (1985)). these Sites have been shown to be reduced in animal models of neurodegenerative disease. Alzha Immer's disease is characterized by extensive neurodegeneration (DeLacoste and White, N. eurobiology of Aging 4 (1): 1-16 (1993)).   To assess the extent of neurodegeneration in PDAPP mice, ouabain binding Determined by mouse brain homogenate. Is the method described by Hauger et al. Adapted. Brain tissue was treated with 200 mM NaCl and 10 mM MgCl_Two100mM Tris HCl containing (pH 7.4) and resuspend in assay buffer to assay Yielded a final concentration of 100 μg protein. Specific binding is 5 mM ATP, 100 mM Tris HCl (pH 7.4), 10 mM MgCl_TwoAnd 1-200 nM [in a solution of 200 mM NaCl.^ThreeH] The determination was made by incubating with ouabain at 37 ° C. for 30 minutes. Non-specific conclusion Were determined in the presence of 100 mM ouabain and in the absence of ATP. Assay Finished by rapid filtration through Whatman GF / B filter. Tube with ice Cold 50 mM Tris HCl (pH 7.4), 15 mM KCl, 5 mM MgCl_TwoAnd washed. Thin filter Transfer to a scintillation vial and check for specific binding by liquid scintillation spectroscopy It was evaluated by the measurement method.   Brain tissue and non-transgene of PDAPP transgenic mice of various ages Na / K-ATPase and Mg-ATPase activities in nick control brain tissue were measured . These results indicate that transgenic samples in older mice Shows some significant difference in activity between non-transgenic samples . From 4, 8, and 12 month old PDAPP transgenic (TG) mice and non- Preparing a mouse brain homogenate from a transgenic (nTG) mouse; and Assays were generally performed as described above. The activity of Mg-ATPase was also determined. The result The results are shown in Tables 7 and 8.   Na / K-ATPase activity between transgenic and non-transgenic tissues Gender differences are significant in the case of 12 month old cerebellum (p <0.05) and Is very significant in the case of the hippocampus (p <0.01). Transgenic organization The difference in Mg-ATPase activity between and non-transgenic tissues was between 8 and 12 months of age. Significant in cortical cases (p <0.05) and in 12-month-old hippocampus And very significant (p <0.01).   E. FIG. In situ hybridization using probes for neurotrophic factors ® down   MR for specific gene products using in situ hybridization The detection and localization of NA is well documented in the literature (Lewis et al., Mole cular Imaging in Neuroscience: A Practical Approach (New York, Oxford Univ. ersity Press. First Edition, 1-21, 1993), Lu and Gillett, Cell Vision 1 (2): 169-1. 76 (1994), Sirinathsinghji and Dunnett, Molecular Imaging in Neuroscienc e: A Practical Approach (New York, Oxford University Press. 1st edition, 43-67, 19 93), Lawrence and Singer, Nuc. Acids Res. 13: 1777-1779 (1985), Zeller And Rogers, Current Protocols in Molecular Biology (New York, John Wiley and Sons. 14.3.1-14.5.5, 1995)). For purposes of illustration, certain embodiments described below include:³⁵ Using an S-radiolabeled oligodeoxyribonucleotide probe, described in Example 6. Transgenic mice and non-transgenic controls Detect BDNF mRNA in cryostat-sectioned mouse brain from mouse Issued. But,³³P-labeled radioactive DNA probe and in vitro transcribed phase Complementary RNA probes can be used as well. Non-radioactive probe labeling is also used ( Knoll, Current Protocols in Molecular Biology (New York, John Wiley and So ns. 14.7.1-14.7.14, 1995)). Hybridization using probes Selection of tissue pretreatment (eg, paraffin-embedded sections) for hybridization and hybridization Washing after dicing depends on the method used and these examples are cited above. It is described in the references. Use known and appropriate precautionary measures against RNAse contamination And are also discussed in the references above.   1. Tissue preparation   Transgenic mice or controls at various stages of development or at post-natal age Freshly dissected whole brains from roll mice, or subregions of interest, are pre-treated at -70 ° C. Freeze quickly in equilibrated isopentane. Move before incision if desired. The material can be perfused with PBS to remove circulating cells from the brain. Brain 1 in isopentane Remove after 5-20 seconds of immersion, wrap in aluminum foil, label appropriately and section For storage at -80 ° C. In situ for cryostat sectioned tissue Signals from hybridization are more sensitive to paraffin-embedded sections But it depends on the time from incision to hybridization Care should be taken. Frozen tissues are preferably probed within 6 weeks. Analyze with redistribution. RNA integrity in tissues degrades after this time . Therefore, if a longer period between incision and analysis is expected, a longer period of -80 Tissues should be fixed prior to storage at ℃ C (see, eg, Lu and Gillett thing).   Prior to sectioning, Probe-On-Plus glass slides (Fisher Scientific, Pittsburg, PA) in absolute ethanol overnight, air-dry briefly in a dust-free environment, and RNAase free can be obtained by drying and heating for 4 hours. After cooling to room temperature, this The slide was treated with 0.01% polylysine (DEPC-treated H_TwoO) for about 5 seconds, Then, air dry in a dust-free area. Transfer coated slides to silica gel or dry It can be stored for up to one month before use in a slide box containing the alite pellet.   For sectioning, the frozen brain stored at -80 ° C was transferred to a -20 ° C cryostat, Weaving (DEPC H_TwoUsing a sterile microtome knife (treated with 70% EtOH in O) Cut into 14 μm thick sections and melt on a polylysine-coated slide. Und Keep slides at -20 ° C until sectioning is complete. Fix this section Dehydrate by continuously immersing slides in the solution described below .   5 minutes in 1.4% paraformaldehyde, 1 × PBS, pH 7.4 at 0 ° C. The solution should be made fresh and may be stored at 4 ° C for up to 1 week) ;   2. 2.5 minutes twice in 1 × PBS each time;   3. DEPC H_TwoOnce in 5% EtOH in O for 5 minutes;   4. DEPC H_TwoOnce in 5% in 70% EtOH in O;   5. DEPC H_TwoOnce in 5% in 95% EtOH in O;   Mount fixed sections in 95% EtOH / DEPC H until use._TwoDip at 4 ° C in O solution Exist. If sections are not fixed immediately, keep them in drylite until use. Can be stored at -80 ° C. In this case, the sections were allowed to reach room temperature before the fixation / dehydration step. Balanced.   2. Probe design and preparation   The sequence of mouse BDNF mRNA / cDNA (Accession # 55573) is based on the Genbank database of nucleic acid sequences. (NCBI, Betheda, MD). Antisense oligode against BDNF Oxynucleotide probe to DNAstar^TMSoftware package primer Many other software packages provide similar capabilities, such as (ymouth, MN). , And also appropriate. Candidate probe 45-55 nucleotides long (about 50% G + C content and encodes a precursor or mature peptide of BDNF mRNA Which hybridizes to the region of interest) was synthesized on an ABI 380B DNA synthesizer. Special As an isomerism control, the sense oligonucleotide corresponding to each probe is Was synthesized. Using the convention that the first nucleotide of the BDNF coding region is position 1, The synthesized BDNF probe was identified at nucleotides 47 to 94 of BDNF (probe 2710 and probe 2710). 2711), nucleotides 158-203 (probes 2712 and 2713), 576-624 nucleotides Otide positions (probes 2714 and 2715) and 644-692 nucleotides (probe 2716 and 2717). The even-numbered oligonucleotide pairs with the sense strand. Odd-numbered oligonucleotides to the antisense strand Probe.   The probe is gel purified on a denaturing acrylamide gel for radiolabeling, and H using standard protocols (Sambrook et al.)_TwoReconstruct in O. Probe (30-35 mg, 2 pmol) with terminal deoxynucleotidyl transferase (Promeg a, Madison WI) and³⁵Production of S-dATP (1000 Ci / mmol, Amersham Inc.) Label according to 3 'homopolymer tailing, using the manufacturer's recommendations. Radiation target Detection probe was used with a size exclusion mini spin column (Biospin-6, Biorad Inc., Hercules). , CA) by column chromatography. Synthesize the specific activity of the probe Quantify by titration counting. A typical specific activity of the probe is 1 × 10⁹~ 5 × 10⁹The range was cpm / μg.   3. Tissue hybridization and post-hybridization washes   In preparing for hybridization, the desired number of slides Remove from storage under a chiller and air dry thoroughly in a slide rack (about 1 hour while). Meanwhile, boiling probe H_TwoO Denature for 2-5 minutes in a bath / H_TwoCool in O bath and 5 × 10^Three~ 10 × 10^ThreeHybridizer to final concentration of cpm / μl Ization buffer (10% dextran sulfate, 50% deionized formamide, 4% × SSC, 5 × Denhardt, 100 μg / ml sheared salmon sperm DNA, 100 μg / ml polyadenylate Dilute acid). DTT is added at a final concentration of 10 mM.   For hybridization, in 100 μl hybridization buffer Diluted probe (0.5 × 10⁶~ 1.0 × 10⁶(corresponding to the cpm probe) Carefully apply to each section that will hybridize with the gel. Pipette tip solution Gently spread the sections using a to cover all section (s) on each slide . The slide containing the probe was then hybridized with the probe overnight. Place in a humidified hybridization chamber at 42 ° C. for localization. C The hybridization chamber has a raised platform for storing slides. Utility box, or acrylic box. This box Put the paper on filter paper (or paper towel) and saturate with 4 × SSC, 50% formamide. And pre-ink for 1-3 hours in a 42 ° C incubator with the lid closed Moisten by immersion, and then place the slides therein. Hybridi Coverslips may be placed on the sections after the application of the This is because the hybridization chamber is properly moist during the procedure. Not necessary. Lower background using prehybridization To obtain the primers, 50 μl of hybridization buffer (probe (None) at 42 ° C. for 1-2 hours. After this time, on An equal volume of hybridization buffer containing the probe at twice the indicated concentration was applied to each section. And hybridization is performed as described above.   Remove slides from hybridization chamber for washing Transfer to ruder. Slide so that the wash solution flows properly over the surface of each section. May be placed every fourth or fifth slot in the slide holder. This arrangement Can significantly reduce background on sections. Medium over the section surface A wash solution with a flow rate of 10% facilitates the removal of unhybridized probe, and To continuously reduce the background. This is a magnetic stirrer bar Suspending the slides in the slide holder on the Best achieved in between. Place the stirrer bar, preferably under the slide holder Place about 1 inch. This is a slide holder from a pipette straddling the washing chamber -Can be done by hanging one or more. Big beaker Pyrex baking dishes at a depth of 4 to 6 inches create a suitable wash chamber You. Place the washing chamber on a hot plate stirrer and set this temperature Calibrate in advance to maintain the wash solution at 55 ° C during. Replace the washing solution This is done using a pre-equilibrated solution. Dehydration after washing and section washing is as follows: Do to:   1. Twice for 5 minutes each time in 1 × SSC at room temperature;   2. Three times for 30 minutes each at 55 ° C. in 1 × SSC;   3. Once per minute at room temperature in 1.times.SSC;   4. Once for 15 seconds at room temperature in 0.1 × SSC;   5. Once in ultrapure water at room temperature for 15 seconds;   6. Once in 50% EtOH for 15 seconds;   7. Once in 15% in 70% EtOH;   8. Once for 15 seconds in 95% EtOH;   The sections are air-dried for 2 hours at room temperature and then for 30 minutes at 55 ° C. Drying on slides The section is placed on an autoradiography cassette and X-ray film (Hyper Film, β-max, Amersham Inc., Arlington Heights, IL) at 4 ° C. for 2-3 days Estimate the exposure time required under the emulsion. X-ray film recommended by manufacturer Develop according to. Immerse sections in emulsion at 42 ° C under appropriate safe light conditions With a photosensitive emulsifier (Amersham LM-1, # RPN40). Coated with photosensitive emulsifier The dried slides are allowed to air dry on a cooled surface for about 30 minutes, and a desiccant (Dryalite Transfer to a plastic slide box containing the pellet). Box seams Can be sealed with black tape, and wrap this box with several layers of aluminum foil To ensure a light tight seal. After 2-4 hours at room temperature to complete drying, 2-6 Transfer box to 4 ° C. for weekly autoradiographic exposure. Photosensitive emulsion coating Prior to developing the overlaid slides, remove the box from the refrigerator and allow to stand for about 1 hour Equilibrate with. The slides are then developed according to the manufacturer's instructions, 1-2 hours Air dry and counterstain with an appropriate counterstain if desired.   Generation of transgenic animal models for testing for Alzheimer's disease and Modifications and variations of the tests will be apparent to those skilled in the art from the foregoing detailed description. this Such modifications and variations are intended to fall within the scope of the following claims.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), UA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AL, AM, AU, BB, BG , BR, CA, CN, CZ, EE, FI, GE, HU, IL, IS, JP, KG, KP, KR, LK, LR, L T, LV, MD, MG, MK, MN, MX, NO, NZ , PL, RO, SG, SI, SK, TR, TT, UA, UZ, VN (72) Inventor Schenk, Dale Be.             United States California 94044,             Pacifica, Sharp Park Road             605 (72) Inventor MacConlogue, Lisa Claire             United States California 94127,             San Francisco, Juanita Way             283 (72) Inventor Soubert, Peter A.             United States California 94080,             South San Francisco, Northwood             Drive 222 (72) Inventors Liddell, Russell E.             United States California 94002,             Belmont, Number 6, Village Co             Tot 2211 [Continuation of summary] Used for screening for compounds.

Claims (1)

[Claims] 1. A method to test compounds for their effect on Alzheimer's disease markers A method comprising the following steps: a) transducing a test compound to a non-human transgenic mammal or the transgenic mammal; Administering to mammalian cells derived from a mammal, wherein the transgenic A milk animal has a nucleic acid construct stably integrated into the genome, wherein the construct is a mammalian cell. Promoter for expression of the construct in vesicles, and Aβ-containing protein And the promoter is operably linked to the region,   Here, the region contains a DNA encoding an Aβ-containing protein, The protein contained is all or a sequence of proteins selected from the group consisting of: It consists of the following parts:   APP770, amino acids 669, 670, 671, 690, 692 and 71 Has a mutation in one or more amino acids selected from the group consisting of APP770, APP751, amino acids 669,670,671,690,6 One or more amino acids selected from the group consisting of 92 and 717; Mutated APP751, APP695, and amino acids 669,670 , 671, 690, 692 and 717. APP695 having a mutation in more than one amino acid,   Here, the Aβ-containing protein comprises amino acids 672 to 714 of human APP. Including   Here, the promoter mediates the expression of the construct as follows: 2-4 months of age Aβ at a level of at least 30 nanograms per gram of the mammalian brain tissue of At least per gram of brain tissue of the mammal, 2-4 months of age, where tot is expressed 2 to 4 month old mammals expressing Aβl-42 at a level of 8.5 nanograms APP and APP at a level of at least 150 picomoles per gram of brain tissue is expressed per gram of brain tissue of the mammal at the age of 2-4 months, wherein the sum of α is expressed APPβ is expressed at a level of at least 40 picomoles and / or from 2 to 4 months Endogenous APP of the transgenic mammal in brain tissue of the mammal at an age Aβ-containing protein at at least twice the level of mRNA encoding MRNA encoding is expressed; and   The transgenic mammal or in the transgenic mammal Markers from mammalian cells of origin and transgenes to which the compound was not administered. Between markers in mammalian cells or mammalian cells derived therefrom To detect or measure Alzheimer's disease markers so that differences in About   Here, the observed difference in the marker indicates that the compound affects the marker. It has an effect. 2. The method according to claim 1, wherein the Aβ-containing protein is selected from the group consisting of: Described method: APP770; amino acids 669, 670, 671, 690, 692 , Encodes one or more amino acids selected from the group consisting of: 717 APP770 with a mutation at the codon; APP751; amino acids 669, 6 70, 671, 690, 692, 717 or one selected from the group consisting of APP751 having a mutation in a codon encoding more than one amino acid; AP P695; consisting of amino acids 669, 670, 671, 690, 692, 717 Changes at codons encoding one or more amino acids selected from the group APP695 having a difference; a protein consisting of amino acids 646 to 770 of APP Quality; protein consisting of amino acids 670 to 770 of APP; amino acids of APP A protein consisting of 672 to 770; and amino acids 672 to 71 of APP A protein consisting of 4. 3. The DNA encoding the Aβ-containing protein is cDNA or cDNA. / Genomic DNA hybrid where the cDNA / genomic DNA hybrid The kit comprises at least one APP intron sequence, wherein the intron sequence is 3. The method of claim 2, which is sufficient for licensing. 4. The promoter is a human platelet-derived growth factor β chain gene promoter The method of claim 1, wherein: 5. Said region further comprises DNA encoding a second protein, wherein the DNA DNA encoding the Aβ-containing protein and encoding the second protein DNA comprising a fusion of the Aβ-containing protein with the second protein Operably linked such that the region encodes an Aβ-containing fusion protein; The method of claim 1. 6. The method according to claim 5, wherein the second protein is a signal peptide. . 7. The Alzheimer's disease marker is a protein, and the observed The difference is in or in the transgenic mammal to which the compound has been administered. Increase or decrease in the amount of the protein present in mammalian cells derived therefrom. The method of claim 1, wherein 8. 8. The method of claim 7, wherein said protein is selected from the group consisting of: CatD, B, Neuronal Thread protein, nicotine receptor , 5-HT2 receptor, NMDA receptor, α2-adrenergic Receptor, synaptophysin, p65, glutamine synthetase, glucose Transporter, PPI kinase, GAP43, cytochrome oxidase, Heme oxygenase, calbindin, adenosine A1 receptor , Choline acetyltransferase, acetylcholinesterase, collagen fibers Acidic protein (GFAP), α1-antitrypsin, C-reactive protein , Α2-macroglobulin, IL-1α, IL-1β, TNFα, IL-6, H LA-DR, HLA-A, D, C, CR3 receptor, MHC I, MHC II , CD31, CR4, CD45, CD64, CD4, spectrin, tau, ubi Chitin, MAP-2, apolipoprotein E, nerve growth factor (NGF), brain-derived Neurotrophic factor (BDNF), advanced glycosylation end product, advanced glycosyl End product receptor, COX-2, CD18, C3, fibroblast growth factor , CD44, ICAM-1, lactotransferrin, C1q, C3d, C4d, C5b-9, gamma RI, Fc gamma RII, CD8, CD59, vitronecti , Vitronectin receptor, beta3 integrin, ApoJ, cluster Phosphorus, type 2 plasminogen activator inhibitor, midkine, mac Lophage colony stimulating factor receptor, MRP14, 27E10, interface Eron-alpha, S100β, cPLA2, c-jun, c-fos, HSP 27, HSP70, MAP5, membrane lipid peroxidase, protein carbonyl Formation (protein carbonyl formation), junB, junD, fosB, fra 1, cyclin D1, p53, NGFI-A, NGFI-B, IκB, NFκB , IL-8, MCP-1, MIP-1α, matrix metalloproteinase, 4-hydroxynonenal-protein conjugate, amyloid P component, laminin, And collagen type IV. 9. The Alzheimer's disease marker is a protein, and the observed Differences exist in the transgenic mammal to which the compound has been administered Reduced or absent amount of the protein in plaque or neurite tissue The method of claim 1. 10. 10. The method of claim 9, wherein said protein is selected from the group consisting of: : CatD, B, protein kinase C, NADPH, C3d, C1q, C5, C4bp, C5a-C9, tau, ubiquitin, MAP-2, neurofilament, Heparin sulfate, chondroitin sulfate, apolipoprotein E, nerve growth factor (N GF), brain-derived neurotrophic factor (BDNF), glycosylation end product, amyloid De-P component, laminin, and collagen type IV. 11. The Alzheimer's disease marker is a protein, and Or in a transgenic mammal to which the compound has been administered or Of the enzymatic or biochemical activity of the protein in mammalian cells derived therefrom. 2. The method of claim 1, wherein the increase or decrease. 12. 12. The method of claim 11, wherein said protein is selected from the group consisting of: Method: nicotine receptor, 5-HT2 receptor, NMDA receptor, α2- Adrenergic receptor, glutamine synthetase, glucose trans Porter, PPI kinase, cytochrome oxidase, heme oxygenase, Calbindin, adenosine A1 receptor, choline acetyltransferase Acetylcholinesterase, collagen fibrillary acidic protein (GFAP), α1- Antitrypsin, C-reactive protein, α2-macroglobulin, IL-1 , TNFα, IL-6, HLA-DR, HLA-A, D, C, CR3 receptor , MHC I, MHC II, CD31, CR4, CD45, CD64, CD4, Spectrin, ubiquitin, and apolipoprotein E. 13. The Alzheimer's disease marker is a nucleic acid encoding a protein, And wherein the difference observed is the transgenic mammal receiving the compound. Increase in the amount of the nucleic acid present in the product or in mammalian cells derived therefrom. 2. The method of claim 1, wherein the reduction is. 14． 2. The encoded protein is selected from the group consisting of: Method according to 3: growth inhibitory factor, CatD, B, neuronal thread protein, d Cotin receptor, 5-HT2 receptor, NMDA receptor, α2-address Narinergic receptor, synaptophysin, p65, glutamine synthetase , Glucose transporter, PPI kinase, GAP43, cytochrome Oxidase, heme oxygenase, calbindin, adenosine A1 receptor, Choline acetyltransferase, acetylcholinesterase, collagen fiber acid Protein (GFAP), α1-antitrypsin, C-reactive protein, α2-macroglobulin, IL-1, TNFα, IL-6, HLA-DR, HL A-A, D, C, CR3 receptor, MHC I, MHC II, CD31, CR 4, CD45, CD64, CD4, spectrin, tau, ubiquitin, MAP- 2. Apolipoprotein E, nerve growth factor (NGF), brain-derived neurotrophic factor (B DN F), advanced glycosylation end product, receptor for advanced glycosylation end product , COX-2, CD18, C3, fibroblast growth factor, CD44, ICAM -1, lactotransferrin, C1q. C3d, C4d, C5b-9, gamma RI, Fc gamma RII, CD8, CD59, vitronectin, vitronectin Receptor, beta3 integrin, ApoJ, clusterin, type 2 plasmin Nogen activator inhibitor, midkine, macrophage colony Stimulatory receptor, MRP14, 27E10, interferon-alpha, S100β, cPLA2, c-jun, c-fos, HSP27, HSP70, MAP5, membrane lipid peroxidase, protein carbonyl formation, junB, j unD, fosB, fra1, cyclin D1, p53, NGFI-A, NGF IB, IκB, NFκB, IL-8, MCP-1, MIP-1α, Matric Smetalloproteinase, 4-hydroxynonenal-protein conjugate, Lloyd P component, laminin, and collagen type IV. 15. The Alzheimer's disease marker is behavior and the observed difference Is observed in the transgenic mammal to which the compound is administered. 2. The method of claim 1, wherein the change is a behavior change. 16. 16. The method of claim 15, wherein the action is selected from the group consisting of: Behavior using memory, behavior using reference memory, locomotor activity, new environment or Emotional responsiveness to new objects, and object recognition. 17． The Alzheimer's disease marker is histopathology and is observed Differences exist in the transgenic mammal to which the compound has been administered 2. The method of claim 1, wherein the reduction is in the extent or severity of the histopathology. 18. 18. The method of claim 17, wherein the histopathological marker is selected from the group consisting of: Methods described: compacted plaque, neurite dystrophy, gliosis , Aβ deposition, decreased synaptic density, and abnormalities in the neural network. 19. The Alzheimer's disease marker is recognition and the observed difference Is the change in the cognition of the transgenic mammal to which the compound has been administered. The method of claim 1. 20. The method of claim 1, wherein the marker is detected or measured using: Methods: RT-PCR, RNase protection, Northern analysis, R-dot analysis, ELISA A, antibody staining, laser scanning confocal imaging, and immunoelectron microscopy Mirror method. 21. 2. The method of claim 1, wherein said mammal is a rodent. 22. A codon encoding amino acid 717 is selected from the group consisting of: 2. The method of claim 1, wherein the mutation is to encode a amino acid: Ile, Ph. e, Gly, Tyr, Leu, Ala, Pro, Trp, Met, Ser, Th r, Asn, and Gln. 23. The codon encoding amino acid 717 is mutated to encode Phe 23. The method of claim 22, wherein the method comprises: 24. If the codon encoding amino acid 670 is the group consisting of Asn and Glu Mutated to encode an amino acid selected from 0 is deleted and / or   The codon encoding amino acid 671 is represented by Ile, Leu, Tyr, Lys, G encodes an amino acid selected from the group consisting of lu, Val, and Ala Or the codon encoding amino acid 671 has been deleted The method of claim 1. 25. The codon encoding amino acid 670 is mutated to encode Asn. And / or the codon encoding amino acid 671 is Leu or 25. The method of claim 24, wherein the method is mutated to encode Tyr. 26. The claim wherein the promoter mediates expression of the construct as follows. Method according to 1: 1 gram of brain tissue of hippocampus or cortex of the mammal 2 to 4 months old 2 to 4 months of age, wherein Aβtot is expressed at a level of at least 30 nanograms per At least 8.5 nanograms per gram of brain tissue of said mammalian hippocampus or cortex The hippocampus of the mammal, 2-4 months old, wherein Aβ 1-42 is expressed at the gram level; APP and APP at levels of at least 150 picomoles per gram of cortical brain tissue. Of the hippocampus or cortex of the mammal at the age of 2 to 4 months, wherein the sum of APPβ is expressed at a level of at least 40 picomoles per gram of brain tissue And / or in the brain tissue of the hippocampus or cortex of the mammal of 2-4 months of age For the mRNA encoding the endogenous APP of the transgenic mammal MRNA encoding Aβ-containing protein is expressed at at least twice the level of Is done. 27. Amyloid plaque that can be stained with Congo Red in the mammalian brain tissue 2. The method of claim 1, wherein is.