CA2222174A1 - Method for identifying alzheimer's disease therapeutics using transgenic animal models - Google Patents

Abstract

The construction of transgenic animal models of human Alzheimer's disease, and methods of using the models to screen potential Alzheimer's disease therapeutics, are described. The models are characterized by pathologies similar to pathologies observed in Alzheimer's disease, based on expression of all three forms of the .beta.-amyloid precursor protein (APP), APP695, APP751, and APP770, as well as various point mutations based on naturally occurring mutations, such as the London and Indiana familial Alzheimer's diseae (FAD) mutations at amino acid 717, predicted mutations in the APP gene, and truncated forms of APP that contain the A.beta. region. Animal cells can be isolated from the transgenic animals or prepared using the same constructs with standard techniques such as lipofection or eletroporation. The transgenic animals, or animal cells, are used to screen for compounds altering the pathological course of Alzheimer's disease as measured by their effect on the amount of APP, .beta.-amyloid peptide, and numerous other Alzeimer's disease markers in the animals, the neuropathology of the animals, as well as by behavioral alterations in the animals.

Description

WO 96/40896 PCT~US~6~ 3 METHOD FOR IDE~ Yl~G ~T~7~ ~ER7s DISEASE
THERAPEUTICS USING TRANSGENIC ANIMAL MODELS
Background of the Illv~llLioll Tldllsgel~ic animal models of Alzheimer's disease are described along with a method of using the transgenic animal models to screen for ther~pelltirs useful for the tr~o~tmlont of ~17hPimPr's disease.
~17h-oim~r's disease (AD) is a degell~ldLi~e disorder of the brain first described by Alios Alzheimer in 1907 after ex~mining one of his patients who suffered drastic reduction in cognitive abilities and had generalized ~lementi~ (The early story of Alzheimer's Disease, edited by Bick et al.
(Raven Press, New York 1987)). It is the leading cause of ~l~menti~ in elderly persons. AD patients have increased problems with memory loss and intellectual functions which progress to the point where they cannot function as normal individuals. With the loss of intellectual skills the patients exhibitpersonality changes, socially illa~lo~l;ate actions and schizophrenia (A
Guide to the Understanding of Alzheimer's Disease and Related Disorders, edited by Jorm (New York University Press, New York 1987). AD is dev~t~tin~ for both victims and their f~mili~s, for there is no effective palliative or preventive treatment for the inevitable neurodegeneration.
The impact of AD on society and on the n~tion~l economy is enormous. It is expected that the demented elderly population in the United States will increase by 41 % by the year 2000. It is expensive for the health care systems that must provide institutional and ancillary care for the AD
patients at an estim~t~ annual cost of $40 billion (Jorm (1987); Fisher, "~l7h~imer's Disease", New York Times, August 23, 1989, page Dl, edited by Fteisberg (The Free Press, New York & London 1983)). These factors imply action must be taken to generate effective tre~tmtont~ for AD.
At a macroscopic level, the brains of AD patients are usually smaller, sometimes weighing less than 1,000 grams. At a microscopic level, the histopathological h~llm~rkc of AD include neurofibrillary tangles (NFT), neuritic plaques, and degeneration of neurons. AD patients exhibit degeneration of nerve cells in the frontal and temporal cortex of the cerebral WO 96/40896 : PCT~US96/09857 cortex, pyramidal neurons of hippocampus, nt:ulolls in the medial, medial central, and cortical nuclei of the amygdala, noradlcnelgic neurons in the locus coeruleus, and the neurons in the basal forebrain cholinergic system.
Loss of neurons in the cholinergic system leads to a consistent deficit in cholinergic ~ ylld~Lic nld~ in AD (Fisl1er (1983); Alzheimer's Disease and Related Disorders, Research and Development edited by Kelly (Charles C. Thomas, SpringfiP~ IL. 1984)). In fact, AD is defined by the n~ul~o~dlllology of the brain.
AD is associated with neuritic plaques m~ lring up to 200 ,um in fli~mP:ter in the cortex, hippocampus, subiculum, hippocampal gyrus, and amygdala. One of the principal con~tit~Pnt~ of neuritic plaques is amyloid, which is stained by Congo Red (Fisher (1983); Kelly (1984)). Amyloid plaques stained by Congo Red are extracellular, pink or rust-colored in bright field, and bilcr~ g~l~L in polarized light. The plaques are composed of polypeptide fibrils and are often present around blood vessels, reducing blood supply to various neurons in the brain.
Various factors such as genetic predisposition, infectious agents, toxins, metals, and head trauma have all been suggested as possible mech~ni~m~ of AD neuropathy. However, available evidence ~LIollgly in~lir~t~s that there are distinct types of genetic predispositions for AD.
First, molecular analysis has provided evidence for mutations in the amyloid precursor protein (APP) gene in certain AD-stricken families (Goate et al.
Nature 349:704-706 (1991); Murrell et al. Science 254:97-99 (1991);
Chartier-Harlin et al. Nature 353:844-846 (1991); Mullan et al., Nature Genet. 1:345-347 (1992)). Additional genes for dominant forms of early onset AD reside on chromosome 14 and chromosome 1 (Rogaev et al., Nature 376:775-778 (1995); Levy-Lahad et al., Science 269:973-977 (1995);
Sherrington et al., Nature 375:754-760 (1995)). Another loci associated with AD resides on chromosome 19 and encodes a variant form of apolipoplotei E (Corder, Science 261:921-923 (1993).
Amyloid plaques are abundantly present in AD patients and in Down's Syndrome individuals ~iUl viving to the age of 40. The o~e.~ s~ion of APP

in Down's Syndrome is recognized as a possible cause of the development of AD in Down's patients over thirty years of age (Rumble et al., New England J. Med. 320:1446-1452 (1989); Mann et al., Neurobiol. Aging 10:397-399 (1989)). The plaques are also present in the normal aging brain, although at S a lower number. These plaques are made up primarily of the amyloid ,~
peptide (A,B; sometimes also referred to in the liLeldLulc as ,B-amyloid peptideor ,~ peptide) (Glenner and Wong, Biochem. Biophys. Res. Comm. 120:885-890 (1984)), which is also the ~ llaly protein con~tit lent in cerebrovascular amyloid deposits. The amyloid is a fil~mentous material that is arranged in 10 beta-pleated sheets. A,~ is a hydrophobic peptide comprising up to 43 amino acids. The rit~termin~tion of its amino acid sequence led to the cloning of the APP cDNA (Kang et al., Nature 325:733-735 (1987); Goldgaber et al., Science 235:877-880 (1987); Robakis et al., Proc. Natl. Acad. Sci. 84:4190-4194 (1987); Tanzi et al., Nature 331:528-530 (1988)) and genomic APP
15 DNA (Lemaire et al., Nucl. Acids Res. 17:517-522 (1989); Yoshikai et al., Gene 87, 257-263 (1990)). A number of forms of APP cDNA have been identified, including the three most abundant forms, APP695, APP751, and APP770. These forms arise from a single precursor RNA by ~ltern~te splicing. The gene spans more than 175 kb with 18 exons (Yoshikai et al.
20 (1990)). APP contains an extracellular domain, a tr~ncmemhrane region and a cytoplasmic domain. A,B consists of up to 28 amino acids just outside the hydrophobic tr~n~memhrane domain and up to 15 residues of this tr~n~m~nnhrane domain. Thus, A~ is a cleavage product derived from APP
which is normally found in brain and other tissues such as heart, kidney and 25 spleen. However, A~ deposits are usually found in ablln-l~n~e only in the brain.
The larger alternate forms of APP (APP751, APP770) consist of APP695 plus one or two additional dom~in~. APP751 consists of all 695 amino acids of APP695 plus an additional 56 amino acids which has 30 homology to the Kunitz family of serine protease inhibitors (KPI) (Tanzi et al. (1988); Weiclem~nn et al., Cell 57:115-126 (1989); Kitaguchi et al., Nature 331:530-532 (1988); Tanzi et al., Nature 329:156 (1987)). APP770 WO 96/40896 PCTrUS96/09857 contains all 751 amino acids of APP751 and an additional 19 amino acid domain homologous to the neuron cell surface antigen OX-2 (Wei-lem~nn et al. (1989); Kitaguchi et al. (1988)). Unless otherwise noted, the amino acid positions referred to herein are the positions as they appear in APP770. The amino acid number of equivalent positions in APP695 and APP751 differ in some cases due to the ~bs~on~e of the OX-2 and KPI (lom~inc. By convention, the amino acid positions of all forms of APP are lc:r~lellced by the equivalent positions in the APP770 form. Unless otherwise noted, this convention is followed herein. Unless otherwise noted, all forms of APP and fr~gmlontc of APP, including all forms of A,~, referred to herein are based on the human APP amino acid sequence. APP is post-translationally modified by the removal of the leader sequence and by the addiLion of sulfate and sugar groups.
Van Broeckhaven et al., Science 248:1120-1122 (1990), have demonstrated that the APP gene is tightly linked to hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) in two Dutch f~milies. This was confirm.o(l by the finding of a point mutation in the APP coding region in two Dutch patients (Levy et al., Science 248:1124-1128 (1990)); The mutation substituted a ~ f;..~ .e for glut~mic acid at position 22 of the A,~ (position 618 of APP695, or position 693 of APP770). In addition, certain families are geneti~ally predisposed to Alzheimer's disease, a condition referred to as f~mili~l Alzheimer's disease (FAD), through mutations rPs--ltin~ in an amino acid replacement at position 717 of the full length protein (Goate et al.
(1991); Murrell et al. (1991); Chartier-Harlin et al. (1991)). These mutations co-segregate with the disease within the f~milies and are absent in f~miliec with late-onset AD. This mutation at amino acid 717 increases the production of the A~l~2 form of A~ from APP (Suzuki et al., Science 264:1336-1340 (1994)). Another mutant form contains a change in amino acids at positions 670 and 671 of the full length protein (Mullan et al.
(1992)). This mutation to amino acids 670 and 671 increases the production of total A~B from APP (Citron et al., Nature 360:622-674 (1992)).

-CA 02222l74 l997-ll-24 WO ~G/1~B36 PCTAJS~'O~

S
There are no robust animal models to study AD, although aging nonhllm~n primates seem to develop amyloid plaques of A~ in brain parenchyma and in the walls of some meningeal and cortical vessels.
Although aged primates and canines can serve as animal models, they are S ~e~iv~ to m~int~in, need lengthy study periods, and are quite variable in the extent of pathology that develops.
There are no spontaneous animal mutations with sllf~lri~nt cimil~ritiPs to AD to be useful as ex.~lilllental models. Various models have been proposed in which some AD-like ~ylllpLullls may be intlllce(l by electrolysis, transplantation of AD brain samples, ~lu,~ "~ chloride, kainic acid or choline analogs (Kisner et al., Neurobiol. Aging 7:287-292 (1986); Mistry et al., JMed Chem 29:337-343 (1986)). Flood et al., Proc. Natl. Acad. Sci.
88:3363-3366 (1986), reported ~mnPstic effects in mice of four ~yllLlleLic peptides homologous to the A,~. Rec~llce none of these share with AD either common ~ylllpLullls, biochemictry or pathogenesis, they are not likely to yield much useful information on etiology or tre~tm~onS
Several transgenic rodent lines have been produced that express either the human APP gene or human APP complement~ry DNA regulated by a variety of promoters. Transgenic mice with the human APP promoter linked to E. coli ~-galactosidase (Wirak et al., The EMBO J 10:289-296 (1991)) as well as transgenic mice expressing the human APP751 cDNA (Quon et al.
Nature 352:239-241 (1991)) or subfr~gmentc of the cDNA inrl~l~ling the A,B
(Wirak et al., Science 253:323-325 (1991); Sandhu et al., J. Biol. Chem.
266:21331-21334 (1991); Kawabata et al., Nature 354:476-478 (1991)) have been produced. Results obtained in the different studies appear to depend upon the source of promoter and the protein coding sequence used. For example, Wirak et al., Science 253:323-325 (1991), found that in transgenic mice expressing a form of the A,~, intracellular ~cc -m~ tinn of "amyloid-like" material, reactive with antibodies prepared against A~ were observed but did not find other histopathological disease symptoms. The intracellular nature of the antibody-reactive material and the lack of other symptoms suggest that this particular transgenic animal is not a faithful model system WO ~G/~ 6 PCTAJS96/09857 for Alzheimer's disease. Later studies have shown that similar st~ining iS
seen in non-transgenic control mice and Wirak et al., Science 253:323-325 (1991) was partially retracted in a commPnt in Science 255:143-145 (1992).
Thus, the sf~ining seen by Wirak et al. appears to be artif~r-tn~l Kawabata et al. (1991) report the production of amyloid plaques, neurofibrillary tangles, and neulullal cell death in their LLdnsg~llic ~nim~
In each of these studies, A~B or a fragment cont~ining A,~ was ~ cssed.
Wirak et al. (1991), used the human APP promoter while Kawabata et al.
(1991) used the human thy-1 promoter. However, Kawabata et al. (1991) was later retracted by Kawabata et al., Nature 356:23 (1992) and Kawabata et al., Nature 356:265 (1992). In transgenic mice expressing the APP751 cDNA from the neuron-specific enolase promoter of Quon et al. (1991), rare, small extracellular deposits of material reactive with antibody prepared against synthetic A,B were observed. A review of the papers describing these early transgenic mice in(lir~te that do not produce char~cteri~tir Alzheimer pathologies (see Marx, Science 255:1200-1202 (1992)).
Transgenic mice e~lessillg APP751 from a neuron-specific enolase (NSE) promoter were recently described by McConlogue et al., Neurobiol.
Aging 15:S12 (1994), Higgins et al., Ann Neurol. 35:598-607 (1995), Mucke et al., Brain Res. 666:151-167 (1994), ~i~gin.~ et al., Proc. Natl. Acad. Sci.
USA 92:4402-4406 (1995), and U.S. Patent 5,387,742 to Cordell. Higgins et al., Ann Neurol. 35:598-607 (1995) describe results with the same mice as described by Quon et al. (1991). Such mice have only sparse A~ deposits which are more typical of very early AD and young Down's syndrome cases.
The deposits seen in this transgenic mouse were also seen, although at a lower abnn~nre, in non-transgenic control animals. Mature lesions such as frequent compacted plaques, neuritic dystrophy and extensive gliosis are not seen in these mice (Higgins et al., Ann Neurol. 35:598-607 (1995)).
McC~onlogue et al. (1994) reported finding no A~ deposits in these mice.
Transgenic mice in which APP is expressed from the neuronal specific ~yllaL~lophysin promoter express APP at low levels equivalent to that in brain WO ~6/~0896 PCT~US9G

tissue from the NSE APP mice described above. These mice were also reported not to display any brain lesions (Higgins et al.).
Transgenic mice cont~ining yeast artificial chromosome (YAC) APP
constructs have also been made (Pearson and Choi, Proc. Natl. Acad. Sci.
USA 90:10578-10582 (1993); Lamb et al., Nature Genetics 5:22-30 (1993);
Buxbaum et al., Biochem. Biophys. Res. Comm. 197:639-645 (1993)). These mice contain the entire human APP genomic gene and express human APP
protein at levels similar to endogenous APP; higher levels of ~ r~,ssion than that obtained in mice using the NSE promoter. None of these mice, however, show evidence of pathology similar to AD.
Alzheimer's disease animal models, including transgenic models, have been recently reviewed by Lannfelt et al., Behavioural Brain Res. 57:207-213 (1993), and Fukuchi et al., Ann. N. Y. Acad. Sci. 695:217-223 (1993).
Lannfelt et al. points out that none of the prior transgenic ~nim~l.c that show ~pale-lL plaques demonstrate neuropathological changes ch~r~eteristic of AD.
Lannfelt et al. also rli~cll~ses possible reasons for the "failure" of previous transgenic animal models. Similarly, Fukuchi et al. tli~cu~es the failure of prior transgenic animal models to display most of the characteristics known to be associated with AD. For example, the transgenic mouse reported by Quon et al. is reported to produce A,~ immllnnreactive deposits that stain only infrequently with thioflavin S and not at all with Congo Red, in contrast to the st~ining pattern of AD A~ deposits.
Alzheimer's disease is characterized by numerous changes in the expression levels of various proteins, the biochemical activity and histopathology of brain tissue, as well as cognitive changes in affected individuals. Such characteristic changes associated with AD have been well doc lmentecl. The most prominent change, as noted above, is the deposition of A,B into amyloid plaques (Haass and Selkoe, Cell 75:1039-1042 (1993)).
A variety of other molecules are also present in plaques, such as apolipoprotein E, l~minin, amyloid P component, and collagen type IV
(Kalaria and Perry, Brain Research 631:151-155 (1993); Ueda et al., Proc.
Natl. Aca. Sci. USA 90:11282-11286 (1993)). Changes in cytoskeletal .lalh~l~ have also been associated with AD, such as the changes in microtubule-associated protein tau, MAP-2 or neurofil~mPnt~ (Kosik et al., Science 256: 780-783 (1992); Lovestone and Anderton, Current Opinion in Neurology & Neurosurgery 5:883-888 (1992); Brandan and Inestrosa, General Pharmacology 24: 1063-1068 (1993); Trojanowski et al., Brain Pathology 3:45-54 (1993); Masliah et al., American Journal of Pathology 142:871-882 (1993)). Alzheimer's disease is also known to stim~ t~ an i"""ll"~i"ll~mm~tQry response, increasing such infl~mm~tory ~ as glial fibrillary acidic protein (GFAP), c~2-macroglobulin, and interleukins 1 and 6 (IL-l and IL-6) (Frederickson and Brunden, Alzheimer Disease and Associated Disorders 8:159-165 (1994); McGeer et al., Canadian Journal of Neurological Sciences 18:376-379 (1991); Wood et al., Brain Research 629:245-252 (1993)). Finally, neuronal and n~:ul~ l changes have been associated with AD, such as the cholinergic, muscarinic, serotinergic, adrenergic, and adensosine receptor systems (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); Masliah and Terry, Clinical Neuroscience 1:192-198 (1993); Greenamyre and Maragos, Cerebrovascular and Brain Metabolism Reviews 5:61-94 (1993); McDonald and Nemeroff, Psychiatric Clinics of North America 14:421-422 (1991); Mohr et al., Journal of Psychiatry & Neuroscience 19:17-23 (1994)).
It is therefore an object of the present invention to provide an animal model for Alzheimer's disease that is constructed using transgenic technology.
It is a further object of the present invention to provide transgenic animals characterized by certain genetic abnormalities in the expression of the amyloid precursor protein.
It is a further object of the present invention to provide transgenic animals exhibiting one or more histopathologies similar to those of Alzheimer's disease.

=

WO 96/40896 PCT~US96/09857 It is a further object of the present invention to provide transgenic ~nim~l~ e~ s~ g one or more A,~-c~ ;.i..i..g ~loleills at high levels in brain tissue.
It is a further object of the present invention to provide a method of S screening potential drugs for the tre~tm~nt of ~17htoimer's disease using transgenic animal models.
S~ of the Invention The construction of transgenic animal models for testing potential tre~tm~ont~ for Alzheimer's disease is described. The models are char~cteri7Pcl by a greater ~imil~rity to the conditions exi~ting in naturally occurring Al_heimer's disease, based on the ability to control t~ les~ion of one or more of the three major forms of the ~-amyloid precursor protein (APP), APP695, APP751, and APP770, or subfr~gm~nt~ thereof, as well as various point mutations based on naturally occurring mutations, such as the lS FAD mutations at amino acid 717, and predicted mutations in the APP gene.
The APP gene constructs are prepared using the naturally occurring APP
promoter of human, mouse, or rat origin, efficient promoters such as human platelet derived growth factor ~ chain (PDGF-B) gene promoter, as well as inducible promoters such as the mouse metallothionine promoter, which can be regulated by addition of heavy metals such as zinc to the animal's water or diet. Neuron-specific expression of constructs can be achieved by using the rat neuron specific enolase promoter.
The constructs are introduced into animal embryos using standard techniques such as microinjection or embryonic stem cells. Cell culture based models can also be prepared by two methods. Cells can be isolated from the transgenic animals or prepared from established cell cultures using the same constructs with standard cell transfection techniques.
The constructs disclosed herein generally encode all or a contiguous portion of one of the three forms of APP: APP695, APP751, or APP770, ~ 30 preferably an A~-cont~ining protein, as described herein. Examples of A,B-cont~ining proteins are proteins that include all or a contiguous portion of APP770, APP770 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, APP751, APP751 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, APP695, and APP695 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, where each of these A,~-colll;.il)i,~g ploleins includes amino acids 672 to 714 of human APP. Some 5 specific constructs that are described employ the following protein coding sequences: the APP770 cDNA; the APP770 cDNA bearing a mllt~til~n at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; the APP751 cDNA cont~inin~ the KPI protease inhibitor domain without the OX-2 domain in the construct; the APP751 cDNA bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; the APP695 cDNA; the APP695 cDNA bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; APP695, APP751, or APP770 cDNA truncated at amino acid 671 or 685, the sites of ~-secretase or Ix-secretase cleavage, respectfully; APP
cDNA truncated to encode amino acids 646 to 770 of APP; APP cDNA
~lullcatcd to encode amino acids 646 to 770 of APP and including at least one intron; the APP leader sequence followed by the A,B region (amino acids 672 to 714 of APP) plus the rem~ining carboxy terminal 56 amino acids of APP;
the APP leader sequence followed by the A,B region plus the rem~ining carboxy terminal 56 amino acids with the addition of a mutation at amino acid 717; the APP leader sequence followed by the A,~ region; the A~ region plus the rem~ining carboxy tennin~l 56 amino acids of APP; the A~ region plus the rem~ining carboxy terminal 56 amino acids of APP with the addition of a mutation at amino acid 717; a combination cDNA/genomic APP gene construct; a combination cDNA/genomic APP gene construct with the addition of a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a combination cDNA/genomic APP gene construct truncated at amino acid 671 or 685; and an APP cDNA construct cont~ining at least amino acids 672 to 722 of APP.
These protein coding sequences are operably linked to leader sequences specifying the transport and secretion of the encoded A,B related protein. A ~lcrellcd leader sequence is the APP leader sequence. These WO 96/40896 PCT~U5~6/O~a/

combined protein coding sequences are in turn operably linked to a promoter that causes high c~ cssion of A~ in transgenic animal brain tissue. A
crcllcd promoter is the human platelet derived growth factor ~ chain (PDGF-B) gene promoter. Additional constructs include a human yeast artificial chromosome construct controlled by the PDGF-B promoter; a human yeast artificial chromosome construct controlled by the PDGF-B
promoter with the addition of a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; the endogenous mouse or rat APP gene modified through the process of homologous recombination between the APP gene in a mouse or rat embryonic stem (ES) cell and a vector carrying the human APP cDNA bearing a mutation at amino acid position 669, 670, 671, 690, 692, 717, or a combination of these mutations, such that sequences in the resident rodent chromosomal APP gene beyond the recombination point (the plcrcllcd site for recombination is within APP exon 9) are replaced by the analogous human sequences bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations. These constructs can be introduced into the ~ldnsgenic ~nim~l~
and then combined by mating of animals C~lCS~illg the dirrclcllL constructs.
The transgenic animals, or animal cells, are used to screen for compounds altering the pathological course of Alzheimer's disease as measured by their effect on the amount and/or histopathology of Alzheimer's disease markers in the animals, as well as by behavioral alterations. These markers include APP and APP cleavage products; A~'; other plaque related molecules such as apolipoprotein E, l~minin, and collagen type IV;
cytoskeletal markers, such as spectrin, tau, neurofil~m~nt~, and MAP-2;
infl~mm~tory llladlkcl~7 such as GFAP, ~2-macroglobulin, IL-1, and IL-6;
and neuronal and synaptic neurotr~n~mht~r related markers, such as GAP43 and synaptophysin, and those associated with the cholinergic, mll~c~rinic, serotinergic, adrenergic, and adensosine receptor systems.
Brief Des~ Lion of the D~edwi~
The boxed portions of the drawings inn'ir~t~- the amino acid coding portions of the constructs. Filled portions in~lir~te the various domains of the WO 96/40896 PCT~US96/098~7 protein as in-lir~tr~l in the Figure,Legend. Lines indicate seq~enres in the clones that are 5' or 3' untran~l~te~l sequences, fl~nking genomic seq~1enres, or introns. The break in the line to the left of the constructs in Figures 7 and8 in-lir~tes the presence of a long DNA sequence.
S Figure la is a srh~m~tir of the APP770 cDNA coding sequence.
Figure lb is a sr,hrm~tir of the APP770 cDNA coding sequence bearing a mllt~tion at position 717.
Figure 2a is a sr,h~m~tir. of the APP751 cDNA coding sequence.
Figure 2b is a schpm~tir of the APP751 cDNA coding sequence bearing a mutation at position 717.
Figure 3a is a srhrm~tic of the APP695 coding sequence.
Figure 3b is a schrm~tir of the APP695 cDNA coding sequence bearing a mutation at position 717.
Figure 4a is a sc,hem~tir, of a coding sequence for the carboxy terminal portion of APP.
Figure 4b is a srhrm~tir, of a coding sequence for the carboxy ~ermin~l portion of APP bearing a mutation at position 717.
Figure 5 is a schr,m~tir, of a coding sequence for the A,B portion of APP.
Figure 6a is a schematic of a combination cDNA/genomic coding sequence allowing ~ltrrn~tive splicing of the KPI and OX-2,exons.
Figure 6b is a srh~ tic of a combination cDNA/genomic coding sequence bearing a mutation at position 717 and allowing alternative splicing of the KPI and OX-2 exons.
Figure 7a is a srh.-m~tic, of a human APP YAC coding sequence.
Figure 7b is a srhem~tir of a human APP YAC coding sequence bearing a mutation at position 717.
Figures 8a and 8b are schematics of genetic alteration of the mouse APP gene by homologous recombination belween the mouse APP gene in a mouse ES cell and a vector carrying the hurnan APP cDNA (either of the wild-type (Figure 8a) or FAD mutant form (Figure 8b)) directed to the exon 9 portion of the gene. As a result of this recombination event, sequences in CA 02222174 l997-ll-24 W 0~6/~8~6 PCT~USg~0~57 the resident mouse chromosomal APP gene beyond the recombination point in exon 9 are replaced by the analogous human sequences.
Figure 9 is a schPm~tic map of the PDAPP vector, a combination cDNA/genomic APP construct.
Figure 10 is a diagram of the genomic region of APP present in the PDAPP construct. The sizes of original introns 6, 7 and 8, as well as the sizes of the final introns are intlic~tPrl on the diagram. The locations of the deletions in introns 6 and 8 present in the PDAPP construct are also in~ t~rl.
Figure 11 is a diagram of the intPrmP~ t~ constructs used to construct the APP splicing cassette and the PDAPP vector.
Figure 12 is a diagram of the PDAPP-wt vector and the pl~mi-l.c used to make the PDAPP-wt vector.
Figure 13 iS a diagram of the PDAPP-Sw/Ha vector and the plasmids 15 and intermediate constructs used to make the PDAPP-Sw/Ha vector.
Figure 14 is a diagram of the PDAPP695V F vector and the plasmids and intermediate constructs used to make the PDAPP695V F vector.
Figure 15 is a diagram of the PDAPP751VF vector and the plasmids and intermediate constructs used to make the PDAPP751VF vector.
Detailed Des~ lion of the Invention The constructs and transgenic ~nim~l~ and animal cells are prepared using the methods and materials described below.
Sources of materials.
Restriction endonucleases are obtained from conventional commercial 25 sources such as New Fngl~n~1 Biolabs (Beverly, MA.), Promega Biological Research Products (Madison, WI.), and Stratagene (La Jolla CA.).
Radioactive materials are obtained from conventional commercial sources such as Dupont/NEN or Amersham. Custom-designed oligonucleotides for site-directed mutagenesis are available from any of several commercial 30 providers of such materials such as Bio-Synthesis Inc., Lewisville, TX. Kits for carrying out site-directed mutagenesis are available from commercial suppliers such as Promega Biological Research Products and Stratagene.

WO 96/40896 . PCT~US96/09857 Clones of cDNA including the APP695, APP751, and APP770 forms of APP
mRNA were obtained directly from Dr. Dmitry Goldgaber, NIH. Libraries of DNA are available from commercial providers such as Str~t~en~-, La Jolla, CA., or Clontech, Palo Alto, CA. PC12 and 3T3 cells were obtained from ATCC (#CRL1721 and #CCL92, respectively). An additional PC12 cell line was obtained from Dr. Charles Marotta of Harvard Medical School, ~c~rhll~ett~ General Hospital, and Mc.T P~n Hospital. Standard cell culture media ~lu~ L~ to the cell line are obtained from convenfinn~l commercial sources such as Gibco/BRL. Murine stem cells, strain D3, were obtained from Dr. Rolf Kemler (Doet~hm~n et al., J. Embryol. Exp. Morphol. 87:27 (1985)). Lipofectin for DNA transfection and the drug G418 for selection of stable L~ ~rollllants are available from Gibco/BRL.
D~finiti~n of APP cDNA clones.
The cDNA clone APP695 is of the form of cDNA described by Kang et al., Nature 325:733-735 (1987), and l~l~sellL~ the most predolllhlall~ form of APP in the brain. The cDNA clone APP751 is of the form described by Ponte et al., Nature 331:525-527 (1988). This form contains an insert of 168 nucleotides relative to the APP695 cDNA. The 168 nucleotide insert encodes the KPI domain. The cDNA clone APP770 is of the form described by Kitaguchi et al. Nature 331:530-532 (1988). This form contains an insert of 225 nucleotides relative to the APP695 cDNA. This insert includes the 168 nucleotides present in the insert of the APP751 cDNA, as well as an addition 57 nucleotide region that does not appear in APP751 cDNA. The 225 nucleotide insert encodes for the KPI domain as well as the OX-2 domain. All three forms arise from the same precursor RNA Llans~liL,t by terTl~tive splicing. The 168 nucleotide insert is present in both APP751 cDNA and APP770 cDNA.
The sequence encoding APP695 is shown in SEQ ID NO:l. This sequence begins with the first base of the initiation codon AUG and encodes a 695 amino acid protein. The region from nucleotide 1789 to 1917 of SEQ
ID NO:l encodes the A,B. The amino acid sequence of APP695 is shown in SEQ ID NO:2. Amino acids 597 to 639 of SEQ ID NO:2 form the A,~. The amino-acid composition of the APP695 is A57, C12, D47, E85, F17, G31, H25, I23, K38, L52, M21, N28, P31, Q33, R33, S30, T45, V62, W8, Y17 reslllting in a c~lclll~t~-l molecular weight of 78,644.45. These sequences are derived from Kang et al. (1988).
The sequence encoding APP751 is shown in SEQ ID NO:3. This sequence begins with the first base of the initiation codon AUG and encodes a 751 amino acid protein. Nucleotides 866 to 1033 of SEQ ID NO:3 do not appear in APP695 cDNA. The region from nucleotide 1957 to 2085 of SEQ
ID NO:3 encodes the A,B. The amino acid sequence of APP751 is shown in SEQ ID NO:4. Amino acids 289 to 345 of SEQ ID NO:4 do not appear in APP695. This 57 amino acid region includes the KPI ~lom~in Amino acids 653 to 695 of SEQ ID NO:4 form the A,~. These sequences are derived from Ponte et al. (1988).
The sequence encoding APP770 is shown in SEQ ID NO:S. This sequence begins with the first base of the initiation codon AUG and encodes a 770 amino acid protein. Nucleotides 866 to 1090 of SEQ ID NO:S do not appear in APP695 cDNA. Nucleotides 1034 to 1090 of SEQ ID NO:5 do not appear in APP751 cDNA. The region from nucleotide 2014 to 2142 encodes the A,~. The amino acid sequence of APP770 is shown in SEQ ID
NO:6. Amino acids 289 to 364 of SEQ ID NO:6 do not appear in APP695.
This 76 amino acid region includes the KPI and OX-2 domains. Amino acids 345 to 364 of SEQ ID NO:6 do not appear in APP751. This 20 amino acid region includes the OX-2 domain. Amino acids 672 to 714 form the A~B. A
probable membrane-spanning region of the APP occurs from amino acid 700 to 723. Unless otherwise stated, all references herein to nucleotide positions refer to the numbering of SEQ ID NO:5. This is the numbering derived from the APP770 cDNA. Unless otherwise stated, all references herein to amino acid positions refer to the numbering of SEQ ID NO:6. This is the numbering derived from APP770. According to this numbering convention, for example, amino acid position 717 refers to amino acid 717 of APP770, amino acid 698 of APP751, and amino acid 642 of APP695. The above sequences are derived from Kang et al. (1988) and Kitaguchi et al. (1988).

CA 02222l74 l997-ll-24 W096/40896 - - PCT~US96~09857 Urlless otherwise noted, all forms of APP and fragments of APP, in~ in~ all forms of A,B, referred to herein are based on the human APP
amino acid sequence. For example, A,B refers to the human A,~, APP refers to human APP, and APP770 refers to human APP770. As used herein, the 5 term cDNA refers not only to DNA molecules actually prepared by reverse L,~s~ ion of mRNA, but also any DNA molecule encoding a protein where the coding region is not hlLt.lu~L~d, that is, a DNA molecule having a continuous open reading frame encoding a protein. As such, the term cDNA
as used herein provides a convenient means of referring to a protein encoding 10 DNA molecule where the protein encoding region is not i.lLtl.u~L~d by intron sequences (or any other sequences not encoding protein).
DPfinition of the APP genoIuic locus.
Chara-;L~ ion of phage and cosmid clones of human genomic DNA
clones listed in Table 1 below originally established a minimnm size of at least 100 kb for the Alzheimer's gene. There are a total of 18 exons in the APP gene (Lemaire et al., Nucl. Acid Res 17:517-522 (1989); Yoshikai et al.
(1990); Yoshikai et al., Nucleic Acids Res 102:291-292 (1991)). Yoshikai et al. (1990) describes the sequences of the exon-intron boundaries of the APP
gene. These results taken together in~ t~- that the minimnm size of the 20 Alzheimer's gene is 175 kb.

-W O~C/qO896 PCT~US96/09857 Table 1. ~l~h~im~r's Cosmid and La nbda Clones.

Name of Insert Library CloneSize (kb) Assigned APP Region 1 GPAPP47A 35 25 kb promoter & 9 kb intron 1 Cosmid 2 GPAAP36A 35 12 kb promoter & 22 kb intron 1 3 GAPP30A30-35 5' coding region 4 GAPP43A30-35 exons 9, 10 and 11 1 GAPP6A 12 exon 6 2 GAPP6B 18 exons 4 and 5 3 GAPP20A20 exon 6 4 GAPP20B17 exons 4 and 5 Lambda 5 GAPP28A18 exons 4 and 5 6 GAPP3A 14 exon 6 7 GAPP4A 19 exon 6 8 GAPPlOA16 exons 9, 10 and 11 9 GAPP16A21 exon 6 Table 2 in~lir~t~s where the 17 introns illL~llu~t the APP coding sequence. The numbering refers to the nucleotide positions of APP770 cDNA as shown in SEQ ID NO:5. The starting nucleotide of exon 1 selll~ the first transcribed nucleotide. It is negative because the + 1 5 nucleotide is the first nucleotide of the AUG initi~tor codon by convention (Kang et al. (1988)). The ending nucleotide of exon 18 represents the last nucleotide present in the mRNA prior to the poly(A) tail (Yoshikai et al.
(1990)). It has been discovered that Yoshikai et al. (1990) and Yoshikai et al. (1991) contain an error in the location of exon 8. Figure 1 of Yoshikai et al. (1991) includes an EcoRI fragment between EcoRI fragments Co,~ i,-g exon 7 and exon 8. In fact, this hlLel~/~nillg EcoRI fragment is actually located immediately after exon 8, so that the EcoRI fragment cont~ining exon 7 and the EcoRI fragment cont~ining exon 8 are adjacent to each other.

W O 96/40896 PCT~US96/09857 Table 2. T.oc~ti~n of Introns in APP Gene Sequence.
Starting Ending Following nucleotidenucleotide Intron Exon 1 -146 57 Intron 1 Exon 2 58 225 Intron 2 Exon 3 226 355 Intron 3 Exon 4 356 468 Intron 4 Exon 5 469 662 Intron 5 Exon 6 663 865 Intron 6 Exon 7 866 1033 Intron 7 Exon 81034 1090 Intron 8 Exon 91091 1224 Intron 9 Exon 101225 1299 Intron 10 Exon 111300 1458 Intron 11 Exon 121459 1587 Intron 12 Exon 131588 1687 Intron 13 Exon 141688 1909 Intron 14 Exon 151910 1963 Intron 15 Exon 161964 2064 Intron 16 Exon 172065 2211 Intron 17 Exon 182212 3432 APP Gene ~ t~ti~ n~.
Certain f~milies are gen~tir~lly predisposed to Alzheimer's disease, a condition referred to as f~mili~l Alzheimer's disease (FAD), through mutations rcsnlting in an amino acid repl~çm~nt at position 717 of the full length protein (Goate et al. (1991); Murrell et al. (1991); Chartier-Harlin et al. (1991)). These mutations co-segregate with the disease within the famili~s. For example, Murrell et al. (1991) described a specific mutation found in exon 17 (which Murrell et al. refers to as exon 15) where the valine of position 717 is replaced by phenyl~l~nin~.
Another FAD mutant form contains a change in amino acids at positions 670 and 671 of the full length protein (Mullan et al. (1992)). In one form of this mutation, the lysine at position 670 is replaced by asparagine and the methionine at position 671 is replaced by leucine. The effect of this mutation is to increase the production of A~ in cultured cells approximately 7-fold (Citron et al., Nature 360: 672-674 (1992); Lai et al., Science 259:514-516 (1993)). Replacement of the methionine at position 671 with leucine by itself has also been shown to increase production of A,~.

~ =

WO ~G/~D~6 PCT~US96/09857 Additional mutations in APP at amino acids 669, 670, and 671 have been shown to reduce the amount of A~ processed from APP (Citron et al., Neuron 14:661-670 (1995)). The APP construct with Val at amino acid 690 produces an increased amount of a Ll.~ erl form of A,B.
APP eA~rcs~ion clones can be constructed that bear a mnt~ti~ n at amino acid 669, 670, 671, 690, 692, or 717 of the full length protein.
The mllt~tinn~ from Lys to Asn and from Met to Leu at amino acids 670 and 671, respectively, are som~tim~os referred to as the Swedish mutation.
Additional mutations can also be introduced at amino acids 669, 670, or 671 which either increase or reduce the amount of A,~ processed from APP.
Mutations at these amino acids in any APP clone or Ll~,lsgel.e can be created by site-directed mutagenesis (Vincent et al., Genes & Devel. 3:334-347 (1989)), or, once made, can be incorporated into other constructs using ~L~dald genetic engineering techniques. Some mutations at amino acid 717 are somPtim~ referred to as the Hardy mutation. Such mutations can include conversion of the wild-type Val717 codon to a codon for Ile, Phe, Gly, Tyr, Leu, Ala, Pro, Trp, Met, Ser, Thr, Asn, or Gln. A ~lcrellcd substitution for Val717 is Phe. These mutations predispose individuals c~lcssing the mutant ~loLcills to develop Alzheimer's disease. It is believed that the mutations affect the expression and/or processing of APP, shifting the balance toward Alzheimer's pathology. Mutations at amino acid 669 can include conversion of the wild-type Val669 codon to a codon for Trp, or deletion of the codon. Mutations at amino acid 670 can include conversion of the wild-type Lys670 codon to a codon for Asn or Glu, or deletion of the codon. Mutations at amino acid 671 can include conversion of the wild-type Met671 codon to a codon for Leu, Val, Lys, Tyr, Glu, or Ile, or deletion of the codon. A ~lcrcllcd substitution for Lys670 is Asn, and a plcrcllcd substitution for Met671 is Leu. These mutations predispose individuals expressing the mutant proteins to develop Alzheimer's disease. The other listed mutations to amino acids 669, 670, and 671 are known to reduce the amount of A~ processed from APP (Citron et al. (1995)). It is believed that WO ~G/4~8~6 PCT~US96/09857 these mutations affect processing of APP leading to a change in A,B
production.
Truncated forms of APP can also be expressed from transgene constructs. For example, APP cDNA truncated to encode amino acids 646 to 770 of APP. The APP cDNA construct Llullcalcd to encode amino acids 646 to 770 of APP, and operatively linked to the PDGF-B promoter, is referred to as PDAPPc125.
Nucleic Acid Constructs ~nl~o(ling A~-c.~ i..i..g I~vleills.
Constructs for use in transgenic animals include a promoter for c~lcs~ion of the construct in a ~ -l-",~ n cell and a region encoding a protein that inl~h~ s all or a contiguous portion of one of the three forms of APP: APP695, APP751, or APP770, with or without specific amino acid mutations as described herein. It is ~Jlcrcll~d that protein encoded is an A,~-cont~ining protein. As used herein, an A~-cont~ining protein is a protein that includes all or a contiguous portion of one of the three forms of APP:
APP695, APP751, or APP770, with or without specific amino acid mutations as described herein, where the protein includes all or a portion of amino acids 672 to 714 of human APP. Preferred A,~-cont~ining ~ tehls include amino acids 672 to 714 of human APP. Preferred forms of such A,B-cont~ining proteins include all or a contiguous portion of APP770, APP770 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, APP751, APP751 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, APP695, and APP695 bearing a mutation in amino acid 669, 670, 671, 690, 692, and/or 717, where each of these A,B-cont~ining proteins includes amino acids 672 to 714 of human APP.
Preferred forms of the above A~-cont~ining proteins are APP770;
APP770 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; APP751; APP751 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; APP695; APP695 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of WO gG/~_ 3~6 PCTrUS96/098~7 amino acid 669, 670, 671, 690, 692, 717; a protein con~i~ting of amino acids 646 to 770 of APP; a protein con~i.cting of amino acids 670 to 770 of APP; a protein con-~i~tin~: of amino acids 672 to 770 of APP; and a protein con.~i.cting of amino acids 672 to 714 of APP.
S In the constructs disclosed herein, the DNA encoding t'ne A,B-co,~ g protein can be cDNA or a cDNA/genomic DNA hybrid, wh~le.
the cDNA/genomic DNA hybrid includes at least one APP intron sequence wherein the intron sequence is sufficient for splicing.
Preferred constructs contain DNA encoding APP770; DNA encoding APP770 bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of DNA
encoding APP770 which encodes an amino acid sequence COlllpli~illg amino acids 672 to 714 of APP770; DNA encoding APP751; DNA encoding APP751 bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of DNA
encoding APP751 which encodes an amino acid sequence COlll~lisillg amino acids 672 to 714 of APP770; DNA encoding APP695; DNA encoding APP695 bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of DNA
encoding APP695 which encodes an amino acid sequence comprising amino acids 672 to 714 of APP770; APP cDNA truncated to encode amino acids 646 to 770 of APP; a combination cDNA/genomic DNA hybrid APP gene construct; a combination cDNA/genomic DNA hybrid APP gene construct bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; or a combination cDNA/genomic DNA hybrid APP gene construct truncated at amino acid 671 or 685.
Preferred forms of such constructs are APP770 cDNA; APP770 cDNA bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of APP770 cDNA encoding an APP amino acid sequence, the amino acid sequence comprising amino acids 672 to 714 of APP770; APP751 cDNA; APP751 . CA 02222174 1997-11-24 6 PCT~US96/09857 cDNA bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations; a fragment of APP751 cDNA encoding an APP amino acid sequence, the amino acid sequence COlll~liSillg amino acids 672 to 714 of APP770; APP695 cDNA; APP695 S cDNA bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, or a combination of these mllt~tion~; a fragment of APP695 cDNA encoding an APP amino acid sequence, the amino acid sequence collll~lisillg amino acids 672 to 714 of APP770; APP cDNA Llunca~ed to encode amino acids 646 to 770 of APP; a combination cDNA/genomic DNA
hybrid APP gene construct; a combination cDNA/genomic DNA hybrid APP
gene construct bearing a mutation in the codon encoding amino acid 669, 670, 671, 690, 692, 717, and a combination of these mutations; and a combination cDNA/genomic DNA hybrid APP gene construct truncated at amino acid 671 or 685.
Construction of Transgenes.
Construction of various APP transgenes can be accomplished using any suitable genetic engineering technique, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y., 1989). Regions of APP clones that have been engineered or mllt~t~-cl can be interchanged by using convenient restriction enzyme sites present in APP cDNA clones. A NruI site starts at position -5 (relative to the first nucleotide of the AUG initiator codon). A KpnI and an Asp718 site both start at position 57 (these are isoschizomers leaving differentsticky ends). A XcmI site starts at position 836 and cuts at position 843. A
ScaI site starts at position 1004. A XhoI site starts at position 1135. A
BamHI site starts at position 1554. A BgllI site starts at position 1994. An EcoRI site starts at position 2020. A SpeI site starts at position 2583.
Another EcoRI site starts at position 3076.
The clones bearing various portions of the human APP gene sequence shown in Figures 1 to 5 can be constructed in a common manner using standard genetic engineering techniques. For example, these clones can be constructed by first cloning the polyA addition signal from SV40 virus, as a WO9C/4~~6 PCT~US~ 93;

253 base pair BclI to BamHI fragment (Reddy et al., Science 200:494-502 (1978), into a modified vector from the pUC series. Next, the cDNA coding sequences (APP770, APP751, or APP695) can be inserted. Correct orient~tion and content of the fr~gmPnt~ inserted can be ~ietPrminprl through 5 restriction endon~lrle~e mapping and limited seql-Pnring. The clones bearing various carboxy termin~l portions of the human APP ~ne sea,uence shown in Figures 4 and 5 can be constructed through several steps in addition to those in~lir~ted above. For example, an APP770 cDNA clone (SEQ ID NO:5) can be digested with Asp718 which cleaves after nucleotide position 57. The 10 res--ltin~ 5' extension is filled in using the Klenow enzyme (Sambrook et al.(1989)) and ligated to a hexanucleotide of the following sequence: AGATCT, the recognition site for BglII. After cleavage with BgllI, which also cuts after position 1994, and re-ligation, the translational reading frame of the protein is preserved. The Llullcal~d protein thus encoded contains the leader 15 sequence, followed by approximately 6 amino acids that precede the A~B, followed by the A~B, and the 56 termin~l amino acids of APP. The clone in Figure 5 is created by converting the nucleotide at position 2138 to a T by site directed mutagenesis in the clone of Figure 4a, thus creating a tPrrnin~Sion codon directly following the last amino acid codon of the A,B.
20 APP cDNA clones naturally contain an NruI site that cuts 2 nucleotides upstream from the initiator methionine codon. This site can be used for chment of the different promoters used to complete each construct.
APP transgenes can also be constructed using PCR cloning techniques. Such techniques allow precise coupling of DNA fr~gmPnt~ in the 25 transgenes.
Combination cDNA/Genomic DNA Clones.
E~ndogenous APP expression results from transcription of precursor mRNA followed by alternative splicing to produce three main forms of APP.
It is believed that this alternative splicing may be important in producing the 30 pattern of APP expression involved in Alzheimer's disease. It is also believed that the presence of introns in ~lcssion constructs can influence the level and nature of expression by, for example, targeting precursor WO ~6/40~96 PCT~US96/09857 mRNA to mRNA processing and transport pdLllwdy~ (Huang et al., Nucleic Acids Res. 18:937-947 (1990)). Accordingly, transgenes combining cDNA
and genomic DNA, which include intron sequences, are a plGfcllGd type of construct.
S The RNA splicing m~çh,1ni~m requires omy a few specific and well known consensus sequences. Such sequences have been i~lentifi~d in APP
genomic DNA by Yoshikai et al. (1990). The disclosed Lldl~7genes can be constructed using one or more complete and intact intron sequences.
However, it is plGrGllGd that the transgenes are constructed using LluncdLGd intron sequences that contain an effective amount of intron seqllenre to allow splicing. In general, truncated intron sequences that retain the splicing donor site, the splicing acceptor site, and the splicing bldllcll~oint seql~on~e will c~n~tit~lt~. an effective amount of an intron. The sufficiency of any LluncdLGd intron sequence can be determined by testing for the presence of correctly spliced mRNA in transgenic cells using m~th~ described below.
Other intron sequences and splicing signals which are not derived from APP gene sequences may also be used in the transgene constructs.
Such intron sequences will enhance expression of the L~ulsgGlle construct. A
~lerGlled heterologous intron is a hybrid between the adenovirus major late region first exon and intron junction and an IgG variable region splice acceptor. This hybrid intron can be constructed, for example, by joining the 162 bp PvuII to HindIII fragment of the adenovirus major late region, cont~ining 8 bp of the first exon and 145 bp of the first intron, and the 99 bp Hindl~l to PstI fragment of the IgG variable region splice acceptor clone-6, as described by Bothwell et al., Cell 24:625-637 (1981). A similar splice signal has been shown to enhance expression of a construct to which it was "ch~-l as described by Manley et al., Nucleic Acids Res. 18:937-947 (1990). It is preferred that the heterologous intron be placed between the promoter and the region encoding the APP.
A ~ler~lled APP combination cDNA/genomic expression clone in~ s an effective amount of introns 6, 7 and 8, as shown in Figure 6.
Such a transgene can be constructed as follows. A plef~ d method of W O 96/40896 PCTrUS96/098~7 construction is described in F.x~mrle 5. A plasmid cont~inin~ the cDNA
portion of the clone can be constructed by first collvel~ g the TaqI site at position 860 in an APP770 cDNA clone to an Xi~oI site by site-directed mutagenesis. Cleavage of the res ~lting plasmid with X~oI cuts at the new S XhoI site and a pre-exi~ting XhoI site at position 1135, and releases the KPI
and OX-2 coding seq~ re. The plasmid thus gt:n~ld~d serves as the acceptor for the KPI and OX-2 alternative splicing cassette.
The all~.llative splicing cassette can be created through a series of cloning steps involving genomic DNA. ~irst, the TaqI site at position 860 in 10 a genomic clone cont~ining exon 6 and the adjacent dowl~Ll~ intron can be converted to an X7zoI site by site-directed mutagenesis. Cleavage of the resl-lting plasmid with X72oI cuts at the new X7zoI site and an X7~oI site within either intron 6 or 7. This fragment, cont~ining a part of exon 6 and at least a part of adjacent intron 6, can then be cloned into the XhoI site in a plasmid 15 vector. Second, a genomic clone cont~ining exon 9 and the adjacent u~Llc~ll genomic sequences is cleaved with X7loI, cleaving the clone at the X7loI site at position 1135 (position 910 using the numbering system of Kang et al. (1987)) and an X7~oI site in either intron 7 or 8. This fr~m~nt, cont~ining a part of exon 9 and at least a part of adjacent intron 8, can then 20 be cloned into the X7~oI site of another plasmid vector. These two exon/intron junction fragments can then be released from their respective plasmid vectors by cleavage with X7loI and either BamHI or BglII, and cloned together into the ~oI site of another plasmid vector. It is ~l~rell~d that the exon/intron junction fragments be excised with BamHI. It is most preferable 25 that BamHI sites are engineered in the intron portion of the exon/intron junction fr~gm~nt~ prior to their excision. This allows the elimin~tion of lengthy extraneous intron sequences from the cDNA/genomic clone.
The XhoI fragment resulting from cloning the two exon/intron junction fragments together can be cleaved with either BamHI or BglII, depending on 30 which enzyme was used for excision step above, and the genomic 6.8 kb BamHI segment, cont~ining the KPI and OX-2 coding region along with their fl~nking intron sequences, can be inserted. This fragment was identified by WO 96/40896 . I PCT~US96/09857 Kitaguchi et al. (1988) using Southern blot analysis of BamHI-digested lymphocyte DNA from one normal individual and eight ~l~hPimPr's disease patients using a 212 bp TaqI-AvaI fragment, nucleotides 862 to 1,073, of APP770 cDNA as the hybri~li7~tion probe. Genomic DNA clones cont~ining 5 the region of the 225 bp insert can be isolated, for example, from a human leukocyte DNA library using the 212 bp TaqI-AvaI fragment as a probe. In the genomic DNA, the 225 bp sequence is located in a 168 bp exon (exon 7) and a 57 bp exon (exon 8), ~,~aldL~d by an intron of approxim~t~ly 2.6 kb (intron 7), with both exons flanked by intron-exon consensus sequences. The exon 7 corresponds to nucleotides 866 to 1,033 of APP770, and the exon 8 to nucleotides 1,034 to 1,090. Exon 7 encodes the highly conserved region of the Kunitz-type protease inhibitor family domain.
After cleavage with X~oI, this ~lt(~rn~tive splicing cassette, cn~ .g both exon and intron sequences, can then be excised by cleavage with XhoI
and inserted into the XhoI site of the modified APP770 cDNA plasmid (the acceptor plasmid) constructed above. These cloning steps gelleldl~ a combination cDNA/genomic expression clone that allows cells in a Lldnsg~l,ic animal to regulate the inclusion of the KPI and OX-2 domains by a natural alternative splicing mech~nicm. An analogous gene bearing a mutation at amino acid 669, 670, 671, 690, 692, 717, or a combination of these mutations, can be constructed either directly by in vitro mutagenesis. A
mutation to amino acid 717 can also be made by using the mllt~tP(l form of APP770 cDNA described above to construct an acceptor plasmid.
Promoters.
Dirr~ promoter sequences can be used to control expression of nucleotide sequences encoding A,l~-cont~ining proteins. The ability to regulate expression of the gene encoding an A,B-cont~ining protein in transgenic animals is believed to be useful in evaluating the roles of the dirrel~ APP gene products in AD. The ability to regulate expression of the gene encoding an A~B-cont~ining protein in cultured cells is believed to be useful in ev~hl~ting expression and processing of the different A,~-cont"inin~
gene products and may provide the basis for cell cultured drug screens. A

WO ~6/10~96 PCT~US96/09857 ~-~r~..ed promoter is the human platelet derived growth factor ,B (PDGF-B) chain gene promoter (Sasahara et al., Cell 64:217-227 (1991)).
Preferred promoters for the disclosed APP constructs are those that, when operatively linked to the protein coding sequences, mP~ tP expression 5 of one or more of the following ~ s~ion products to at least a specific level in brain tissue of a two to four month old animal transgenic for one of the disclosed APP constructs. The products and their expression levels are A,B,ot to a level of at least 30 ng/g (6.8 pmoles/g) brain tissue and preferablyat least 40 ng/g (9.12 pmoles/g) brain tissue, A,~l42 to a level of at least 8.5ng/g (1.82 pmoles/g) brain tissue and preferably at least 11.5 ng/g (2.5 pmoles/g) brain tissue, full length APP (FLAPP) and APPcY combined (FLAPP+APP~) to a level of at least 150 pmoles/g brain tissue, APP~ to a level of at least 42 pmoles/g brain tissue, and mRNA encoding human A,B-cont~ining protein to a level at least twice that of mRNA encoding the endogenous APP of the transgenic animal. A,~,ot is the total of all forms of A~. A,~l42 is a form of A~ having amino acids 1 to 42 of A~B (corresponding to amino acids 672 to 714 of APP). FLAPP+APP(x refers to APP forms cont~ining the first 12 amino acids of the A~ region (corresponding to amino acids 672 to 684 of APP). Thus, FLAPP+APPcY leL,lcsen~ a llli~u~c: of full length forms of APP and APP cleaved at the ~-secretase site (Esch et al., Science 248:1122-1124 (1990)). APP,~ is APP cleaved at the ~B-secretase site (Seubert et al., Nature 361:260-263 (1993)).
It is inren-lP~l that the levels of expression described above refer to amounts of expression product present and are not limited to the specific units of measure used above. Thus, an expression level can be measured, for example, in moles per gram of tissue, grams per grams of tissue, moles per volume of tissue, and in grams per volume of tissue. The equivalence of these units of measure to the measures listed above can be determined using known conversion methods.
The levels of expression described above need not occur in all brain tissues. Thus, a promoter is considered p-cfe--c:d if at least one of the levelsof expression described above occurs in at least one type of brain tissue.

CA 02222l74 l997-ll-24 WO 96/40896 PCT~US96/09857 Where ~ ssion is tissue-specific, it is understood that if the ~ ion level is sufficient in the specific brain tissue, the promoter is considered p.~r~ d even though the expression level in brain tissue as a whole may not, and need not, reach a threshold level. It is ~lt;Ç~llcd that this level of expression is observed in hippocampal and/or cortical brain tissue. The promoter can m~ t~ expression of the above expression products to the levels described above either col~LiluLiv~ly or by intlllction Tn~ ction can be accomplished by, for example, a-lminictration of an activator molecule, by heat, or by expression of a protein activator of ~lal~ ion for the promoter operatively linked to the gene encoding an A~-cont~ining protein. Many inducible expression systems which would be suitable for this purpose are known to those of skill in the art.
It is ~rerell~d that, in making the above measurements, the brain tissue is ~ al~d by the following method. A brain from a transgenic test animal is ~ ected and the tissue is kept on ice throughout the homogenization procedure except as noted. The brain tissue is homogenized in 10 volumes (w/v) of 5 M gn~nitlin~-Hcl~ 50 mM Tris-HCl, pH 8.5. The sample is then gently mixed for 2 to 4 hours at room temperature.
Homogenates are then diluted 1:10 in cold casein buffer #1 (0.25%
casein/phosphate buffered saline (PBS) 0.05% sodium azide, pH 7.4, lX
protease inhibitor cocktail) for a final 0.5 M gll~ni~lin-? concentration and kept on ice. 100X protease inhibitor cocktail is composed of 2 mg/ml a~l~lil~ill7 0.5 M EDTA, pH 8.0, 1 mg/ml lt:~ep~ill. Diluted homogenates are then spun in an Eppendorf microfuge at 14,000 lpm for 20 mimltes at 4~C. If further dilutions are required, they can be made with cold gl~ni~lin~ buffer #2 (1 part guanidine buffer #1 to 9 parts casein buffer #1).
It is ~.ler~:lled that the following assay be used to identify ~ rel.~d promoters for their ability to m~rli"tt~ expression of A,~ to the levels described above. Antibody 266 (Seubert et al., Nature 359:325-327 (1992)) iS
dissolved at 10 ,ug/ml in buffer (0.23 g/L NaH2PO4-H20, 26.2 g/L NaHPO4-7H2O, 1 g/L sodium azide adjusted to pH 7.4) and 100 ~l/well is coated onto 96-well immllnt ~ y plates (Costar) and allowed to bind overnight. The WO ~G/4C836 PCT~US96/09857 plate is then aspirated and blocked for at least 1 hour with a 0.25% human serum albumin solution in 25 g/L sucrose, 10.8 g/L Na2HPO4-7H2O, 1.0 g/L
NaH2PO4-H2O, 0.5 g/L sodium azide adjusted to pH 7.4. The 266 coated plate is then washed lX with wash buffer (PBS/0.05% Tween 20) using a S Skatron plate washer. 100 ,~Ll/well of A,B1-40 standards and brain tissue samples are added to the plate in triplicate and inrnh~tPrl overnight at 4~C.
A,B1-40 standards are made from 0.0156, 0.0312, 0.0625, 0.125, 0.250, 0.500, and 1.000 ,llg/ml stocks in DMSO stored at -40~ C as well as a DMSO only control for background tlet~rrnin~tion. A~ ~ndalds consist of 1:100 dilution of each ~L~lldard into gll~nirlin.o buffer #3 (1 part BSA buffer to 9 parts gll~nitlin~ buffer #1) followed by a 1:10 dilution into casein buffer#1 (Note: the final A,~ concentration range is 15.6 to 1000 pg/ml and the final gl~niclin.o concentration is 0.5 M). BSA buffer consists of 1 % bovine serum albumin (BSA, immllnnglobulin-free)lpBslo 05% sodium azide. The plates and casein buffer #2 (0.25% casein/PBS/0.05% Tween 20/pH 7.4) are then brought to room t~ elaLulc~ (RT). The plates are then washed 3X with wash buffer. Next, 100 /ll/well of 3D6-biotin at 0.5 ~g/ml in casein buffer #2 is added to each well and in~nhat~d at 1 hour at RT.
Monoclonal antibody 3D6 was raised against the synthetic peptide DAEFRGGC (SEQ ID NO:10) which was conjugated through the cysteine to sheep anti-mouse immnn~globulin. The antibody does not recognize secreted APP but does recognize species that begin at A~ position 1 (Asp). For biotinylating 3D6, follow Pierce's NHS-Biotin protocol for labeling IgG (cat.
#20217X) except use 100 mM sodium bicarbonate, pH 8.5 and 24 mg NHS-biotin per ml of DMSO.
The plates are then again washed 3X with wash buffer. Then, 100 ,ul/well of horseradish peroxidase (HRP)-avidin (Vector Labs, cat. # A-2004) diluted 1:4000 in casein buffer #2 is added to each well and incubated for 1 hour at RT. The plates are washed 4X with wash buffer and then 100 ~l/well of TMB substrate (Slow TMB-ELISA (Pierce cat. # 34024)) at RT is added to each well and incnh~ted for 15 mimlt~s at RT. Finally, 25 ,ul/well WO 96/40896 . PCTAJS96/09857 of 2 N H2SO4 is added to each well to stop the enzymatic reaction, and the plate is read at 450 nm to 650 nm using the Molecular Devices Vmax reader.
It is plcrell~d that the relative levels of mRNA encoding human A,~-cont~ining protein mRNA encoding the endogenous APP of the Lldns~t;n-c 5 animal be measured in the manner described by Bordonaro et al., Biotechniques 16:428430 (1994), and Rockel~L~in et al., J. Biol. Chem.
270:28257-28267 (1995). Preferred methods for measuring the ~ s~ion level of A,BI 42, FLAPP +APPcY, and APP,~ are described in Example 8.
Yeast Artificial Chr.J...o.so...~c.
The constructs shown in Figure 7 can be constructed as follows.
Large segments of human genomic DNA, when cloned into certain vectors, can be propagated as autonomously-replicating units in the yeast cell. Such vector-borne segments are referred to as yeast artificial chromosomes (YAC;
Burke et al. Science 236:806 (1987)). A human YAC library is commercially available (Clontech, Palo Alto, CA) with an average insert size of 250,000 base pairs (range of 180,000 to 500,000 base pairs). A YAC
clone of the Alzheimer's gene can be directly isolated by screening the library with the human APP770 cDNA. The inclusion of all of the ~-~senti~
gene regions in the clone can be confirm~-l by PCR analysis.
The YAC-APP clone, shown in Figure 7a, can be established in embryonic stem (ES) cells by selecting for neomycin resistance encoded by the YAC vector. ES cells bearing the YAC-APP clone can be used to produce transgenic mice by established methods described below under "Transgenic Mice" and "Embryonic Stem Cell Methods". The YAC-APP
gene bearing a mutation at amino acid 717 (Figure 7b) can be produced through the ~ell~ldlion of a YAC library using genomic DNA from a person affected by a mutation at amino acid 717. Such a clone can be identified and established in ES cells as described above.
Genetic Alteration of the Mouse APP Gene.
The nucleotide sequence homology between the human and murine Alzheirner's protein genes is approxim~t~.ly 85%. Within the A,l~-coding region, there are three amino acid differences between the two sequences.

CA 02222l74 l997-ll-24 WO 9"4C~6 PCTAJS96/09857 Amino acids Lys 670, Met671, and Val717,which can be mllt~te~l to alter APP processing, are conserved between mouse, rat, and man. Wild-type rodents do not develop Alzheimer's disease nor do they develop deposits or plaques in their central nervous system (CNS) analogous to those present in S human Alzheimer's patients. Therefore, it is possible that the human but not the rodent form of A,l~ is capable of causing disease. Homologous recombination (Capecchi, Science 244: 1288-1292 (1989)) can be used to convert the mouse Alzheimer's gene in situ to a gene encoding the human A,B
by gene repl~r~-m~ont This recombination is directed to a site dow~ e~l-10 from the KPI and OX-2 domains, for example, within exon 9, so that the natural ~ v~ splicing m~-rh~ni.cm.c a~plop.late to all cells within the transgenic animal can be employed in ~re~illg the final gene product.
Both wild-ype (Figure 8a) and mutant (Figure 8b) forms of human cDNA can be used to produce transgenic models ~ lcsshlg either the wild-15 ype or mutant forms of APP. The recombination vector can be constructedfrom a human APP cDNA (APP695, APP751, or APP770 form), either wild-ype, mutant at amino acid 669, 670, 671, 690, 692, 717, or a combination of these ml-t~tionc. Cleavage of the recombination vector, for example, at the XhoI site within exon 9, promotes homologous recu...bil~lion witbin the 20 directly adjacent sequences (Capecchi (1989)). The endogenous APP gene resulting from this event would be normal up to the point of recombination, within exon 9 in this example, and would consist of the human cDNA
sequence thereafter.
Preparation of Constructs for Transfections and Microinjections.
DNA clones for microinjection are cleaved with en7ymes ap~.u~liate for removing the bacterial plasmid sequences, such as SalI and NotI, and the DNA fragments electrophoresed on 1% agarose gels in TBE buffer (Sambrook et al. (1989)). The DNA bands are vi.cl-~li7P~l by staining with ethi~ m bromide, and the band cont~ining the APP expression sequences is - 30 excised. The excised band is then placed in dialysis bags cont~ining 0.3 M
sodium acetate, pH 7Ø DNA is electroeluted into the dialysis bags, extracted with phenol-chloroform (1:1), and precipitated by two volumes of WO 96/40896 . PCTAJS96/09857 ethanol. The DNA is redissolved in 1 ml of low salt buffer (0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on an Elutip-DTM column.
The column is first primed with 3 ml of high salt buffer (1 M NaCl, 20 mM
Tris, pH 7.4, and 1 mM EDTA) followed by washing with 5 ml of low salt 5 buffer. The DNA solutions are passed through the column for three times to bind DNA to the column matrix. After one wash with 3 ml of low salt buffer, the DNA is eluted with 0.4 ml of high salt buffer and precipitated by two volumes of ethanol. DNA concentrations are measured by absorption at 260 nm in a UV spectrophotometer. For microinjection, DNA concentrations are adjusted to 3 ~g/ml in 5 mM Tris, pH 7.4 and 0.1 mM EDTA. Other methods for puri~lc~tinn of DNA for microinjection are also described in Hogan et al., Manipulating the Mouse Embryo (Cold Spring Harbor Labo.~tuly, Cold Spring Harbor, NY, 1986); in Palmiter et al., Nature 300:611 (1982); in The Qiagenologist, Application Protocols, 3rd edition, published by Qiagen, Inc., Chatsworth, CA.; and in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).
Construction of Transgenic ~nim~lc, A. Animal Sources.
Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA), Taconic (Gerrn~ntown, NY), and Harlan Sprague Dawley (Tn~ n~polis, IN).
Many strains are suitable, but Swiss Webster (Taconic) female mice are p-~fe-lc:d for embryo retrieval and l-dl~rel. B6D2FI (Taconic) males can be used for mating and vasectomized Swiss Webster studs can be used to stimul~t~ pseudopregnancy. Vasectomized mice and rats can be obtained from the supplier.
B. Microinjection Procedures.
The procedures for manipulation of the rodent embryo and for microinjection of DNA are described in detail in Hogan et al., Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory, Cold Spring Harbor, WO 96/40896 PCTrUS96109857 NY, 1986), the teaching.~ of which are generally known and are incorporated herein.
C. Tl al~Yel~ic Mice.
Female mice six weeks of age are intlllre~:l to superovulate with a S IU
injection (0.1 cc, ip) of pregnant mare serum gonadotropin (PMSG; Sigma) followed 48 hours later by a 5 IU injection (0.1 cc, ip) of human chorionic gonadotropin (hCG; Sigma). Females are placed with males immediately after hCG injection. Twenty-one hours after hCG injection, the mated females are s~rrifire~l by CO2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts and placed in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin (BSA; Sigrna).
Sull~ulldillg cumulus cells are removed with hyaluronidase (1 mg/ml).
Pronuclear embryos are then washed and placed in Earle's b~l~nred salt solution cont~ining 0.5% BSA (EBSS) in a 37.5~C inrllb~tor with a hllmiclifie~l atmosphere at 5 % CO2, 95 % air until the time of injection.
Embryos can be implanted at the two cell stage.
~n-lomly cycling adult female mice are paired with vasectomized males. Swiss Webster or other comparable strains can be used for this purpose. Recipient females are mated at the same time as donor females. At the time of embryo transfer, the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body weight. The oviducts are exposed by a single midline dorsal incision. An incision is then made through the body wall directly over the oviduct. The ovarian bursa is then torn with w~tchmzlkers forceps. Embryos to be transferred are placed in DPBS (Dulbecco's phosphate buffered saline) and in the tip of a LlalL~r~l pipet (about 10 to 12 embryos). The pipet tip is insertedinto the infundibulum and the embryos Ll~llsrt;ll~d. After the transfer, the incision is closed by two sutures.
D. Transgenic Rats.
The procedure for generating transgenic rats is similar to that of mice (~mmer et al., Cell 63:1099-112 (1990)). Thirty day-old female rats are given a subcutaneous injection of 20 IU of PMSG (0.1 cc) and 48 hours later WO ~G/~0~6 . PCT~US96/09857 each female placed with a proven male. At the same time, 40-80 day old females are placed in cages with vasectomi_ed males. These will provide the foster mothers for embryo llal~ir~.. The next morning females are checked for vaginal plugs. Females who have mated with v~ce~ d males are 5 held aside until the time of ~ ~rel. Donor females that have mated are s~crifire~l (CO2 asphyxiation) and their oviducts removed, placed in DPBS
(Dulbecco's phosphate buffered saline) with 0.5% BSA and the embryos collected. Cumulus cells surrounding the embryos are removed with hyaluronidase (1 mg/ml). The embryos are then washed and placed in EBSS
(Earle's bal~nre-l salt solution) cont~ining 0.5% BSA in a 37.5~C incubator until the time of microinjection.
Once the embryos are injected, the live embryos are moved to DPBS
for L-~rt:l into foster mothers. The foster mothers are ~n~sth~ti7P~l with k~ ...i..r (40 mg/kg, ip) and xylazine (5 mg/kg, ip). A dorsal midline 15 incision is made through the skin and the ovary and oviduct are exposed by an incision through the muscle layer directly over the ovary. The ovarian bursa is torn, the embryos are picked up into the transfer pipet, and the tip ofthe LLdl~rei pipet is inserted into the infundibulum. Approximately 10 to 12 embryos are L-~l~r~ d into each rat oviduct through the infundibulum. The 20 incision is then closed with sutures, and the foster mothers are housed singly.
E. Embryonic Stem (ES) Cell Methods.
1. Introduction of cDNA into ES cells.
Methods for the culturing of ES cells and the subsequent production of transgenic animals, the introduction of DNA into ES cells by a variety of 25 methods such as electroporation, calcium phosphate/DNA precipitation, and direct injection are described in detail in Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, ed. E.J. Robertson, (IRL Press 1987), the teachings of which are generally known and are incorporated herein.
Selection of the desired clone of transgene-cont~ining ES cells can be 30 accomplished through one of several means. For random gene integration, an APP clone is co-precipitated with a gene encoding neomycin resi~t~nre.
Transfection is carried out by one of several methods described in detail in WO 96/40896 PCTrUS96J09857 Lovell-Badge, in Teratocarcinomas and Embryonic Stem Cells, A Practical Approach, ed. E.J. Robertson, (IRL Press 1987), or in Potter et al., Proc.
Natl. Acad. Sci. USA 81:7161 (1984). Lipofection can be performed using reagents such as provided in commercially available kits, for example S DOTAP (Boehringer-~ nnht~im) or lipofectin (BRL). C~lcillm phosphate/DNA precipitation, lipofection, direct injection, and electroporation are the ~lefelled methods. In these procedures, 0.5 X 106 ES cells are plated into tissue culture dishes and transfected with a mixture ofthe linearized APP clone and 1 mg of pSV2neo DNA (Southern and Berg, J.
10 Mol. Appl. Gen. 1:327-341 (1982)) precipitated in the presence of 50 mg lipofectin (BRL) in a final volume of 100 ,Ll. The cells are fed with selection mf ~ m cont~inin~ 10% fetal bovine serum in DMEM supplementefl with G418 (between 200 and 500 ,ug/ml). Colonies of cells resistant to G418 are isolated using cloning rings and expanded. DNA is extracted from drug 15 resistant clones and Southern blots using an APP770 cDNA probe can be used to identify those clones carrying the APP sequences. PCR detection methods may also used to identify the clones of interest.
DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination, described 20 by Capecchi (1989). Direct injection results in a high efficiency of integration. Desired clones can be id~ntifie-l through PCR of DNA prepared from pools of injected ES cells. Positive cells within the pools can be identified by PCR subsequent to cell cloning (Zimmer and Gruss, Nature 338:150-153 (1989). DNA introduction by electroporation is less efficient 25 and requires a selection step. Methods for positive selection of the recombination event (for example, neo resistance) and dual positive-negative selection (for example, neo resistance and gancyclovir reci~nre) and the subsequent identification of the desired clones by PCR have been described by Joyner et al., Nature 338: 153-156 (1989), and Capecchi (1989), the 30 tezlehin~~ of which are generally known and are incorporated herein.

CA 02222l74 l997-ll-24 WO ~6/108~6 : PCT~US96/05~a 2. Embryo Recovel~ and ES cell Injection.
Naturally cycling or superovulated female mice mated with males can be used to harvest embryos for the implantation of ES cells. It is desirable to use the C57BL/6 strain for this purpose when using mice. Embryos of the S a~)plo~lidl~ age are recovered approximately 3.5 days after s~lrceccful mating.
Mated females are sacrificed by CO2 asphyxiation or cervical dislocation and embryos are flushed from excised uterine horns and placed in Dulbecco's modified es,centi~l mPrlinm plus 10% calf serum for injection with ES cells.
Approximately 10 to 20 ES cells are injected into blastocysts using a glass 10 microneedle with an internal rii~mPt~pr of approximately 20 ~m.
3. Transfer of Embryos to Pseudopregnant Females.
Randomly cycling adult female mice are paired with v~ce~;lo.,.i~
males. Mouse strains such as Swiss Webster, ICR or others can be used for this purpose. Recipient females are mated such that they will be at 2.5 to 3.5 15 days post-mating when required for implantation with blastocysts cont~ining ES cells. At the time of embryo Lldl~.rel, the recipient females are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body weight. The ovaries are exposed by making an incision in the body wall directly over the oviduct and the ovary and uterus are externalized.
20 A hole is made in the uterine horn with a 25 gauge needle through which the blastocysts are lldll~r~lled. After the transfer, the ovary and uterus are pushed back into the body and the incision is closed by two sutures. This procedure is repeated on the opposite side if additional tldl~.r~l.. are to be made.
25 Irl~ntific~tion, Characterization, and Utili7~tinn of Transgenic Mice and Rats.
Transgenic rodents can be idPntifiPd by analyzing their DNA. For this purpose, tail samples (1 to 2 cm) can be removed from three week old animals. DNA from these or other samples can then be prepared and 30 analyzed by Southern blot, PCR, or slot blot to detect transgenic founder (Fo) animals and their progeny (Fl and F2).
A. Pathological Studies.

CA 02222l74 l997-ll-24 W O~G/1C~96 PCTAJS96/09857 The various Fo~ F" and F2 animals that carry a transgene can be analyzed by immlmc)histology for evidence of A~B deposition, ~ ssion of APP or APP cleavage products, neuronal or neuritic abnorm~liti~s, and infl~ tol~ responses in the brain. Brains of mice and rats from each S transgenic line are fixed and then sectioned. Sections are stained with antibodies reactive with the APP and/or the A,B. Secondary antibodies conjugated with fluorescein, rho-l~minP, horse radish peroxidase, or alk~linP
phosphatase are used to detect the primary antibody. These methods permit i-lentifir~tion of amyloid plaques and other pathological lesions in specific areas of the brain. Plaques ranging in size from 9 to >50 ,um ch~r~cteri~tir~lly occur in the brains of AD patients in the cerebral cortex, but also may be observed in deeper grey matter including the amygdaloid nucleus, corpus striatum and diencephalon. Sections can also be stained with other antibodies diagnostic of Alzheimer's plaques, recognizing antigens such as APP, Alz-50, tau, A2B5, neurofil~mPnt~, ~,yllaptophysin, MAP-2, ubiquitin, complement, neuron-specific enolase, and others that are characteristic of Alzheimer's pathology (Wolozin et al., Science 232:648 (1986); Hardy and Allsop, Trends in Pharm. Sci. 12:383-388 (1991); Selkoe, Ann. Rev. Neurosci. 12:463-490 (1989); Arai et al., Proc. Natl. Acad. Sci.
USA 87:2249-2253 (1990); Majocha et al., Amer. Assoc. Neuropathology Abs 99:22 (1988); Masters et al., Proc. Natl. Acad. Sci. 82:4245-4249 (1985); Majocha et al., Can J Biochem Cell Biol 63:577-584 (1985)).
St~ining with thioflavin S and Congo Red can also be carried out to analyze the presence of amyloid and co-localization of A~ deposits within neuritic plaques and NFTs.
B. Analysis of APP and A,~ Expression.
1. mRNA.
Messenger RNA can be isolated by the acid gll~nitlinillm thiocyanate-phenol:chloroform extraction method (Chomaczynski and Sacchi, Anal Biochem 162: 156-159 (1987)) from cell lines and tissues of transgenic ~nim~l~ to determine expression levels by Northern blots, RNAse and mlr~ e protection assays.

2. Protein.
APP, A~, and other fr~gm,ontc of APP can and have been detected by using polyclonal and monoclonal antibodies that are specific to the APP
extra-cytoplasmic domain, A,B region, A~l42, A,l~l40, APP~, FLAPP+APP~, 5 and C-t~, ,;""c of APP. A variety of antibodies that are human seq~lenre specific, such as 10D5 and 6C6, are very useful for this purpose (Games et al. (1995)).
3. Western Blot Analysis.
Protein fractions can be isolated from tissue homogenates and cell lysates and subjected to Western blot analysis as described by, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor, NY, 1988); Brown et al., J. Neurochem. 40:299-308 (1983); and Tate-Ostroff et al., Proc Natl Acad Sci 86:745-749 (1989).
Briefly, the protein fractions are denatured in T~mmli sample buffer and electrophoresed on SDS-Polyacrylamide gels. The ~ s are then ~re~ d to nitrocellulose filters by electroblotting. The filters are blocked, inrllbatr(l with ~ llaly antibodies, and finally reacted with enzyme conjugated secondary antibodies. Subsequent inr~lb~tion with the ~ lia chromogenic substrate reveals the position of APP derived proteins.
C. p~thologir~l and Behavioral Studies.
1. Pathological Studies.
Tmmlmohictology and thioflavin S staining are con~ cte~1 as described elsewhere herein.
ln situ Hybri~ tionc: Radioactive or enzym:~tir~lly labeled nucleic acid probes can be used to detect mRNA in situ. The probes are degraded or prepared to be approximately 100 nucleotides in length for better penetration of cells. The hybridization procedure of Chou et al., J. Psych. Res. 24:27-50 (1990), for fixed and paraffin embedded samples is briefly described below although similar procedures can be employed with samples sectioned as frozen material. Paraffin slides for in situ hybridization are dewaxed in xylene and rehydrated in a graded series of ethanols and finally rinsed in phosphate buffered saline (PBS). The sections are post-fixed in fresh 4%

W 096/40896 PCTrUS96/09857 paraformaldehyde. The slides are washed with PBS twice for 5 .I.i.~ PS to remove paraform~lcl.-hyde. Then the sections are permeabilized by tre~trnl~nt with a 20,~4g/ml proteinase K solution. The sections are re-fixed in 4%
paraform~ hyde, and basic molecules that could give rise to background 5 probe binding are acetylated in a 0.1 M tri~th~n~lamine~ 0.3 M acetic anhydride solution for 10 mimlt~c. The slides are washed in PBS, then dehydrated in a graded series of ethanols and air dried. Sections are hybridized with antisense probe, using sense probe as a control. After a~lo~liate washing, bound radioactive probes are ~iet~cte~l by 10 autoradiography or enzym~ti~lly labeled probes are ~ t~cte~l through reaction with the app,~ iate chromogenic substrates.
2. Behavioral Studies.
Behavioral tests (lesign~d to assess learning and memory deficits are employed. An example of such as test is the Morris water maze (Morris, Learn Motivat. 12:239-260 (1981)). In this procedure, the animal is placed in a circular pool filled with water, with an escape platform submerged just below the surface of the water. A visible marker is placed on the platform so that the animal can find it by navigating toward a proximal visual cue.
Al~ ldLively, a more complex form of the test in which there are no local cues to mark the platform's location will be given to the ~nim~lc. In this form, the animal must learn the platform's location relative to distal visual cues, and can be used to assess both reference and working memory. A
learning deficit in the water maze has been demonstrated with PDAPP
transgenic mice. An example of behavioral analysis for acsec.cing the effect of transgenic expression of A,B-cont~ining proteins is described in Example 9.
Operant behavior studies of memory function: Memory function of the disclosed transgenic animals can be ~cses.ced by testing memory-related feeding behavior (Dunnett, "Operant delayed m~t~hing and non-matching position in rats" in Behavioral Neuroscience, Volume I: A Practical Approach (Sagal, ed., IRL Press, N.Y., 1993) pages 123-136; Zornetzen, Behav. Neur. Biol. 36:49-60 (1982)). Transgenic and non-transgenic mice, are trained to earn food rewards in a two component operant procedure. One WO 96/40896 PCT~US96/09857 component features a delayed spatial ~lt~rn~tion schedule. Under this schlo~lnle, the mouse must remember over a variable time delay which lever it has pressed in the previous trial so that it can earn a reward by pressing the z~ltern~t~ lever on the current trial. This provides a measure of the animal's 5 recent or "working" memory. The second component rea~ s a disel;...;..;.~ion spatial ~lt~rn~tion srh~tlllle. Under this schedule, the mouse earns a reward by pressing what~v~l lever is illl....;..~lt~(l This tli~(~. ;...i..i.~ion behavior is an example of lert.c;llce memory. These two groups of mice, transgenic and non-transgenic, can be chronically studied 10 over time, for example, from 3 months of age until the end of their useful life span, in order to assess the development of st;l~iLivi~y to cholinergic antagonists and behavioral imp~irml~nt on these memory tasks. It is expected that the disclosed transgenic mice will model the cognitive deficits of Alzheimer's disease with enh~nre-l se~ ivity to the memory-disrupting 15 effects of cholinergic antagonists and imp~irTn~nt on "wolhing" and referencememory tasks. Dose-response ch~llenges with the cholinergic antagonist can be con~ ctto-l at various ages. These memory behavioral tests can also be used to compare the effect of compounds on the behavioral imr~irm~nt of the disclosed transgenic animals. In this case, the two groups of mice are 20 transgenic mice to which a test compound is a-lmini~t~-red and transgenic mice to which the compound is not ~lmini~tered Emotional reactivity and object recogni~ion: Various functions of the disclosed transgenic animals can be a~.sesse(l by testing locomotor activity, emotional reactivity to a novel environment or to novel objects, and 25 object recognition. A first set of ~c~es~m~ntc are performed in the same animals at ~lirrelt;l~t ages (each animal is its own control) in order to test their performance in terms of locomotor activity, emotional reactivity to a novel environment or to novel objects, and object recognition, a form of memory which is severely impaired in AD patients. On the first day, transgenic and 30 non-transgenic control mice are individually placed in a square open field with a central platform. For 30 minutes, horizontal and vertical activity, and crossings of the platform, are recorded by blocks of 5 mimltes for each WO 9~M08~6 PCTAJS96/09857 animal. On the second day, each animal is submitted to two trials with an i lLelL~ial of 1 hour. On the first trial, two i~lentir~l objects are placed in the open field and the animal is allowed 3 mimltes of exploration. On the second trial, one of the objects is replaced by a new object and the time spent by the 5 animal in exploring the f~miliar and novel object is recorded during the next 3 min--t~s (Fnnacellr and Delacour, Behav. Brain Res. 31.47-59 ~988)2.
Animals are then tested for neophobic behavior, which is considered as an index of anxiety, in a free exploration ~ ation, in which ~nimal~ are given the oppol~unily to move freely between a famili~r and a novel ellvilul...lent.
Thereafter, the same animals are submitted to various learning tasks to investigate their learning and memory capacities. They are first tested for spatial recognition memory in a T-maze delayed alternation task at 6 hour and 24 hour delays. This form of memory has been shown to be very sensitive to hippocampal damage. One half of the animals of each group is then trained in a positively reinforced lever-press task as described above.
This can be used to measure post training improvement in pt:lrollllance of the ~nimal~, which has been shown to involve hippocampal activation. The other half of the animals is trained in spatial disclilllina~ion in an 8 arm radial maze (Oltons and .~mnelson, J. Ejcp. Psychol. [Animal Behav.~ 2:97-116 (1976)) in order to evaluate working and reference memory and to analyze their strategies (angle preference), which give a better index of memory capacities.
The animals trained and tested in the bar-lever press task at 2 to 3 months old can be trained and tested in radial maze at 9 to 10 months old, and vice-versa. A working memory deficit has been demonstrated in PDAPP
transgenic mice in the radial arm maze.
Two additional groups can also be submitted to the same behavioral tests as above at 9 to 10 months old in order to determine whether behavioral screening performed at 2 to 3 months old influ~nr~ecl further le~rning and memory capacities.
These memory behavioral tests can also be used to compare the effect of compounds on the behavioral imp~irm~-nt of the disclosed transgenic animals. In this case, the two groups of mice are transgenic mice to which a CA 02222l74 l997-ll-24 W096/40896 : PCT~US96/09857 test eompound is ~lminictered, and transgenie miee to whieh the eompound is not ~rlminictt?red.
The proeedures applied to test transgenie miee are similar for ~ISgCLlC rats.
D. Preferred Characteristics.
The above phenotypie eharaeteristies of the diselosed Lldlls2~,cll~c ~nim~l.c ean be used to identify those forms of the diselosed ~ ,.cgel~ie ~nim~le that are plcfcnlcd as animal models. Additional phenotypie ehar~etericti~c, and assays for m~cllrin~ these ~h~r~etto.risties, that ean also10 be used to identify those forms of the diselosed ~ldlls~ e animals that are ~lcrcllcd as animal models, are deseribed in Example 6. These ehar~e.teri.cties are preferably those that are similar to phenotypie eharaeteristies observed in Alzheimer's disease. APP and A,B markers whieh are also useful for identifying those forms of the diselosed transgenie animals 15 that are plcfellcd as animal models are deseribed below. Any or all of the these l.lalhel~, or phenotypie eharaeteristies ean be used either alone or in eombination to identify ~lcfcllcd forms of the diselosed Lldlls ,cl~ie animals.
For example, the presenee of plaques in brain tissue that ean be stained with Congo red is a phenotypie eharaeteristie whieh ean identify a diselosed 20 transgenie animal as ~lcrcllcd. It is int~.ntlt~ that the levels of c~L~les~,ion of eertain APP-related proteins present in plcr~llcd transgenie animals (~li.ccllccerl above) is an independent eharaeteristie for identifying ~lcrcllcdtransgenie ~nim~lc. Thus, the most plcfcllcd transgenie animals will exhibit both a diselosed c~lcssion level for one or more of the APP-related proteins 25 and one or more of the phenotypie eharaeteristies tli.cellcced above.
Espeeially plcfcllcd phenotypie eharaeteristies (the presenee of whieh identifies the animal as a preferred transgenie animal) are the presenee of amyloid plaques that ean be stained with Congo Red (Kelly (1984)), the presenee of extraeellular amyloid fibrils as identified by eleetron mieroseopy 30 by 12 months of age, and the presenee of type I dystrophie neurites as identifit-d by eleetron mieroseopy by 12 months of age (eomposed of spherieal neurites that eontain synaptie proteins and APP; Diekson et al., Am CA 02222l74 l997-ll-24 WO ~614~6 PCTAJS96/09857 J Pathol 132:86-101 (1988); Dickson et al., Acta Neuropath. 79:486493 (1990); Masliah et al., J Neuropathol E~p Neurol 52: 135-142 (1993);
Masliah et al., Acta Neuropathol 87:13~-142 (1994); Wang and Munoz, J
Neuropathol E~p Neurol 54:548-556 (1995)). Examples of the detection of S these ch~r~rteristics is provided in Example 6. It is most plc;felled that theL.c,lls~ellic ~nim~l~ have amyloid plaques that can be stained with Congo Red as of 14 months of age.
S~lee~ g of Compounds for Tre~trnent of ~l7h~impr~s Disease.
The transgenic ~nim~, or animal cells derived from transgenic 10 anim~ , can be used to screen compounds for a potential effect in the tre~tm~on~ of Alzheimer's disease using standard methodology. In such AD
screening assays, the compound is ~lmini.~tered to the animals, or introduced into the culture media of cells derived from these anim~l~, over a period of time and in various dosages, then the ~nim~ or animal cells are ex~min.o-15 for alterations in APP expression or processing, expression levels orlocalization of other AD markers, histopathology, and/or, in the case of ~nim~l~, behavior using the procedures described above and in the .o~mples below. In general, any improvement in behavioral tests, alteration in AD-associated markers, reduction in the severity of AD-related histopathology, 20 reduction in the expression of A,~ or APP cleavage products, and/or changes in the presence, absence or levels of other compounds that are correlated with AD which are observed in treated animals, relative to untreated animals, is indicative of a compound useful for treating Alzheimer's disease. The specific proteins, and the encoding transcripts, the enzymatic or biochemical 25 activity, and/or histopathology of those proteins, that are associated with and characteristic of AD are referred to herein as markers. Expression or loc~li7~tion of these markers characteristic of AD has either been rletecte~l, or is expected to be present, in the disclosed transgenic animals. These markers can be measured or (letected, and those measurements compared between 30 treated and untreated transgenic animals to determine the effect of a tested compound.

WO 96/40896 ~ PCTAJS9~/09857 Markers useful for AD screening assays are selectecl based on ~let~cf~hle changes in these lllalk~l~, that are associated with AD. Many such lllalht;l., have been iclentifiecl in AD and have either been ~l~tecte~l in the disclosed transgenic animals or are expected to be present in these animals.
S These lll~h~, fall into several categories based on their nature, location, orfunction. Preferred examples of markers useful in AD screening assays are described below, group as A,B-related nlalh~, plaque-related lualh~ "
cytoskeletal and neuritic markers, infl~mm~1ory markers, and ~ ulollal and llt:ul~ .-related markers.
A. A~-related Markers.
Expression of the various forms of APP and A,(~ can be directly measured and compared in treated and untreated transgenic animals both by immllnohi~tochemistry and by qll~ntit~tive ELISA measurements as described above and in the examples. Cullclllly, it is known that two forms of APP
products are found, APP and A,(~ (Haass and SeLkoe, Cell 75:1039-1042 (1993)). They have been shown to be illL-ilLsichlly associated with the pathology of AD in a time dependent manner. Therefore, ~ r~ d assays compare age-related changes in APP and A,(~ expression in the transgenic mice. As described in Example 6, increases in A,B have been demonstrated during aging of the PDAPP mouse.
Preferred targets for assay measurement are A,~ markers hnown to increase in individuals with Alzheimer's disease are total A~B (A,B~o,)7 A,B 142(A,BI42; A~B with amino acids 1-42), A~l40 (A~ with amino acids 1-40), A~
N3(pE) (A,~N3(pE)); A~B X-42 (A,~X42; A,~ forms ending at amino acid 42);
A,l~ X-40 (A~X40; A,~ forms ending at amino acid 40); insoluble A,~
(A,~l,soluble); and soluble A,B (A~so~Uble; Kuo et al., J. Biol. Chem.
271(8):4077-4081 (1996)). A,BN3(pE) has pyrogl~lt~mic acid at position 3 (Saido, Neuron 14:457-466 (1995)). A,~X42 refers to any of the C-terminal forms of A~ such as A~l342. A~l~,soluble refers to forms of A~B that are recovered as described in Gravina, J. Biol. Chem. 270:7013-7016 (1995).
APP,B can also be specifically measured to assess the amount of ,B-secretase activity (Seubert et al., Nature 361:260-263 (1993)). Several of these A,B

W O 96/40896 PCT~US96/09857 forms and their association with A~lzheimer's disease are described by Haass and SeLkoe (1993). Detection and mea~ulGlllG~L of A~tot~ A~l42, and A~X42 are described in Example 6. Generally, specific forms of A,B can be assayed, either q~ tivGly or qualitatively using specific antibodies, as ~lescrihe(l 5 below. When rGrt.li,.g to amino acid positions in forms of A,~, the positions correspond to the A~ region of APP. Amino acid 1 of A~ corresponds to amino acid 672 of APP, and amino acid 42 of A,B corresponds to amino acid 714 of APP.
Also l~rGrellGd as targets for assay measurement are APP lllalhGl~.
10 For example, dirr~lGllL forms of secreted APP (termed APPc~ and APP,~) can also be measured (Seubert et al., Nature 361:260-263 (1993)). Other APP
forms can also serve as targets for assays to assess the potential for compounds to affect Alzheimer's disease. These include FLAPP+APP~, full length APP, C-termin~l fr~gm~-nt~ of APP, especially C100 (the last 100 amino acids of APP) and C57 to C60 (the last 57 to 60 amino acids of APP), and any forms of APP that include the region corresponding to A,~l40.
APP forms are also pl~:rGllGd targets for assays to assess the potential for compounds to affect Alzheimer's disease. The absolute level of APP and APP transcripts, the relative levels of the dirrelell~ APP forms and their cleavage products, and loc~li7~tinn of APP expression or processing are all markers associated with Alzheimer's disease that can be used to measure the effect of tre~tment with potential therapeutic compounds. The loc~li7~tion of APP to plaques and neuritic tissue is an especially ~ r~ d target for these assays.
Ql-~ntit~tive measurement can be accomplished using many standard assays. For example, transcript levels can be measured using RT-PCR and hybridization methods including RNase protection, Northern analysis, and R-dot analysis. APP and A,~ levels can be assayed by ELISA, Western analysis, and by comparison of immnnnhistoch~mir~lly stained tissue sections. Tmmnnohistochrmi~al st~ining can also be used to assay localization of APP and A~ to particular tissues and cell types. Such assays were described above and specific examples are provided below.

WO 96/40896 PCT~US96/09857 B. Plaque-related Markers.
A variety of other molecules are also present in plaques of individuals with AD and in the disclosed transgenic ~nim~l.c, and their presence in plaques and nPllritir tissue can be ~letectp~l- The amount of these ..,~.k~
5 present in plaques or neuritic tissue is expected to increase with the age of ullLlcaled Lldllsge..ic animals. Preferred plaque-related ...i..k~; are apoli~rolei-l E, glycosylation end products, amyloid P component, advanced glycosylation end products (Smith et al., Proc. Natl. Acad. Sci.
USA 91:5710 (1994)), growth inhibitory factor, l~minin, collagen type IV
(Kalaria and Perry (1993); Ueda et al. (1993)), receptor for advanced glycosylation products (RAGE), and ubiquitin.
While the above markers can be used to detect specific culllpollell~ of plaques and neuritic tissue, the location and extent of plaques can also be ~l~Le. ,..i.~Prl by using well known histochPmic~l stains, such as Congo Red and15 thioflavin S, as described above and in some examples below.
C. Cyto~k~ l and Neuritic Markers.
Many changes in cytoskeletal markers associated with AD have also been ~let~PctPrl in transgenic PDAPP mice. These markers can be used in AD
screening assays to determine the effect of c~,lll~ounds on AD. Many of the 20 changes in cytoskeletal markers occur either in the neurofibrillary tangles or dystrophic neurites associated with plaques (Kosik et al. (1992); Lovestone and Anderton (1992); Brandan and Inestrosa (1993); Trojanowski et al.
(1993); Masliah et al. (1993)).
The following are preferred cytoskeletal and neuritic markers that 25 exhibit changes in and/or an association with AD. These ~ .k~ can be tPct~p~l~ and changes can be determinP~l, to measure the effect of compounds on the disclosed transgenic animals. Spectrin exhibits increased breakdown in AD. Tau and neurofilaments display an increase in hyperphosphorylation in AD, and levels of ubiquitin increase in AD. Tau, ubiquitin, MAP-2, 30 neurofilaments, heparin sulfate, and chrondroitin sulphate are localized to plaques and neuritic tissue in AD and in general change from the normal localization. GAP43 levels are decreased in the hippocampus and abnormally W O ~6/408~6 PCTrUS96/09857 phosphorylated tau and neurofil~m~nt~ are present in PDAPP L~ sgenic miee.
D. Tnfl~ Markers.
~ ~17htoimPr's disease is also known to stim~ t~ an S i,,."~ i"ll~mm~tory response, with a eorresponding increase in infl~mm~tory llldl~ 7 (Freclerirk~on and Brunden (1994); MeGeer et al.
(1991); Wood et al. (1993)). The following are ~re~lled infl~"",~ oly alh~, that exhibit changes in and/or an association with AD. Detection of changes in these markers are useful in AD scl~ g assays. Acute phase ~loleills and glial lllal~" such as cYl-allLiLly~sill, C-reactive protein, ~2-macroglobulin (Tooyama et al., Molecular & Chemical Neuropathology 18:153-60 (1993)), glial fibrillary acidic protein (GFAP), Mac-l, F4/80, and cytokines, sueh as IL-l~ and ,l~, TNF~, IL-8, MIP-lc~ (Kim et al., J.
Neuroimmunology 56: 127-134 (l99S)), MCP-l (Kim et al., J. Neurological Sciences 128:28-35 (l99S); Kim et al., J. Neuroimmunology 56:127-134 (1995); Wang et al., Stroke 26:661-665 (l99S)), and IL-6, all inerease in AD
and are expeeted to increase in the disclosed transgenic ~nim~
Complement lllal~l." such as C3d, Clq, C5, C4d, C4bp, and CSa-C9, are localized in plaques and neuritic tissue. Major histocompatibility complex (MHC) glycuploLeills, such as HLA-DR and HLA-A, D,C increase in AD.
Microglial markers, such as CR3 receptor, MHC I, MHC II, CD 31, CDlla, CDllb, CDllc, CD68, CD45RO, CD45RD, CD18, CD59, CR4, CD45, CD64, and CD44 (Akiyama et al., Brain Research 632:249-259 (1993)) increase in AD. Additional infl~mm~tory markers useful in AD screening assays include ~2 macroglobulin receptor, Fibroblast growth factor (Tooyama et al., Neuroscience Letters 121:155-158 (1991)), ICAM-l (Akiyama et al., Acta Neuropathologica 85:628-634 (1993)), LactoLlal~,re~ (Kawamata et al., American Journal of Pathology 142:1574-85 (1993)), Clq, C3d, C4d, CSb-9, Fc gamma RI, Fc gamma RII, CD8 (McGeer et al., Can J Neurol Sci 16:516-527 (1989)), LCA (CD45) (McGeer et al. (1989); Akiyama et al., Journal of Neuroimmunolog~ 50:195-201 (1994)), CD18 (beta-2 integrin) (Akiyama and McGeer, Journal of Neuroimmunology 30:81-93 (1990)), WO 96/40896 = . PCT~US96/09857 CD59 (McGeer et al., Brain Research 544:315-319 (1991)), Vitronectic (McGeer et al., ~anadian Journal of Neurological Sciences 18:376-379 (1991); Akiyama et al., Journal of Neuroimmunology 32:19-28 (1991)), ViL.olle~;Lill receptor, Beta-3 integrin (Akiyama et al. (1991)), Apo J, clusterin (McGeer et al., Brain Research 579:337-341 (1992)), type 2 pl~minngen activator inhibitor (Akiyama et al., Neuroscience Letters 164:233-235 (1993)), CD44 (Akiyama et al., Brain Research 632:249-259 (1993)), Midkine (Yasuhara et al., Biochemical & Biophysical Research Communications 192:246-251 (1993)), Macrophage colony stim~ ting factor receptor (Akiyama et al., Brain Research 639:171-174 (1994)), MRP14, 27E10, and ulL~lreivu-alpha (Akiyama et al., Journal of Neuroimmunology 50:195-201 (1994)). Additional lllalhel~ which are associated with infl~mm~tion or oxidative stress include 4-hydroxynonenal-protein conjugates (Uchida et al., Biochem. Biophys. Res. Comm. 212: 1068-1073 (1995);
Uchida and St~dtm~nJ Methods in En~ymology 233:371-380 (1994); Yoritaka et al ., Proc. Natl. Acad. Sci. USA 93 :2696-2701 (1996)), IKB, NFKB
(K~lt~r~lmi~lt et al., Molecular Aspects of Medicine 14: 171-190 (1993)), cPLA2 (Stephenson et al., Neurobiology Dis. 3:51-63 (1996)), COX-2 (Chen et al., Neuroreport 6:245-248 (1995)), Matrix metalloploteillases (Backstrom et al., J. Neurochemistry 58:983-992 (1992); Bignami et al., Acta Neuropathologica 87:308-312 (1994); Deb and Gottschall, J. Neurochemistry 66: 1641-1647 (1995); Peress et al., J. Neuropatholog;y & Experimental Neurology 54:16-22 (1995)), Membrane lipid peroxidation, Protein oxidation (Hensley et al., J. Neurochemistry 65:2146-2156 (1995); Smith et al., Proc.
Natl. Acad. Sci. USA 88:10540-10543 (1991)), and llimini.~h~d ATPase activity (Mark et al., J. Neuroscience 15:6239 (1995)). These markers can be cletecte~l, and changes can be determin.--l, to measure the- effect of compounds on the disclosed transgenic animals.
E. Neuronal and Neu,~,ll;...~...il~r-related Markers.
Changes in neuronal and n~uloLlA~ "~ el biochemistry have been associated with AD and in the disclosed PDAPP animals. In AD there is a ofoulld reduction in cortical and hippocampal cholinergic innervation. This CA 02222l74 l997-ll-24 W O 96/40896 PCT~US96/09857 is evidenced by the dramatic loss of the ~yllLll~lic enzyme choline ac~LylL.~l~r~dse and decreased acetylcholinersterase, synaptosomal choline uptake (as measured by hemirholinium binding) and synthesis and release of acetylcholine (Rylett et al. (1983); Sims et al. (1980); Coyle et al., Science 219:1184-1190 (1983); Davies and Maloney, Lancet 2:1403 (1976); Perry et al., Lancet 1:189 (1977); Sims et al., J. Neurochem. 40: 503-509 (1983)) all of which are useful markers. These lllalkel~ can be used in AD screening assays to ~lel~ "-i"~ the effect of compounds on AD. There is also a loss of basal forebrain neurons and the galanin system becomes hy~ lLlo~hic in AD.
In addition to changes in the ",~,k~ described above in AD, there is also atrophy and loss of basal forebrain cholinergic neurons that project to the cortex and hippocampus (Whitehouse et al., Science 215:1237-1239 (1982)), as well as alterations of ellLolllhlal cortex neurons (Van Hoesan et al., Hippocampus 1:1-8 (1991). Based upon these obs~ Lions measurement of these enzyme activities, neuronal size, and neuronal count numbers are expected to decrease in the disclosed transgenic animals and are therefore useful targets for detection in AD screening assays. Basal forebrain llC~Ul~Jn~Sare dependent on nerve growth factor (NGF). Brain-derived ~ uloLl~hic factor (BDNF) may also decrease in the hippocampus in the disclosed transgenic ~nim~ls and is therefore a useful target for detection in AD
screening assays.
It has also been shown that APP and A,B release are affected by stimnl~ion of muscarinic receptors both in vitro in tissue culture as well as inbrain slices. Similar fin-ling.s have also been obtained with application of other agonists linked to phosphoinosital turnover (Nitsch et al. (1992); Hung et al., J. Biol. Chem. 268:22959-22962 (1993); Nitsch et al., Proceedings of the Eighth Meeting of the International Study Group on the Pharmacology of Memory Disorders Associated with Aging 497-503 (1995); Masliah and Terry (1993); Greenamyre and Maragos (1993); McDonald and Nemeroff (1991);
Mohr et al. (1994); Perry, British Medical Bulletin 42:63-69 (1986); Masliah et al., Brian Research 574:312-316 (1992); Schwagerl et al., Journal of Neurochemistry 64:443-446 (1995)). Based upon these observations, it is WO ~G/4~96 PCT/U~GI~5357 possible that llculol~ rl agonists will reduce the pro(lllcti~n of A,~ in the disclosed transgenic animals. Based on this reasoning, screening assays that measure the effect of compounds on l~Ulot~ .J receptors can possibly be used to id~llLifyillg compounds useful in treating AD.
In addition to the well-docl-m~ntP-l changes in the cholinergic system, dy~ru~ ion in other receptor systems such as the seluLi~ gic, adl~nclgic, ~len- sinP, and nicotine receptor systems, has also been do~;.-...~..~rcl Markers characteristic of these challges, as well as other llculullal lll~h~
that exhibit both metabolic and structural changes in AD are listed below.
Changes in the level and/or loc~li7~ti-)n of these lllalk~l~ can be measured using similar techniques as those described for mP~cnrin~ and ~etPctin~ the earlier lllalh~
The following are ~lcrellcd cytoskeletal and neuritic lll~l~ that exhibit cl~nges in and/or an association with AD. Levels of cathepsin (cat) D,B and Neuronal Thread Protein, and phosphorylation of elongation factor-2, increase in AD. Cat D,B, protein kinase C, and NADPH are localized in plaque and n,~llritir tissue in AD. Activity and/or levels of nicotine receptors, S-HT2 receptor, NMDA lcce~Lo~ 2-adlcllc~gic receptor, ~ylla~Lo~hysin, p65, glllt~minP synfh.ot~.~e, glucose Llal~ulL~l, PPI kinase, drebrin, GAP43, cytochrome oxidase, heme oxygenase, calbindin, adenosine Al receptors, mono amine metabolites, choline act;LylL dl~rel~se, acetylcholinesterase, and symptosomal choline uptake are all reduced in AD.
Additional markers that are associated with AD or after tre~tmPnt of cells with A,~ include (1) cPLA2 (Stephenson et al., Neurobiology of Diseases 3:51-63 (1996)), which is upregulated in AD, (2) Heme oxygenase-l (Premkl-m~r et al., J. Neurochemistry 65:1399-1402 (1995); Schipper et al., Annals of Neurology 37:758-768 (1995); Smith et al., American Journal of Pathology 145:42-47 (1994); Smith et al., Molecular & Chemical Neuropathology 24:227-230 (1995)), cjun (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-W 096/40896 PCTAJS~G~3 519 (1994); Renkawek et al., Ne~roreport 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 (1991); T~k~h~hi et al., Acta Neuropathologica 81:626-631 (1991)), which are inrlllred in AD and in 5 cortical cells after A,B tre~tm~?nf, and (3) junB, junD, fosB, fral (Estus et al., J. CeU Biology 127:1717-1727 (1994)), cyclin D1 (F~ ul et al., Neuron 12:343-355 (1994); Kranenburg et al., EMBO Journal 15:46-54 (1996)), p53 (Chopp, Current Opinion in Neurology & Neurosurgery 6:6-10 (1993); Sakhi et al., Proc. Natl. Acad. Sci. USA 91:7525-7529 (1994); Wood and Youle, J.
10 Neuroscience 15:5851-5857 (1995)), NGFI-A (Vaccarino et al., Molecular Brain Research 12:233-241 (1992)), and NGFI-B, which are intlllced in cortical cells after A~ tr~o~tm.ont F. Mea~ul;ll~ the Amounts and T o~li7~ti~n of AD Markers.
Qll~ntit~tive measurement can be accomplished using many standard 15 assays. For example, ~ scli~l levels can be measured using RT-PCR and hyblidi~lion methods including RNase protection, Northern analysis, and R-dot analysis. Protein marker levels can be assayed by ELISA, Western analysis, and by comparison of immnnnhistoch.onnit~lly stained tissue sections. Tmmnnohi~torhl mir~l st~ining can also be used to assay 20 loc~li7~tion of protein markers to particular tissues and cell types. The localization and the histopathological association of AD markers can be ~ot~rminl-~l by histochemical detection methods such as antibody st~ining, laser sc~nning confocal im~ging, and immnnnelectron micrography.
Fx~mples of such techniques are described in M~cli~h et al. (1993) and in 25 Example 6 below.
In the case of receptors and enzymatic markers, activity of the receptors or enzymes can be measured. For example, the activity of n~ul~ LlA l-~. "itter metabolizing enzymes such as choline acetyltransferase andacetylcholine esterase can be measured using standard radiometric enzyme ~ 30 activity assays.
The activity of certain ~ uroLl~ e~ receptors can be determined by m~cllring phosphoinositol (PI) turnover. This involves m~llring the -WO ~6/~0~96 PCT~US9~03 ~ccnmlll~tion of inositol after stimnl~tion of the receptor with an agonist.
Useful agonists include carbachol for cholinergic l~ce~Lols and o,~ ,hrinP for glllt~min.orgic receptors. The llulllbeL of receptors present in brain tissue can be a~s~ed by qn~ iLi livt:ly m~ nrin~ ligand binding to 5 the receptors.
The levels and turnover of receptor ligands and n~uloLl,~ , can be ti~ .o.l by qn~..lil;llivt; assays tahen at various time points. Dopamine turnover can be measured using DOPAC and HVA. MOPEG sulfate can be used to measure nol~i..~hrinlo turnover and 5-HIAA can be used to measure 0 ~ UI~ turnover. For example, l~l~ill~hrine levels have been shown to be reduced 20% in the hippocampus of 12 to 13 month old PDAPP
Ll~lsgenic mice relative to controls. Generally, the above assays can be pelrolllled as described in the li~ldLul~, for example, in Rylett et al. (1983);Sims et al. (1980); Coyle et al., Science 219:1184-1190 (1983); Davies and Maloney, Lancet 2:1403 (1976); Perry et al., Lancet 1:189 (1977), Sims et al., J. Neurochem. 40: 503-509 (1983). These lllalh~l~ are also described by Bymaster et al., J. Pharm. Exp. Ther. 269:282-289 (1994).
G. Screening Assays Using Cultured Cells.
Screening assays for ~le~ g the therapeutic potential of compounds can also be performed using cells derived from animals transgenic for the disclosed APP constructs and cell cultures stably transfected with the disclosed constructs. For example, such assays can be performed on cultured cells in the following manner. Cell cultures can be transfected generally in the manner described in Tnt--rn~tional Patent Application No. 94/10569 and Citron et al. (1995). Derived transgenic cells or tr~n~fected cell cultures can then be plated in Corning 96-well plates at 1.5to 2.5 x 104 cells per well in Dulbecco's minim~l essenti~l media plus 10%
fetal bovine serum.
Following overnight inr~lbation at 37~C in an inr~lbator equilibrated with 10% carbon dioxide, media are removed and replaced with media cont~ininp~ a compound to be tested for a two hour ~lc;L~c~llllent period and cells were inrllh~t~d as above. Stocks co.,l;.i"i"g the compound to be tested CA 02222l74 l997-ll-24 W O96/1~-96 PCTAUS~6~'03 are first prepared in 100% dimethylsulfoxide such that at the final concentration of compound used in the tre~tml-nt the concentration of dimethylsulfoxide does not exceed 0.5%, preferably about 0.1%.
At the end of the ~ Ll~ nt period, the media are again removed 5 and replaced with fresh media co~ the compound to be tested as above and cells are hl ;u~aL~d for an additional 2 to 16 hours. After tre~tmrnt plates are cellLliruged in a Rec~m~n GPR at 1200 rpm for five mimltes at room t~ ldLul~: to pellet cellular debris from the conditioned media. From each well, 100 ,uL of conditioned media or ~l~lo~liale dilutions thereof are Ll~l~r~ d into an ELISA plate precoated with antibody 266 (an antibody directed against amino acids 13 to 28 of A,B) as described in TntPrn~tional Patent Application No. 94/10569 and stored at 4~C overnight. An ELISA
assay employing labelled antibody 6C6 (against amino acids 1 to 16 of A~) can be run to measure the amount of A,B produced. Dirre,c,lL capture and detection antibodies can also be used.
Cytotoxic effects of the compounds are measured by a mo~ifir~tion of the method of T-T~n~en et al., J. Immun. Method. 119:203-210 (1989). To the cells rem~ining in the tissue culture plate, 25 ~L of a 3,(4,5-dimethylthiazol-2-yl)2,5-dipht:llylL~Lldzolium bromide (MTT) stock solution (5 mg/mL) is added to a final concentration of 1 mg/mL. Cells are inrllh~trd at 37~C for one hour, and cellular activity is stopped by the addition of an equal volume of MTT lysis buffer (20% w/v sodium dodecylsulfate in 50%
dimethylform~mi~le, pH 4.7). CompletP extraction is achieved by overnight ~h~king at room temperature. The difference in the OD562l"l, and the OD650~
is measured in a Molecular Device's UV,I,a,, microplate reader, or equivalent, as an indicator of the cellular viability.
The results of the A,B ELISA are fit to a standard curve and expressed as ng/mL A~. In order to normalize for cytotoxicity, these results are divided by the MTT results and expressed as a percentage of the results from - 30 a control assay run without the compound.

W096/~0~6 : PCTrUS~6~0~357 All publications cited herein are hereby illcol~ulaLed by lGr~ ce.
The illrolLudlion contained in these publications for which the publications arecited is generally known.
F,Y ~ 1,1C 1: Ex~ ion of pMTAPP-1 in NIH3T3 and PC12 Cells.
The clone pMTAPP-1 is an example of an APP770 ~:A~ression CO~LL~1~;L as shown in Figure la where the promoter used is the metallothionine promoter. Stable cell lines were derived by transfecting NIH3T3 and PC12 cell lines (ATCC #CCL92 and CRL1721). Five hundred thousand NIH3T3 or PC12 cells were plated into 100 mm dishes and Ll,ll.~r~c~d with a 111iA~U1C: of S mg of the Sall fragment and 1 mg of pSV2neo DNA (Southern and Berg (1982)) precipitated in the presence of 50 mg lipofectin (Gibco, BRL) in a final volume of 100 ,ul. Polylysine-coated plates were used for PC12 cells, which normally do not adhere well to tissue culture dishes. The cells were fed with selection mrrlillm co-.l;.illillg 10%
fetal bovine serum in DMEM or RPMI and supplemrntr(l with G418. Five hundred mg/ml (biological weight) and 250 mg/ml of G418 were used to select colonies form NIH3T3 and PC12 cells, respectively. Fifteen days after transfection, colonies of cells resistant to G418 were isolated by cloning ringsand e~p~n~le-l in T flasks. The presence of APP cDNA in the cells was ~l~trctrrl by PCR using the procedure of Mullis and Faloona, Methods Enymol. 155:335-350 (1987), the tr~rhing~ of which are generally known and are incorporated herein.
Expression of APP in 25 colonies from each cell line was analyzed by immun~st~ining (Majocha et al. (1988)). Cells were grown to subconflllrnre and fixed in a solution cont~ining 4% parafnrm~l~lehyde, 0.12 M NaCl, and 20 mm Na3PO4, pH 7Ø They were incubated overnight with a plllllaly monoclonal antibody against a synthetic A,~ sequence (Masters et al. (1985);
Glenner and Wong) provided by Dr. Ronald Majocha, M~,s~rhll~ettc General Hospital, Boston, MA, followed by a generalized anti-mouse antibody conjugated to biotin (Jackson TmmllnnResearch Labs, PA). Tmmlml~st~ining was then performed by adding avidin-horse:radish peroxidase (HRP) (Vector Labs, Bllrling~me, CA) and ~ mint~ben7.iclin~ as the chromogen (Majocha et WO 96/40896 PCT~US9G/~5~JI

al. (1985)). The results in-lir~tt-cl that the pMTAPP-l vector was e~ cs~hlg APP in both NIH3T3 and PC12 cells.
F.Y~ 2: E~.e~ion of pEAPP-l in PC12 Cells.
pEAPP-l is an example of an APP770 c~le~ion construct as shown in Figure la where the promoter used is the 25 kb human APP gene promoter. DNA from this construct was transfected into PC12 cells as described above. Certain clones of pEAPP-l transfected cells exhibited a dirrc.ellLidtion phenotype morphologically similar to that exhibited by PC12 cells treated with nerve growth factor (NGF). PC12 cells normally are fairly round and flat cells. Those l~dl~rc~;led with pEAPP-l have cytoplasmic extensions resembling n~ollrit~s. PC12 cells treated with NGF
extend very long nPuriti~ extensions. Thirteen PC12 cell clones tr~n~fect with pEAPP-l were selected and propagated. Eight of these cell clones exhibited the spontaneous dirrelc.lliation phenotype with clones 1-8, 1-1, and 14 exhibiting the strongest phenotypes. Staining of pEAPP-l transfected PC12 cells with antibody against the A~ as described in Example 1 inf1ir~tt~-1 that those cells exhibiting the dirrclcllliation were also C~lCSsillg APP.
Rec~ e PC12 cells lldl~ire-;ted with the pMTAPP-l clone did not exhibit this phenotype even though the APP770 cDNA is expressed, these results suggest that expression of APP770 from the human promoter has novel ~lu~clLies regarding the physiology of the cell.
F,Y~mple 3: Ex~res~ion of pMTA4 in PC12 Cells.
pMTA4 is an example of the type of construct shown in Figure 4a where the promoter used is the metallothionine promoter. The protein encoded by this construct differs slightly from that depicted in Figure 4a. An APP770 cDNA clone was digested with Asp718 which cleaves after position 57 (number system of Kang et al. (1987)). The resulting S' extension was filled in using the Klenow enzyme (Sambrook et al. (1989)). The same DNA
~lc~aldlion was also cleaved with EcoRI which also cuts after position 2020 - 30 and the resulting 5' extension was filled in using the Klenow enzyme (Sambrook et al. (1989)). Self-ligation of this molecule results in an expression clone in which the truncated protein thus encoded contains the W096/40896 PCTrUS96/09857 leader seq ~enre~ followed by a shortened version of the A,~ starting with the seq ~nre Phe-Arg-Val-Gly-Ser-of the A,B followed by the 56 terminal amino acids of APP. DNA from this construct was transfected into PC12 cells as described above.
FY~n-rlf~ 4: Generation of Tr~ c Mice ~ APP under the control of the MT-1 promoter.
Transgenic mice were made by microinjecting pMTAPP-1 vector DNA into pronuclear embryos. pMTAPP-1 is an example of the type of construct shown in Figure la in which the APP770 coding sequence is 10 operably linked to the metallothionine promoter. The procedures for microinjection into mouse embryos are described in Manipulating the Mouse Embryo by Hogan et al. (1986). Only a brief description of the procedures is described below.
Mice were obtained from Taconic Laboratories (GPrm~n Town, New 15 York). Swiss Webster female mice were used for embryo retrieval and implantation. B6D2Fl males were used for mating and v~ectomi7~cl Swiss webster studs were used to ~imlll~te pseudopregnancy.
A. Embryo Recovel ~.
Female mice, 4 to 8 weeks of age, were in-illred to superovulate with 20 5 IU of pregnant mare's serum gonadotropin (PMSG; Sigma) followed 48 hours later by 5 IU of human chorionic gonadotropin (hCG; Sigma).
Females were placed with males immr~ t~.ly after hCG injection. Embryos were recovered from excised oviducts of mated females 21 hours after hCG
in Dulbecco's phosphate buffered saline with 0.5% bovine serum albumin 25 (BSA; Sigma). Surrounding cnm~ c cells were removed with hyaluronidase (1 mg/ml). Pronuclear embryos were then washed and placed in Earle's bal~nre-i salt solution cont~ining 0.4% BSA (EBSS) in a 37.5~C incubator with a hllmi~1ificrl atmosphere at 7% CO2, 5% ~2~ and 88% N2 until the time of injection.
B. Microinjection.
Elutip-DTM purified Sall DNA was dissolved in S mM Tris (pH 7.4) and 0.1 mM EDTA at 3 ,ug/ml concentration for microinjection.

WO 96/40896 PCT~US~61U9~;/

Microneedles and holding ~ipeLLes were pulled from Fisher coagulation tubes (Fisher) on a DKI model 720 pipette puller. Holding pipettes were then broken at ~rn~ ely 70 ~m (O.D.) and fire polished to an I.D. of about 30 ~m on a Narishige miclofo,~e (model MF-83). Pipettes were mounted on Narishige miclu~ ulators which were ~tt~rhP~l to a Nikon Diaphot miciusco~e. The air-filled injection pipette was filled with DNA solution through the tip after breaking the tip against the holding pipette. Embryos, in groups of 30 to 40, were placed in 100 ~l drops of EBBS under p~.,.rr oil for mi~;lullla~ulation. An embryo was oriented and held with the holding pipette. The injection pipette was then inserted into the male pronucleus (usually the larger one). If the pipette did not break through the membrane immPtli~tely the stage was tapped to assist in penetration. The nucleus was then injected and the injection was monitored by swelling of the nucleus. Following injection, the group of embryos was placed in EBSS until Lldl~r~l to recipient females.
C. T~ rel.
p~n-lomly cycling adult female mice were paired with vase~;L~ ,ed Swiss Webster males. Recipient females were mated at the same time as donor females. At the time of Lldl~rel, the females were ~n~sthPti7P(l with avertin. The oviducts were exposed by a single midline dorsal incision. An incision was then made through the body wall directly over the oviduct. The ovarian bursa was then torn with watch makers forceps. Embryos to be Llal~r.,.lcd were placed in DPBS and in the tip of a Lldl~rel pipet (about 10 to 12 embryos). The pipet tip was inserted into the infundibulum and embryos were Lldl~rellcd. After the Lldl~rcl, the incision was closed by two sutures.
D. Analysis Of Mice For Trdlls~elle Illle~ldlioll.
At three weeks of age or older, tail samples about 1 cm long were excised for DNA analysis. The tail samples were digested by inrllb~ting with ~h~king overnight at 55~C in the presence of 0.7 ml 5 mM Tris, pH 8.0, 100 mM EDTA, 0.5% SDS and 350 ~g of proteinase K. The digested material was extracted once with an equal volume of phenol and once with an equal W 096/40896 ~ PCTrU~,~G~uZ~a volume of phenol:chloroform (1:1 lLIibLtul~). The ~7u~ i were mixed with 70 ~4l 3 M sodium acetate (pH 6.0) and the DNA was L~lccipiL~Led by adding equal volume of 100% ethanol. The DNA was spun down in a microfuge, washed once with 70% ethanol, dried and dissolved in 100 ~l TE
buffer (10 mM Tris pH 8.0 and 1 mM EDTA).
Ten to twenty microliters of DNA from each sample was rli~este(l with BamHI, electrophoresed on 1% agarose gels, blotted onto nitrocellulose paper, and hybridized with 32P-labeled APP cDNA frslgm~?nt T.dns~ellic ~nim~lc were i~ientifi~tl by ~ntor~liography of the hybridized nitrocellulose filters. The DNAs were also analyzed by PCR carried out by synthetic primers to g~ dLe an 800 bp fr~gm~nt of APP DNA.
A total of 671 pronuclear embryos were microinjected out of which 73 live and 6 dead pups were born. DNA analysis itit~ntifiP~l 9 L.~gellic mice (5 females and 4 males) which were bred to generate Fl and F2 transgenics. These animals can be analyzed for ~ ssion of mRNA and protein of APP in diLre-~llL tissues and for analysis of behavioral and pathological abnorm~liti.os as described above. Tlal~,g~l.ic mice with this construct express Ll~sgellic RNA.
~Y~mrle 5: Construction of APP construct co~;;~ a combination cDNA/genomic coding sequence.
A cDNA/genomic APP construct cont~ining introns 6, 7 and 8 was prepared by combining APP cDNA encoding exons 1-6 and 9-18 with genomic APP sequences encoding introns 6, 7 and 8, and exons 7 and 8 (see Figure 6). In order to create a splicing cassette small enough for convenient insertion in a pUC vector, two deletions in intronic sequences were made. A
deletion was made in intron 6 from position 143 of intron 6 to the BamHI
site located upstream of the beginning of exon 7 (1658 bp before the beginning of exon 7). Another deletion was made in intron 8 from the first BamHI site in intron 8 to a site at 263 bp before the beginning of exon 9.
To avoid confusion, these truncated forms of APP introns 6 and 8 are referred to herein as intron ~\6 and intron ~8. BamHI sites were engineered at the sites of these deletions, so that they are marked by the presence of WO 9~/'C3~5 PCTAJS9GI~5 BamHI sites. In this construct, referred to as PDAPP, exons 7 and 8 and intron 7 are intact genomic sequences, except that the unique XhoI site in intron 7 was destroyed.
DNA fr:~gment~ cont~ining the Ll....~ l introns were ge~ d as 5 follows: a BamHI site was engineered 143 bp into intron 6 nucleotide by PCR mutagenesis ("Mutagenesis by PCR" in PCR Technolo~7: Current Innovations (Griffith and Griffith, eds., CRC Press, 1994) pages 69-83) and another BamHI site was engin~-ered by PCR mutagenesis 263 bp prior to the beginning of exon 9. These sites were engineered into sepal~te APP genomic 10 DNA clones co,~ i"i~ the junctions of exon 6 and intron 6, and intron 8 and exon 9, respectively, reslllting in modified APP genomic DNA clones.
The entire cassette was assembled in the APP cDNA clone as follows (Figure 11). The 889 bp BamHI to XcmI fragment of APP cDNA cont~ining exons 1 through 5 and part of exon 6 (including nucleotides 1 to 843 of SEQ
15 ID NO:5) was cloned into a vector cont~ining BamHI and XhoI sites dow~,L.~ll from the insertion site to make APP770x-oligo-x. APP770x-oligo-x was then cut with XcmI and BamHI. Then two fr~gm~ont~ were obtained from the modified APP genomic DNA clone cont~ining the junction of exon 6 and intron 6 described above by cutting with XcmI and BamHI.
20 The rçsnltin~ 34 bp fragment from the XcmI in exon 6 to the XcmI in intron 6, and 131 bp fragment from the XcmI in intron 6 to the artificially created BamHI site at position 143 bp of intron 6 were ligated into APP770x-oligo-x in a three-way ligation step to make APP770x-E6O1igo-x. The orientation of the fragments was confirm~l by sequencing. APP770x-E6O1igo-x was then 25 cut with BamHI and XhoI. Then the 313 bp BamHI and XhoI fragment from the modified APP genomic DNA clone conf~ining the junction of intron 8 and exon 9 was ligated into APP770x-E601igo-x to make APP770xE6E9x.
APP770xE6E9x was then cut with BamHI and the 6.8 kb BamHI
fragment of APP genomic DNA encoding the KPI and OX-2 rlom~in~ (exons 30 7 and 8) was inserted at this site. This fragment starts at the BamHI site 1658 bp Up~ ,alll of the start of exon 7 and extends to the first BamHI site in intron 8. This BamHI fragment was obtained from a lambda phage genomic CA 02222l74 l997-ll-24 WO 9G/1~896 PCT~US96/09857 clone encoding this portion of the APP gene, that was obtained from a Human Placental genomic library in the T ~mh~l~ FIXII vector obtained from Stratagene. This BamHI fragment originally contained an XhoI site which was destroyed by cutting, filling in, and relig~tion The locations of the S ~elçtion~ are diagramed in Figure 10. This clone, co~ i"i~-g exons 1-8 and part of 9, and introns 6, 7 and 8, was termed the "APP splicing cassette."
The APP splicing cassette was cut out with NruI and X7~oI and used to replace the NruI to XhoI cDNA fr~gm~nt of APP cDNA bearing the Hardy mllt~tion This mutant form of APP cDNA was produced by col.v~:lLillg the 10 G at nucleotide position 2145 to T by site directed mutagenesis. This changes the encoded amino acid from Val to Phe. The resllltin~ construct is a combination cDNA/genomic APP "minigene."
Sequencing of the 6.8 kb BamHI fragment cont~ining APP exons 7 and 8 derived from the APP genomic clone used to generate this construct 15 showed that intron 7 is 2.6 kb long, and that the first BamHI site in intron 8, the u~sLl~ site of the deletion in intron 8 engin.oered into the APP
minigene construct, is 2329 bp dowl~Llcalll from the end of exon 8. This does not coincide with the restriction map of the APP gene published by Yoshikai et al. (1990) and Yoshikai et al. (1991). Comparison of their map 20 to our sequence jn~ tes that Yoshikai et al. switched the order of two EcoRI fr~menf~ in their restriction mapping. The 1.60 kb EcoPI fragment Co~ i"i"g exon 8 iS actually ups~ l of the 1.48 kb EcoRI fragment and the 1.48 kb EcoRI fragment Yoshikai et al. mapped in intron 7 is actually in intron 8. We have con~lrm~d this location for the EcoRI fragment cont~ining 25 exon 8 by sizing of PCR generated fr~gmentc from hum-an DNA.
This APP minigene was operatively linked to the PDGF-B promoter to provide e~ ssion of the APP cDNA/genomic construct in m~mm~ n cells. The PDGF ~-chain S' fl~nking sequence was inserted upstream of the NruI site at the beginning of the APP minigene. This fragment includes 1.3 30 kb upsL c:aln of the L-a-L~ ion initiation site, where the PDGF-B promoter resides, and approximately 70 bp of 5' untr~n~l~t~d region, ending at the AurII site (Higgins et al. (1994)). The late SV40 polyadenylation signal, CA 02222l74 l997-ll-24 W 09C/~0~96 PCTIUS~J~Z

carried on a 240 bp BamHI to BclI fr~m~nt, was added dowl~L~ of the APP minigene. This col~Ll,l~;L, combining the PDGF-B promoter, the APP
splicing cassette, the Hardy mllt~tion, and the SV40 polyadenylation signal is referred to as PDAPP (Figure 9).
S ~y~npl~ 6: Trd-l~g~ic mice co~ the PDAPP construct.
T1AI~ ell;C mice were gc;ne.aL~d using the PDAPP construct described in Fx~mrle 5. Tl~nsgellic mice were gen~r~t~o~l by microinjection using standard techniques as described above. PDAPP DNA was microinjected into the embryos at the two-cell stage. Plasmid sequences (pUC) were 10 removed by SacI and NotI digestion before miclohlje~lion. Seven founder mice were g~n~aLed and line 109 was used for ~L~llsive analysis. Only heterozygous anim~l~ were used. Southern analysis of 104 ~nim~l~ from four g~neldLions showed that a~ L"nately 40 copies of the L-~lsgelle were inserted at a single site and Ll~ in a stable manner. Human APP
15 me~s.onger RNA was produced in several tissues of the L-~sgellic mouse, but at especially high levels in brain. RNase protection assays revealed at least 20-fold more APP e~ression in the brains of line 109 ~nim~l~ than in the mouse lines expressing neuron-specific enolase (NSE)-promoter-driven APP
transgenes previously described by Quon et al. (1991), Mucke et al., Brain 20 Res. 666:151-167 (1994), McConlogue et al., Neurobiol. Aging 15:S12 (1994), and Higgins et al., Ann Neurol. 35:598-607 (1994).
A. E~ e~ion of APP Tralls~ Ls and Protein.
RNA was isolated from brain tissue as described by Chomaczynski and Sacchi, Analyt. Biochem. 162:156-159 (1987), and subjected to RT-PCR
25 as described by Wang et al., Proc. Natl. Acad. Sci. U.S.A. 86:9717-9721 (1989), using human-specific APP primers (S'-CCGATGATGACGAGGACGAT-3', SEQ ID NO:7;
S'-TGAACACGTGACGAGGCCGA-3', SEQ ID NO:8) using 40 cycles of 1 minute at 94~C, 40 seconds at 60~C, and 50 seconds at 72~C. RT-PCR
- 30 analysis demo~ dt~d the presence of ll~nscli~ encoding the 695, 751 and 770 isoforms of human APP in transgenic animal brains but not in brains from non-transgenic littermates. The identities of the human APP RT-PCR

CA 02222l74 l997-ll-24 WO 96/1D ~.4~ PCTrUS96/09857 bands from the transgenic mouse RNA were verified by subcloning and seqll.on-~ing.
The relative levels and alL~ dLiv~ splicing of APP LldnS~ L~ in brains of PDAPP Lldllsgel~ic mice, NSE-APP Lldn~ ~ic mice, non-Lldnsg~l,ic mice, and humans with and without AD were compared in RNase protection assays (Rockt~ et al., J. Biol. Chem. 270:28257-28267 (1995)). PDAPP mice essed approximately 5-fold higher total APP mRNA levels than non-I~A~gel~ic controls, and at least 20-fold higher human APP mRNA levels than most NSE-APP transgenic mice. While NSE-driven human APP
~ ion does not affect the levels of murine APP rnRNA, PDAPP
transgenic mice showed a signifir~nt 30% decrease in murine APP
Ll~llS~ L~. While the relative ablm-l~n~es of murine APP770:751:695 mRNAs in non-Lldnsg~-.ic mouse brains were roughly 1:1:35, the corresponding human APP mRNA levels in PDAPP tr~n~geni- mouse brains were 5:5:1.
Analysis of holo-APP was performed by brain homo~c;lli~;~lion in 10 volumes of PBS co"l;.i"i"g 0.5 mM EDTA, 10 ~ug ml~ U~Li11 and 1 mM
PMSF. Samples were spun at 12,000g for 10 min and the pellets resuspended in RIPA (150 mM NaCl, 50 mM Tris, ph 8.0, 20 mM EDTA, 1.0% deoxycholate, 1.0% Triton X-100, 0.1% SDS, 1 mM PMSF and 10 ~g ml~l leupeptin). Samples (each co"l;.i"i"g 30 ,ug total protein) were analyzed by SDS-PAGE, Lldl~r~ d to Immobilon membranes and reacted with either the holo-APP antibody, anti-6 (anti Bx 6), described by Oltersdorf et al., J.
Biol. Chem. 285:4492 4497 (1990), or 8E5 monoclonal antibody. 8E5 was prepared against a bacterial fusion protein encompa~ing human APP residues 44~1-692 (Oltersdorf et al. (1990)) and is human-specific, showing essenti~lly no crossreactivity against mouse APP. Tmmlmoblot analysis of total APP
expression (human and mouse) in transgenic mouse line 109 and control lhterm~t~ brain tissue using C-terminal APP antibody anti-6 showed much higher levels of expression in the Lldnsgellic mice. Tmmllnoblot analysis of brain homogenates using either the holo-APP polyclonal antibody anti-6 or the human-specific APP monoclonal antibody 8E5 revealed human APP over-W O ~6t~836 PCT~US96/09~;

ession in the llA~gel~;c mouse at levels at least 3-fold higher in hippocampus than either endogenous mouse APP levels or those in AD brain.
For immlmnblot analysis of A~B, a 9-month-old mouse brain was homogenized in S ml 6 M gu~niclin~ HCl, 50 mM Tris, pH 7.5. The homogenate was centrifuged at 100,000g for 15 min and the ~u~ was dialyzed against H20 overnight adjusted to PBS with 1 mM PMSF and 25 ~g ml~l leupeptin. This material was immnno~ ci~ildled with antibody 266 resin, and immlm~blotted with the human-specific A,B antibody, 6C6, as described by Seubert et al., Nature 359:325-327 (1992). Using this human-specific A,~ antibody (6C6), a 4 kD ,~ amyloid-imml-nr)reactive peptide was isolated from the brains of the transgenic animals, which corresponds to the relative molecular mass of A~. Brain levels of A,~ were at least 10-fold higher in line 109 animals than in the previously described human APP
transgenic mice. Embryonic day 16 cortical cell cultures from ~ ic ~nim~l~ col~Li~uLively secreted human A,~, including a sllbst~nti~l fraction of A,~ 142 (5 ng ml~' total A~; 0.7 ng ml~l A~ 142), as ~ilotectecl in media by human-specific A,~ enzyme-linked immlmnsorbent assays, as described by Seubert et al. (1992) and McConlogue et al. (1994), and as described in Example 8. Thus, line-109 animals greatly ovelc~lcssed human APP
mRNA, holo-APP and A,~ in their brains.
B. Histop~thQIogy of PDAPP Transgenic Mice.
Brains from 180 transgenic and 160 age-m~tchtocl non-transgenic age-m~trh-o-1 controls (4 to 20 months old) representing five ~ ions of the line 109 pedigree were extensively ex~min.ofl histopathologically. Some mouse brains were removed and placed in alcohol fixative (Arai et al., Proc.
Natl. Acad. Sci. U.S.A. 87:2249-2253 (1990)) for 48 hours before paraffin embedding. Other mice were perfused with saline followed by 4%
pdldfollllaldehyde in 0.1 M sodium phosphate. For paraffin embedded brains, 6 ~m coronal or parasaggital sections from transgenic and non-transgenic mice were placed adjacent to each other on poly-L-lysine coated slides. The sections were deparaffini7~-l, rehydrated and treated with 0.03%
H202 for 30 min before overnight incubation at 4~C with a 1:1,000 dilution - ~ =

wo g~/~r ~6 ; PCT~US96/09857 of the A,B antibody, R1280 (T~m~nk~ et al., Proc. Natl. Acad. Sci. U.S.A.
89: 1345-1349 (1992)). For absorption studies, synthetic human A,~ 140 peptide (Games et al., Neurobiol. Aging 13:569-576 (1992)) in 10% aqueous dimethyl~lllphnxi~le was added to a final concentration of 7.0 ,uM to the diluted antibody and inr~lh~trrl for 2 hours at 37~C. The diluent was applied to the sections and processed under the same conditions as the standard antibody solution. Peroxidase rabbit IgG kit (Vector Labs) was then used as c~collllllended, with 3~3~ minnben7irlin~ (DAB) as the chromogen.
Similarly fixed human AD brain was processed .cimnlt~n~ously under itlentir~l conditions.
Before 4 months of age, no obvious A~ deposition was cletect~l However, by approximately 4 months of age, the lldnS~ l]iC animals began to exhibit deposits of human A,~ in the hippocampus, corpus callosum and cerebral cortex. These A~B plaques increased with age, and by eight months many deposits of 30 to 200 ~Lm were seen. As the animals aged beyond 9 months, the density of the plaques increased until the A~B-st~ining pattern resembled that of AD. Vascular amyloid, another feature of AD pathology, developed in older mice. Robust pathology was also seen in another lldnsg~llic line gell~lcLtt:d from the PDAPP vector (line 35).
A~ deposits of varying morphology were clearly evident as a result of using a variety of A,B antibodies, inrl~l~ling well char~rteri7r~l human-specific A,B antibodies and antibodies specific for the free amino and carboxy termini of A,l~ 1-42. Antibody 9204, described by Saido et al., J. Biol. Chem.
289:15253-15257 (1994), is specific to A~ 1-5 and was used at a concentration of 7.0 ,ug ml~'. Antibody 277-2, specific for A,(~ 1-42, was prepared by i,n~ .g New 7e~l~nrl white rabbits with the peptide cysteine-aminoheptanoic acid-A,B 33-42 conjugated to cationized BSA ('Super Carriers'; Pierce) using a standard i----~,l",i,;11ion protocol (500 ~g per injection). Specific antibodies were affinity-purified from serum against the immllnngen irnmobili_ed on agarose beads. Before inrnhation with antibody 277-2, sections were treated for 1 to 2 min with 80% formic acid. For detection, the antibody was reacted using the peroxidase rabbit IgG kit W O9~/40~96 PCT~US~G/053~7 (Vector Labs). The product was then vi~ li7~o~1 using DAB as the chromogen, Some sections were then inr-lb~te~l overnight at 4~C with a 1:500 dilution of polyclonal anti-GFAP (Sigma). The GFAP antibody was reacted using the ~lk~lin~ phosphatase anti-rabbit IgG kit and ~lk~linP
S pho~srh~t~ substrate kit 1 (Vector Labs; used according to the m~mlfartllrer's recomm~o-n-l~tions). Additional sections were incubated overnight with the F480 antibody (Serotec) used at a 1:40 ~iiluti~m to visualize microglial cells. The mouse peroxidase kit (Vector Labs) was then used according to the m~nnfartllrer~s rec~-mm~n~l~tions. Some sections were stained with thioflavin S using standard procedures (Dickson et al., Acta Neuropath. 79:486-493 (1990)) and viewed with ultraviolet light through an FITC filter of m~ximllm wavelength 440 nm.
Serial sections demonstrated many plaques were positively stained with both the 9204 and 277-2 antibodies. The forms of the A,l~ deposition ranged from diffuse irregular types to comp~cttorl plaques with cores.
Roughly spherical, and wispy, irregular deposits, were labelled with antibody 9204 specific for the free amino terminus of A,B. Astrocytic gliosis associated with A~ deposition was evident after double immnn~labelling with antibodies to glial fibrillary acidic protein (GFAP) and human A~B. A
compacted A,B core and 'halo' was evident in several plaques. Non-transgenic littermates showed none of these neuropathological changes.
Immunost~ining was fully absorbable with the relevant synthetic peptide, and was ~pa.~llL using a variety of processing conditions, including fixation with paraformaldehyde and Trojanowski methods. Many plaques were stained with thioflavin S, and some were also stained using the Bielschowsky silver method and were birefringent with Congo Red, in-lir~ting the true amyloid nature of these deposits.
The majority of plaques were i~ y ~u~ unded by GFAP-positive reactive astrocytes, similar to the gliosis found in AD plaques. The - 30 neocortices of the transgenic mice contained diffusely activated microglial cells, as defined by their amoeboid appearance, shortened processes, and staining with Mac-l antibody. Staining by antibodies recognizing CA 02222l74 l997-ll-24 WO 96/1~96 PCTAUS~G/0~357 phosphorylated neurofil~mtont~ and phosphorylated tau in-1ir~to~1 that aberrant phosphorylation occurred in PDAPP brain that was similar 'to AD. These pho~pholylations are seen in AD and are thought to preclude formation of neurofibrillary tangles. Although paired helical fil~m~nt~ (PHF) have not yet S been ~let~oct~d in PDAPP mice, the detection of abnormally phosphorylated neurofilaments and tau are thought to be associated with, and the initial step in, the formation of PHF in AD.
Clear evidence for neuritic pathology was ~a~ using both conventional and confocal immnnomici~Jsco~y. Forty ~m thick vibratome sections were inrllb~tocl overnight at 4~C with R1280 (1:1,000) in combination with polyclonal anti-~yll~ophysin (1:150; Dako) or 8ES (7.0 ,ug -1). Some sections were inrubatt~rl with anti-~ylld~Lophysin or monoclonal anti-MAP 2 (1:20, Boehringer-Mannheim), and then reacted with a goat anti-rabbit biotinylated antibody (1:100) followed by a lll~Lulc; of FITC-conjugated horse anti-mouse IgG (1:75) and avidin D Texas red (1:100) (Vector Labs). The double-immlmolabelled sections were viewed on a Zeiss Axiovert 35 microscope with ~tt~h.o~l laser confocal sc~nning system MRC
600 (Bio-Rad). The Texas red channel collected images of the R1280 or ~y~Lophysin labelling, and the FITC channel collected ~yl~Lophy~ill, 8E5, 20 or MAP 2 labelling. Optical z-sections 0.5 ,um in thi~l~n~os~ were collected from each region, similar to the image processing and storage described by Masliah et al., J. Neuropath. Exp Neurol. 52:619-632 (1993).
Many A,B plaques were closely associated with distorted neurites that could be cletecte~l with human APP-specific antibodies and with anti-25 ~yll~o~hysin antibodies, suggesting that these neurites were derived in partfrom axonal sprouts, as observed in the AD brain. The plaques colllpLessed and distorted the surrounding neuropil, also as in the AD brain. Synaptic and dendritic density were also reduced in the molecular layer of the hippocampal dentate gyrus of the transgenic mice. This was evident by 30 reduced immlmnstaining for the ~lc~yll~Lic marker ~yl~p~L)hysin and the dendritic marker MAP-2 in AD brain (Masliah et al., Am. J. Path. 138:235-246 (1991)).

W O 96t40896 PCT~US96/09857 .

Col . ri . " ~til~n of the presence of extracell~ r A~B was obtained using immlln-~electron microscopy. For imml-nnelectron microscopy, mice were perfused with saline followed by 2.0% pcuarOI "~k1ehyde and 1.0%
glllt~r~k~ yde in cacodylate buffer. Forty ~m thick vihr~t-)m~ sections were S inrllb~t~d with the R1280 antibody, and reacted using a peroxidase rabbit IgG
kit (Vector Labs). Tmmllnl~labelled sections with A,~ deposits were then f~ed in 1.0% ammonium tetraoxide and embedded in epon/araldite before viewing llltr~thin sections with a Jeol CX100 electron micl~scope (Masliah et al., Acta Neuropath. 81:428-433 (1991)).
Table 3. Ullr~l-u~u~al Similarities and Differences Between AD and PDAPP Tl ~I:~g(cnic Plaques.

Alzheimer's PDAPP
Disease Amyloid fibrils size 9-11 nm 9-11 nm electron density moderate high pinocytic vesicles abundant occ~ion~l D~ ul' '- neurites dense laminar bodies abundant abundant synaptic vesicles and contacts yes yes neurofilament ~c-~m~ tir~n yes yes TYPE II
paired helical filaments yes none?
Cells s ~o ~ d with amyloid f microglia abundant occasional neurons uc~ ;o~ l abundant nculuse~lcL~Jly granules abundant abundant rough endoplasmic reticulum abundant abundant coated pits yes yes Tables 3 and 4 present a summary of the above results, showing cytological and pathological similarities between AD and PDAPP mice. For every feature ex~mint-cl, with the exception of paired helical filaments, the PDAPP mice exhibited pathology characteristic of AD. These fintlin~ show s that production of human APP in transgenic (TG) mice is sufficient to cause not only amyloid deposition, but also many of the complex subcellular de~elle.dLi~e changes associated with AD.
SUBSTITUTE Stl~ 1- (RULE 26) W 096/40896 PCT~US96/09857 Table 4. p~th~logy in ~l~hpim~r~s Disease and the PDAPP Mouse.
.A17hlqim~r's PDAPP
Disease A~ Deposition into Plaques Diffuse + +
Neuritic + +
Vascular + +
Brain Region Specificity + +
Neuritic Dystrophy + +
Synaptic Loss + +
Tnfl~"""~loly Response Astrocytosis + +
Microgliosis + +

Cytoskeletal Alterations Phosphorylated Neurofil~m~nt~ + +
Phosphorylated Tau + +
PHF/Tangles + -(?) The most notable feature of these Lldnsgenic mice is their Alzheimer-20 like ll~:ulu~athology, which inrlllcles extracellular A~ deposition, dystrophic neuritic components, gliosis, and loss of synaptic density with regional specificity resembling that of AD. Plaque density increases with age in these transgenic mice, as it does in humans (Selkoe, Rev. Neurosi. 17:489-517 (1994)), irnplying a progressive A,~ deposition that exceeds its clearance, as 25 also proposed for AD (Maggio et al., Proc. Natl. Acad. Sci. U.S.A. 89:5462-5466 (1992)). The PDAPP transgenic mice provide strong new evidence for the primacy of APP ~ es~ion and A,(~ deposition in AD neuropathology.
Such mice also provide a sufficiently robust AD model in which to test whether compounds that lower A~ production and/or reduce its neurotoxicity 30 in vitro can produce beneficial effects in an animal model prior to advancing such drugs into human trials.

W 096/40896 PCTAJS96~0~a Example 7: Construction APP l~ s ~A~J~ g APP from the PDGF-B promoter.
PDAPP Ll~ls~ ~ic mice contain a splicing cassette that permits ,ssion of all three major human APP isoforms, where t~ ession is 5 driven by the PDGF-B promoter, and which includes a mllt~tion in amino acid 717, the site of f~mili~l AD mutations. It is expected that these fe~Lulc;s, and others described above, can be used independently to produce tr~n~genir mice useful as models of Alzheimer's disease. Some specific examples of such constructs are described below.
A. Construction of PDAPP-wt.
A wild type version of the cDNA/genomic clone PDAPP was constructed in which the mutation to amino acid 717 was replaced with the wild type. This was accomplished by replacing the 1448 bp X7toI to SpeI
fragment of PDAPP, which inrl~ es the part of the APP cDNA seq~l~onre that encodes the Hardy mutation in which Val717 is replaced by Phe, with the 1448 bp X7zoI to SpeI fragment of a wild type APP clone. This fr~gm/ont corresponds to the region from position 1135 to 2588 of SEQ ID NO:S.
None of the intron sequences of PDAPP are replaced or removed by this substitution. This construct is referred to as PDAPP-wt. A sch~omz~tir of PDAPP-wt and its construction is shown in Figure 12.
B. Construction of PDAPP-SwHa.
Another version of the cDNA/genomic clone PDAPP was constructed in which the Swedish mutant at amino acids 670 and 671 was introduced.
Plasmid pNSE751.delta3'spl.sw contains cDNA of the human APP751 which includes the Swedish mutation of Lys to Asn and Met to Leu at amino acids 670 and 671, respectively. A 563 bp EcoRI to SpeI fragment from this plasmid was replaced with the corresponding 563 bp EcoRI to SpeI fragment of PDAPP, which includes an identical part of the APP cDNA sequence with the exception of Phe717 of the Hardy mutation. This fragment corresponds to the region from position 2020 to 2588 of SEQ ID NO:5. This results in pNSE.delta3'spl.sw/ha, which contains both the Swedish mutation at amino acids 670 and 671, and the Hardy mutation at amino acid 717.

W0~6/1_~96 PCT~US96/09857 The 1448 bp XhoI to SpeI fragment of PDAPP was then replaced with the 1448 bp XhoI to SpeI fr~gm~nt of pNSE752.delta3'spl.sw/ha, which contains both the Swedish mutation and the Hardy mutation, to form PDAPP-Sw/Ha. A srhPm~tir of PDAPP-Sw/Ha and its construction is s shown in Figure 13.
C. Construction of PDAPP695v F.
A construct ~-n~o~ling only APP695, but let~i";~-g the Hardy mutation, PDGF-B promoter, and vector sequences of PDAPP, can be made. This can be accomplished by ligating the 6.6 kb XhoI to Nr~I fragment from PDAPP, which contains the C-termin~l part of the APP sequences, and the polyadenylation, pUC, and PDGF-B promoter sequences, to the 1.2 kb XhoI
to BclI fragment of pCK695, which contains a hybrid splice signal and the rem~ining N-terminal portion of the APP sequences (on a 911 bp XhoI to NruI fr~gm~nt of APP695 cDNA). The hybrid splice signal is the same as was described earlier and is also present in vector pohCK751, which is described by Dugan et al., J Biological Chem. 270: 10982-10989 (1995).
pCK695 is identical to pohCK751 except that the herpes simples virus replication and p?~k~in~ sequences of pohCK751 were removed, and the plasmid encodes APP695 instead of APP751.
In this vector the PDGF-B promoter drives the ~ s~ion of APP695 cont~inin~ the mutation of Val717 to Phe. The hybrid splice signal is in~ ded to potentially enh~n~e expression. Additional vectors derived from this may be constructed which lack any splice signals, or into which other splice signals have been added to obtain this same function.
D. Construction of PDAPP7!;1v F.
A construct encoding only APP751, but ret~ining the Hardy mutation, PDGF-B promoter, and vector sequences of PDAPP, can be made. This can be accomplished by ligating the 6.65 kb XhoI to KpnI fragment of PDAPP, including part of the APP sequences, the polyadenylation signals, pUC and PDGF-B promoter sequences to the 1.0 kb KpnI to XhoI fragment cont~ining the rem~in~l~or of the human APP751 cDNA sequences (nucleotides 57 to 1084 of SEQ ID NO:3) to make the inr~rrnto~ te plasmid PDAPP~sp751vl.

WO 9G1408~6 PCT~US9610~

The 1.0 kb KpnI to X7loI fragment encoding a portion of human APP751 can be obtained from the plasmid poCK751, which is i~lenti~ to pohCK751 except that the herpes simplex viral sequ~nres were removed.
To introduce splicing sequences, the first intron from PDAPP, which s is intron ~6, is then inserted into PDAPP~sp751vF to make PDAPP751VF.
To accomplish this, the 2,758 bp Asp718 to ScaI fragment of PDAPP
co~ i,.;"g exons 2 through 6, intron ~6, and part of exon 7, is ligated to the 6,736 bp fr~gmPnt obtained by complete digestion of PDAPP~sp751vF with Asp718 and partial digestion with ScaI. This 6,736 bp fragment provides the r~m~inin~ additional APP sequences (part of exon 1, the rest of exon 7, and exons 9 through 18), polyadenylation signals, pUC and PDGF-B promoter seq~len~es. The res-lting construct is referred to as PDAPP751VF.
In this vector the PDGF-B promoter drives the expression of APP751 c~"li.;"i"g the mutation of Val717 to Phe. One splice signal (derived from intron 6) is included to potentially enh~n~e ~ cs~ion. Additional vectors derived from this may be constructed which lack any splice signals, or into which other splice signals have been added to obtain this same function.
E. Construction of PDAPP770V-F-A construct encoding only APP770, but ret~ining the Hardy mutation, PDGF-B promoter, and vector sequences of PDAPP, can be made. This can be accomplished by replacing the KpnI to X7loI fragment of PDAPP751VF
cont~ining APP exons 2-7 and a part of exon 9, with the KpnI to X~oI
fragment of APP770 cDNA, which contains exons 2-8 and a part of exon 9.
This fragment corresponds to nucleotides 57 to 1140 of SEQ ID NO:S. The 2s resnlting construct is referred to as PDAPP770V F.
In this vector the PDGF-B promoter drives the expression of APP770 cont~ining the mutation of Val717 to Phe. PDAPP770V F contains the same intron sequences present in PDAPP751V F. Additional vectors derived from this may be constructed into which a splice signals have been added to obtain enh~nred expression.

CA 02222l74 l997-ll-24 W O 96/40896 PCTAUS~6/'~3 ~mp'C 8: Ex~J.t:~ion Levels of APP Ex~ s~ion Products in Brain Tissue of PDAPP Mice.
The PDAPP mouse line described in Example 6 was ~x;.~ od for the levels of several dt;liv~liv~s of the APP in hippoc~mpal, cortical, and s cerebellar brain regions of mice of various ages. Levels of APP cleaved at the beta-secretase site (APP,l~) and APP cont~ining at least i2 amino acids of A,B (FLAPP+APPc~; a mixture of APP~ and full length APP (FLAPP)) were found to be nearly constant within a given brain region at all ages ev~ln~t~d The _ippocampus expressed the highest level of all APP forms. In contrast, 0 gu~ni-linP extractable levels of A,~ showed rcm~rk~ble age-dependent eases in a manner that mirrored the amyloid plaque deposition observed immlm~hi~toch~Tnic~lly. Specifically, A,~ levels in hippocampus increased 17-fold by 8 months of age and 106-fold by 1 year of age, compared to that found in 4 month old animals. At 1 year of age A,B co~ s approximately 1% of the total protein in hippocampus. The cerebral cortex also showed large increases in A,B with age. In contrast, the mean level of A~B in cerebellum across all age groups was co~ dldLively low and nnrh~nging.
Further analysis of the A~ in these brains using an ELISA specific for A~ 2 showed that this longer version made up 27% of the 19 pmoles/g of the A,l~ present in the brains of young animals; this percentage increased to 97% of the 690 pmoles/g in 12 month old animals. The selective deposition of A,~l12 and the spacial distribution of the A~ deposits are further evidence that the pathological processes ongoing in the PDAPP transgenic mice parallel the human Alzheimer's fli~e~e(l condition.
Levels of A~-cont~ining proteins were measured through the use of ELISAs configured with antibodies specific to A,B, A~ 2~ APP cleaved at the ~-secretase site (Seubert et al. (1993)), and APP cont~inin~ the first 12 amino acids of A,B (FLAPP+APP(x; a mixture of full length APP and c~-secretase cleaved APP (Esch et al.)). Striking similarities in both the regional variation and depositing form of A,B are noted between the mouse model and the human AD condition. The results also show that, because of WO ~G/10~96 PCTAJS961~~3 the mAgnitllcle and temporal predictability of A,~ deposition, the PDAPP
mouse is a practical model in which to test agents that either inhibit the procescing of APP to A,B or retard A,B amyloidosis.
A. Materials and Methods.
1. Brain Tissue I~ion.
The heL~lo;~y~oL~ Lldl~gellic (Line 109, Games et al.; I2ocLr~ rill et al.) and non-Ll~genic animals were An~Sth~ti7~rl with Nembutol (1:5 solution in 0.9% saline) and perfused intracardially with ice cold 0.9%
saline. The brain was removed and one hrmi.cpht-re was prepared for lo immlmohistoch~?mir~l analysis, while four brain regions (cerebellum, hippocampus, thAlAmllc, and cortex) were tliccecte~l from the other hemicph-ore and used for A,B and APP measures.
To prepare tissue for ELISAs, each brain region was homogenized in 10 volumes of ice cold gnAniflinP buffer (5.0 M g~lAni-lin.--HCl, 50 mM Tris-Cl, pH 8.0) using a motorized pestle (Kontes). The homogenates were gently mixed on a Nutator for three to four hours at room Lelll~ lule, then either assayed directly or stored at -20~C prior to ~ll.A.,IilAlion of A~ and APP. Prel;",i,IA,y experiments showed the analytes were stable to this storage condition.
2. A,~ Mea~ur~
The brain homogenates were further diluted 1:10 with ice-cold casein buffer (0.25% casein, phosphate buffered saline (PBS), 0.05% sodium azide, 20 ,u~/ml aploLi~ , 5 mM EDTA pH 8.0, 10 ,ug/ml leupeptin), reducing the final concentration of guanidine to 0.5 M, before centrifugation (16,000 x g 2s for 20 mim-Ies at 4~C). The A~ standards (1-40 or 1-42 amino acids) were prepared such that the final composition included 0.5 M gllAni~line in the presence of 0.1 % bovine serum albumin (BSA) .
The "total" A,B sandwich ELISA consists of two monoclonal antibodies (mAb) to A,B. The capture antibody, 266, is specific to amino acids 13-28 of A,~ (Seubert et al. (1992)); while the antibody 3D6, which is specific to amino acids 1-5 of A,(~, was biotinylated and served as the reporterantibody. The 3D6 biotinylation procedure employs the mAmlfArtllrer's WO ~G/~C~96 PCTAJS9G~

(Pierce) protocol for NHS-biotin labeling of immlmnglobulins except 100 mM
sodium bicdll)ulldtc, pH 8.5 buffer was used. The 3D6 antibody does not recognize secreted APP or full-length APP but detects only A,~ species with amino termin~l aspartic acid. The assay has a lower limit of se~iLivily of s a~lo~iludLcly 50 pg/ml (11.4 pM) and showed no cross-reactivity to the endogenous murine A,B peptide at concentrations up to 1 ng/ml.
The configuration of the A,Bl 12-specific sandwich ELISA employs the mAb 21F12, which was gel~eldtcd against amino acids 3342 of A,~. The antibody shows less than 0.4% cross-reactivity with A,BI40 in either ELISA or 0 colll~cLiLivc radioi.~ n~ y (RIA). Biotinylated 3D6 is also the reporter antibody in this assay which has a lower limit of sensiLiviLy of approximately 125 pg/ml (28.4 pM).
The 266 and 21F12 mAbs were coated at 10 ~g/ml into 96-well immlmn~s~y plates (Costar) overnight at rûom Lclll~eldLulc. The plates were then aspirated and blocked with 0.25% human serum albumin in PBS buffer for at least 1 hour at room Lclll~cldLulc, then stored de~irc~t~d at 4~C until use. The plates were rehydrated with wash buffer prior to use. The samples and standards were added to the plates and inruhat~-l at room Lclll~ dLulc for 1 hour. The plates were washed at least 3 times with wash buffer (Tris buffered saline, 0.05% Tween 20) bcLwcen each step of the assay.
The biotinylated 3D6, diluted to 0.5 ,ug/ml in casein assay buffer (0.25% casein, PBS, 0.05% Tween 20, pH 7.4), was incubated in the well for 1 hour at room LclllpeldLulc. Avidin-HRP (Vector, B--rling~m~, CA), diluted 1:4000 in casein assay buffer, was added to ehe wells and inr~lbatt?d 2s for 1 hour at room temperature. The colorimetric ~ub~Lldlc (100 ,ul), Slow TMB-ELISA (Pierce), was added and allowed to reace for 15 mimlt~s, after which the enzymatic reaction is stopped with 25 ~l of 2 N H2SO4. Reaction product was quantified using a Molecular Devices Vmax m~llring the difference in absorbance at 450 nm and 650 nm.
3. APP ELISAs.
Two dirrclc-l~ APP assays were nfili7~ The first recognizes APPa and full length forms of APP (FLAPP+APPa), while the second recognizes W O ~6/~96 PCTAUS96/~

APP,~ (APP ending at the methionine preceding the A,~ domain (Seubert et al. (1993)). The capture antibody for both the FLAPP+APP~x and APP,~
assays is 8ES, a monoclonal antibody raised to a b~ct~ri~lly expressed fusion protein collcs~onding to human APP amino acids ~ 592 (Games et al.).
s The lcpol~el mAb (2H3) for the FLAPP+APPcY assay was gellcl~Lcd against amino acids 1-12 of A~. The lower limit of sel~iliviLy for the 8E5/2H3 assay is approximately 11 ng/ml (150 pM). For the APP~B assay, the polyclonal antibody 192 was used as the reporter. This antibody has the same specificity as antibody 92 (Seubert et al. (1993)), that is, it is specific0 to the carboxy-tcllllillus of the ,~-secretase cleavage site of APP. The lower limit of ~c~iliviLy for the ,~-secretase 8E5/192 assay is approximately 43 ng/ml (600 pM).
For both APP assays, the 8E5 mAb was coated onto 96-well Costar plates as described above for 266. Purified recombinant secreted APP~x (the APP751 form) and APP596 were the rcrclcllce standards used for the FLAPP+APP~ and APP,~ assays, respectively. APP was purified as described previously (Esch et al.) and APP concentrations were ~ d by amino acid analysis. The 5 M gu~nirlinf brain homogenate samples were diluted 1:10 in specimen diluent for a final buffer composition of 0.5 M
NaCl, 0.1% NP40, 0.5 M gll~nillinr. The APP standards for the respective assays were diluted into buffer of the same final composition as for the samples. The APP standards and samples were added to the plate and inrubate-l for 1.5 hours at room temperature. The plates were thoroughly washed between each step of the assay with wash buffer. Reporter antibodies 2H3 and 192 were biotinylated following the same procedure as for 3D6 and were inrllb~t~rl with samples for 1 hour at room temperature. Streptavidin-~lk~lin~ phosphatase (Boehringer Mannheim), diluted 1:1000 in specimen diluent, was incubated in the wells for 1 hour at room L~lllpt;:ldLul~;:. The fluorescent substrate 4-methyl-umbellipheryl-phosphate, was added, and the plates read on a CytofluorTM 2350 (Millipore) at 365 nm excitation and 450 nm emisslon.

4. Monoclonal Antibody Pro~l~rti~ n.
The immnnogens for 3D6, 21F12, and 2H3 were s~pdldlt:ly conjugated to sheep anti-mouse immlmnglobulin (Jackson T,...~.."..,csedl~;h Labs) using maleimicl--h~-x~nnyl-N-hydroxysuccinimide (Pierce). A/J mice 5 (Jackson Laboratories) were given illL d~cliLolleal injections (IP) of 100 ,ug of the a~loplidL~ immlln~gen emlll~ifito~l in Freund's complete adjuvant (Sigma) and two s--bseq~ent IP injections of 100 ,~g immllnr~gen were given on a biweekly basis in Freund's incomplete adjuvant (Sigma). Two to three weeks after the third boost, the highest titer mouse of a given immlln~gen was injected hlLldve~ usly and intraperitoneally with 50-100 ~4g of immnn-)gen in PBS. Three days post injection, the spleen was removed, splenocytes were isolated and fused with SP2/0-Agl4 mouse myeloma cells. The hybridoma SUp~ were screened for high affinity monoclonal antibodies by RIA as previously described (Seubert et al. (1992)). Purified monoclonal antibodies 15 were prepared from ascites.
5. ~mmnnoh;~torh~
The tissue from one brain h~ ph~re of each mouse was drop-fixed in 4% paraformzlkl~hyde and post-f~ed for three days. The tissue was mounted coronally and 40 ~m sections were collected using a vibratome.
20 The sections were stored in anti-freeze at -20~C prior to st~ining~ Every sixth section, from the posterior part of the cortex through the hippocampus, was immnn~st~in~l with biotinylated 3D6 at 4~C, overnight. The sections were then incubated with horseradish peroxidase avidin-biotin complex (Vector) and developed using 3~3~ min~bçn7i~lin~ (DAB) as the 25 chromogen.
B. Rf~clllt.~.
1. A,B and APP Assays.
The FLAPP+APP~ assay recognizes secreted APP inrlll~ling the first 12 amino acids of A,~. Since the reporter antibody (2H3l l2) is not specific to 30 the alpha clip site occurring between A~ amino acids 16 and 17 (Esch et al.),this assay also recognizes full length APP. Preliminary experiments using immobilized APP antibodies to the cytoplasmic tail of full length APP to WO 9~/lC~96 PCT~US96/09857 deplete the llli~ul~ suggest that approximately 30 to 40% of the FLAPP+APP~ is full length. The APP,~ assay recognizes only the APP
clipped imm~ t~ly amino-termin~l to the A~ region due to the specificity of the polyclonal reporter antibody 192 (Seubert et al. (1993)).
s The specific nature of the A~ activity was further characterized as follows. Gll~ni~lin.o homogenates of brain (excluding cerebellum and brain stem) were subjected to size e~rl~ n chromatography (Superose 12) and the reslllting fractions analyzed using the total A,B assay.
Comparisons were made of 2, 4, and 12 month tr~n~genir mouse brain homogenates and a non-transgenic mouse brain homogenate to which A,~l40 had been spiked at a level roughly equal to that found in the 12 month old tr~n~genic mice. The elution profiles of the transgenic brain homogenate were similar in that the peak fractions of A,~ illllllllll~reactivity occurred in the same position, a single broad symmrtric peak which was coincident with the immllnoreactive peak of spiked A,~l40. Attempts were then made to immlmt-~leplete the A~ eactivity using resin bound antibodies against A,~ (mAb 266 against A,~'l328), the secreted forms of APP (mAb 8E5 against APP444592 of the APP695 form), the carboxy-trrmiml~ of APP (mAb 13G8 against APP676695 of the APP695 form), or heparin agarose. Only the 266 resin captured A,~ immunoreactivity, demol~,LldLhlg that full length APP or carboxy-terminal fragments of APP are not contributing to the A,B
measurement. The A,l~,42 ELISA employs a capture antibody that recognizes A~,42 but not A,~,40. The A,BI42 assay, like the total A,B assay, is not affected by the full length or carboxy-terminal forms of APP cont~ining the 2s A~B region in the homogenates as shown by similar immlmodepletion studies.
2. Total A,B and APP Measures.
Table S shows the levels of total A,B, FLAPP+APPcY, and APP,~ in the hippocampus, cortex, cerebellum, and th~l~mn~ of transgenic mice as a function of age. Each data point represents the mean value for each age group. The relative levels of FLAPP+APP~ and APP,B in all four brain regions remain relatively constant over time. The hippocampus expresses the highest levels of FLAPP+APPo~ and APP,~ followed by the th~l~mll~, cortex, W O96/40896 . . : PCT~US96/09857 and cerebellum, respectively. In the hippocampus, the levels of FLAPP+APPcY are approxim~tely 3.5 to 4.5-fold higher tnan APP,~ at all ages. The mean value of all ages for FLAPP+APPc~ and APP,B assays in tne nippocampus were 674 (i465) pmoles/gram and 175 (+11) pmoles/gram, S ~ e~;Liv~ly. From this it can be estim~tP~l that the pool of brain APP
consists of approximately 50% APPo~, 30% full length APP, and 20% APP,~.
TABLE 5. PDAPP T~ ,e Cohort Animal Data Total A,5 & APP Measures in pmoles/gram of Brain Tissue.

AGE INA~ & APPf'F.RFRF.T T UM HIPPOCAMPUS CORTEX THALAMUS
MONTHSFORM
2 A~ 4.03 il.08 35.41 i6.38 14.256.41 il.59 (n=8) (n=8) i2.27 (n=8) (n = 8) 2 FLAPP+ ND ND ND ND
APP~
2 APP,~ ND ND ND ND
4 A,~ 4.10 iO.61 38.08 i6.51 15.957.60 il.52 (n=14) (n=14) i2.60(n=14) (n= 14) 4 FLAPP+ 395 il20 703 il06 446 i706.37 il66 APP~ (n=14) (n=14) (n=14)(n=14) 4 APP,578 i38 (n=14)198 i30 (n-14)126 i23 70 il7 (n= 14)(n= 14) 6 A,~ 4.55 il.3887.48 i30.33 30.198.34 i2.40 (n=10) (n=10) i8.33-(n=10) (n = 10) 6 FLAPP+ 403 +77 694 ilO7 506 i97670 il56 APPa~ (n=10) (n=10) (n=10)(n=10) 6 APP,~ 51 i87 (n=10)194 i35 (n=10) 129 i25 56 i33 (n= 10)(n= 10) 6.5 A~ 5.42 il.08133.63 i57.10 33.278.83 il.l9 (n= 10) (n= 10) i 12.19(n= 10) (n = 10) 6.5FLAPP+ 346 i74 580 i 115 436 i63553 i 123 APP~ (n= 10) (n= 10) (n= 10)(n= 10) 6.5 APP,~27 i77 (n=10)169 i41 (n=10) 108 il6 58 i22 (n = 10)(n = 10) 7 A,~ 4.44 i0.56200.77 i94.68 60.558.94 il.l9 (n=10) (n=10) i27.13(n=10) (n= 10) WO 9G/4~96 PCTAUS96/09857 7 FLAPP+378 i70656 i73 (n=10)469 i62604 ilO7 APP~(n=10) (n=10) (n=10) 7 APP,~56 i52 (n=10) 176 i27 (n=10) 101 i20 56 i28 (n= 10)(n= 10) 7.5 A,~5.14 il.39461.35 i345 95 81.839 10.84 iS.22 (n=10) (n=10) iS3.00 (n=10) (n= 10) 7.5 FLAPP+362 iS4554 i77 (n=10)409 i44503 i80 APPcr(n=10) (n=10) (n=10) 7.5 APP,~20 iS8 (n=10) 168 i27 (n=10) 118 i21 57 i22 (n = 10)(n = 10) 8 A,~4.42 iO.73635.52 i302.45 128.68 10.87 i3.39 (n= 13) (n= 13) i62.80 (n= 13) (n= 13) 8 FLAPP+386 iS2 660 ilO2 494 i87672 ilSO
APPCY(n=13) (n=13) (n=13) (n=13) 8 APP~64 i77 (n=13) 174 i27 (n=13) 102 i26 57 i30 (n= 13)(n= 13) 8.5 A,65 54 il.ll633.11 i363.14 118.39 13.96 i7.34 (n=10) (n=10) iS9.91 (n=10) (n = 10) 8.5 FLAPP+439 i79 764 ill4 558 i80750 il32 APP~r(n = 10)(n = 10) (n= 10)(n = 10) 8.5 APP,~28 iS9 (n=10) 185 i34 (n=10) 108 i42 47 i28 (n= 10)(n= 10) 9 A,~5.52 il.ll1512.39 254.8319.46 i8.99 (n = 10) i624.286 i 105.927(n= 10) (n = 10) (n = 10) 9 FLAPP+500 ill2763 il25 549 i78815 il67 APPCY(n = 10)(n = 10) (n = 10)(n = 10) 9 APP,64 i83 (n=10) 169 i25 (n=10) 121 i32 49 i26 (n = 10)(n = 10) A,~4.04 il.O22182.21 343.49 15.46 (n=ll) ill94 49 il65.531il3.38 (n=ll) (n=ll) (n=ll) FLAPP+452 il30678 i93 (n=ll)491 ilO2 693 il66 APPCY(n=ll) (n=ll) (n=ll) APP,652 i32 (n=ll) lS9 i22 (n=ll) 87 ilS 46 ilO
(n=ll) (n=ll) 12 ~3.26 iO.35 4356.23 691.17 18.08 (n=9)il666.44 (n=9) i360,93il3.50 (n=9) (n=9) 12 FLAPP+385 il66638 i272 444 il71708 i278 APP~(n=10) (n=10) (n=10) (n=10) CA 02222174 1997-ll-24 WO 96/40896 PCTrUS96/09857 12 APP~ ¦ 41 +29 (n=10) ¦ 134 +47 (n=10) ¦ 76 i31 ¦ 35 ~19 ll ¦ (n=10) ¦ (n=10) ¦¦
~D = not~ d In contrast to APP levels, A,B levels increased dr~m~tir~lly with age in the _ippocampus and cortex. However, no such increase was noted in the - cerebellllm of the PDAPP Lldnsg~nic mice, and only a moderate increase was seen in th~l~mll~ (Table 5). The increase of A,B is greater in t_e hippocampus relative to the cortex, which also correlates with the 3D6 immllnohi~toch~mir~l results (see ~liccll~inn below). Compared to the cortex levels of 4 month old mice, A,B levels increase 10-fold by 8 months of age and 41-fold at 12 months old (660 + 380 pmoles A~B/gram tissue at age 12 months). The corresponding i~ ,ases in A,B observed in hippocampus are o even more il~ ssive, as the 8 month value is 15 times that at 4 months oldand increases to 106-fold at 12 months old (4,040 ~ 1750 pmoles A,B/g tissue at 12 months). At 12 months of age, A,~ co~ e~, appr~ xim~t~ly 1%
of protein in hippocampus of the PDAPP mice.
To see if the dramatic rise in brain A,B concentration is due to amyloid deposition, we next vi.~;u~1i7.-.cl A,B deposits immllnohistorh~mir~lly,using the opposite hemisphere of the same mice used for A,~ measurements.
Notably, a parallel increase in A,B plaque burden and A,~ level exists. These fin~1ing.~ strongly argue that the rise in brain A,B concentration llrt~rmin~ byELISA is due to the age-dependent amyloidosis.
3. A~ 2 Measures in Tl~lsge,lic Mouse Brain.
Concentrations of A~l42 in the cortex of Lldnsgt;nic mice were evaluated at dirr~ ages. As shown in Table 6, the percentage of A,B
which is A,~l42 in the cortex of transgenic mice, also increases with age. The ELISA data suggest that A~l42 is preferentially depositing in the transgenic mice, and that the deposits ~letecterl by mAb 3D6 immnnosf~ining are primarily A~l42.
Table 6. A,Bl42 Levels in the Cortex of Transgenic Brain.
Age (months) A,~l42 (pmoles/g) 4 4.71 8 75.65 CA 02222l74 l997-ll-24 W Og~ r~6 PCTrUS96/09857 247.43 12 614.53 4. A~B Tmmnnn,~ in PDAPP T~ ic Brain.
Transgenic ~nim~l~ with A~B values ~ sellLil,g the mean A~ value of the age group were used for 3D6im mlmost~ining. A progression of A~
deposition is seen in the 4, 8, 10, and 12 months old ~nim~l~. At four months of age, transgenic brains cont~in~-l small, rare punctate deposits, 20 ,um in rli~mtoter, that were only infrequently observed in the hippocampus and frontal and cingulate cortex. By eight months of age, these regions contained o a number of thioflavin-positive A~ aggregates that formed plaques as large as 150 ~m in ~ m~ter. At ten months of age, many large A,~ deposits were found throughout the frontal and cingulate cortex, and the molec3ll~r layers of the hippocampus. The outer molecular layer of the dentate gyrus receiving rO~ pathway arrelellL~ from the t:llLoll~ al cortex was clearly delin~te~
s by A,B deposition. This general pattern was more pronounced by heavier A,B
deposition at one year of age. The ~n~tomir.~l loc~li7~tion of A~B deposition is l~ .k;1~1y similar to that seen in Alzheimer's disease.
C. Discussion.
A,B arnyloidosis is an established diagnostic criteria of Alzheimer's disease (Mirra et al., Neurology 41:479486 (1991)) and is con~i~t~nrly seen in higher cortical areas as well as the hippocampal formation of the brain in affected subjects. It is believed that A~ amyloidosis is a relatively early event in the pathogenesis of AD that subsequently leads to neuronal dysfunction and ~lem~nti~ through a complex cascade of events (Mann et al., Neurodegeneration 1:201-215 (1992); Morris et al., Neurology 46:707-719 (1996)). Vario~s pat~hways of APP processir.g have been described (reviewed in Schenk et al., J. Med. Chem. 38:4141-4154 (1995)) including the major cY-secretase pathway where cleavage of APP occurs with A,~ (Esch et al.) and the amyloidogenic or ,B-secretase pathway where cleavage of APP occurs at the N-terrnin--~ of A,~ (Seubert et al. (1993)). Further cleavage of APP leads to the constitutive production of A~ forms including those ending at position 40 (A~5l 40) or 42 (A,Bl~2). ELISAs that detect specific APP products arising CA 02222l74 l997-ll-24 WO ~6/1~996 : PCT~US96/09857 from these individual lJaLllwdy~7 in the PDAPP mouse brain allow ,."i",.linn of whether dirrel~llLial processing of APP co~ ibuLes to the regional or l~lllpoldl specificity of amyloid formation and deposition.
A,~ amyloid deposition seen in the PDAPP mouse brain is highly age and region specific. Amyloid deposition begins at around 7 months of age, and by 12 months of age, amyloid deposition is very ~loroulld throughout the hippocampus and in the rostral region of the cortex. The age dependent increases in amyloid deposition correlate well with the drarnatic rise in A~B
levels in these brain regions as measured by ELISA assay. An increase in o A,B is measurable by 7 months of age and by 10 months the hippocampus as 2180 pmoles/g of A,~, a concentration equivalent to that of my cytoskeletal s and comparable to the levels found in the cortex of human AD brain (Gravina et al., J. Biol. Chem. 270:7013-7016 (1995)). A~ levels in the cerebellum, an unaffected brain region, still are at 4 pmoles/g -- ess~onti, lly~ rllAl~ged relative to the levels at 4 months of age -- again correlating with amyloid deposition measured by histological analyses. These results in-lirAt~
that in aged PDAPP mice, brain A,~ levels reflects amyloid burden and therefore direct immllnnAs~"y measurement of brain A,~ levels can be used to test for compounds that reduce amyloid plaque burden.
In the PDAPP mouse, individuals suffering Down's Syndrome, and individuals with certain forms of FAD, overproduction of A,B is almost certaimy an accelerating factor not only in A,B deposition but in subsequent neuropathology (Citron et al., Mann et al., Miller et al., Archives of Biochem. Biophys. 301:41-52 (1993)). A comparison of the A,~
measurements seen in the PDAPP mouse with those reported for AD brain tissue reveals several striking similarities. For example, in the PDAPP
mouse, the relative levels of A,B peptide in hippocampus from young (2 months of age) versus old (10 months of age) mice is nearly a hundred fold.
Similar finfling~s were noted by Gravina et al. in colllpal"lg control brain tissue relative to that of AD. The rise in brain A~ levels in the PDAPP
mouse is rather pronounced between the ages of six to nine months of age.
Again, this timecourse parallels, in an accelerated manner, that seen in CA 02222174 1997-ll-24 Down's SylldLume brain tissue, where amyloid deposition begins at a~ xi~ t~ly 30 years of age and increases subst~nti~lly until a~ro,~ aL~ly age 60 (Maml).
In s lmm~ry, the above results show that a reproducible hlcl~ase in 5 m~ nr~ble A,~ occurs in the brain tissue of the PDAPP mice and that this increase correlates with the S~ ily of armyloid deposition. These ri.~
in~ te that these mice can be used to identify agents or compounds that ph~rm~rologically reduce A,B peptide production or affect its deposition.
Example 9: Behavioral Differences in PDAPP Tr~ ..ic Mice.
o Alzheimer's disease is characterized by cognitive deficits inrlllrling memory loss, and imp~irment of memory functions. To cl~ot.ormine if the disclosed transgenic mice exhibit similar deficits, LldLISg~ C (TG) and non-transgenic (nTG) mice were evaluated for task perfcllllallce in three types of maze a~aldLus used to test working and reference memory; the Y maze, the radial arm maze (RAM), and the water maze. The transgenic mice tested le~ selll the fifth gt~ dLion derived from the PDAPP mice described in Example 6. The Y maze and the radial arm maze are used to assess spontaneous ~ltern~tion which is a function of working memory. For the Y
maze task, the mouse is placed in the stem of a Y maze twice, each time allowing a choice entry into one of the arms. Entering both arms is a successful alternation, requiring memory of the previously entered arm, while entering the same arm on both trials is a failure. Chance ~clÇollllallce is 50% alternation, that is, 50% of the mice alternate.
For the radial arm maze task, the mouse is placed at the center of a maze with multiple arms r~ ting from the center. In the testing described below, a radial eight-arm maze was used. ~ltern~tion perfollllallce is measured by allowing only eight entries, with the number of dirrel~ arms entered being the measure of performance. The number of different-arm entries can be compared to the number of different-arm entries expected by chance, which is 5.25 (Spetch and Wilkie, "A program that stiml-l~tt~s random choices in radial arm mazes and similar choice situations" Behavior ~esearch Methods & Instrumentation 12:377-378 (1980)). Performance WO 96/40896 : PCT~US96/09857 above chance, that is, above 5.25, requires memory of the previously entered arms.
The water ma_e used for the tests described below co~isL~ of a pool of water in which a submerged platform is placed. This hidden plafform 5 (HP) can be found by ~wil~ g mice either by chance (first trial) or through memory of positional clues visible from the tank (sl-hseqn~nt trials). Subject mice were trained in the hidden platform task according to standard procedures. Briefly, mice were first pretrained in a small pool (47 cm mt~ter~ 20 cm platform), which teaches them how to navigate in water, lO that the platform is the goal, that there is no other escape, and that to find it they must resist their natural inf lin~tion to stay along the sides of the pool.They were then trained to find a single platform position in the hidden platform task using a larger pool and smaller platform (71 cm pool, 9 cm platfform).
During the HP task, visual cues were located inside the pool (intram~7~ cues; black pieces of cardboard - circle, plus, or hol~ollL~l lines -located in three quadrants at the top of the wall, which was 38 cm high above water level), and various room cues were visible outside the pool (e~Ll~lla_~
cues).
The mice accign~-l to the chara-;Le~ Lion cohort study were tested on the behavioral tasks described above over 3 days during the week or two before ellthanacia. Their transgenic status was not known to the tester. Non-transgenic littermatt~s were used for cnmp~ricon. Each morning the subject mice were run in the Y maze and RAM as described above. They were then 2s tested for general strength on the inclined plane (INP) test. For this, micewere placed in a 10-cm-wide runway lined with ridged plastic and elevated with the head up at 35~. The angle was then increased until the mouse slid off, and the angle was recorded. This was repeated three times each day.
The average scores for the three days were calculated for each mouse for the Y maze (0=repeat, l=alternate), RAM (number of dirrelc;ll- arms and time to finish, 10 minute limit), and INP (average of all nine trials). General activity was also rated on the first day of testing. Each mouse was observed CA 02222174 1997-ll-24 WO 9C/~0~96 PCT~US96/09857 in the cage, and picked up and held. A mouse that rem~inr~ calmly in the hand was scored 1, with progressively greater activity and reaction to h~ntllin~ scored up to 4.
Following the above tests each day, mice were tested in the water 5 maze as described above. Briefly, mice were pretr~inr-l in a small pool to climb on a large submerged platform as their only means of escape from the water. They were then given six blocks of four trials each to learn the location of a small platform in a large pool. For analysis, all four trials within each block were averaged. The exception was the first hidden 10 platform block, for which only the last three trials were averaged. The firsttrial was analyzed s~l~dldL~ly, because it is the only one for which platform location could not be known, and thus did not relate to spatial l.-~rning. It isthus used as a control for non-spatial factors, such as motivation and ~w;.,....i.-g speed. The p~lroll-lal-ce effects between blocks were analyzed as 15 a repeated measure for the hidden platform task. Standard analysis of v~ial~ce (ANOVA) c~lr~ ti~ ns were used to assess the ~ipnifir~nre of the results.
Results in the RAM show that TG performed si~..;r.r~..lly worse than nTG across all ages (Group effect: p=0.00006). The time to finish was also 20 ~ignifir~ntly different between TG and nTG mice (Group effect: p=0.005).
The correlation between the time to finish and the number of arms chosen was small (R= -0.15, p=0.245 in each group). This suggests that the con~i~t~nt imp~irmt~nt in the RAM is not accounted for by the increased time to complete the task taken by TG mice. Results in the Y maze were also 25 significantly different for TG and nTG mice (Group effect: p=0.011).
Validation studies performed on non-transgenic mice intlir~te that the Y maze is a less sensitive measure than the RAM.
Measures of strength (INP) and activity in-lic~te no differences between TG and nTG mice. These are considered very rough measures, with 30 only large differences being ~etrct~hle. There was, however, a decrease in the activity score for all mice over time (Age effect: p=0.070). There was a difference in body weight, with TG weighing 8% less than controls (Group WO ~6/40~96 ~ PCTAUS~6~ 357 effect: p=0.0003), primarily in female TG mice. However, this does not seem to have an effect on the results, as shown by the lack of any dirrclcllce in ~Lle~l (see above) or ~Willllllillg speed (see below) bc~wccll TG and nTG
mice.
Results of the hidden platform task, considered here a test of lcrclellce memory, show a co~ Lcll~ dirr~.cllce bcLwcell TG and nTG mice.
ANOVA reveals that the effects of transgenic status (Group effect:
p=0.00016) and trial blocks (Block effect: p<0.00001) are .cignifir~nt The effect of transgenic status on pclrollll~llce is accounted for by slower o ~clr . ,~.~re by TG mice across all trial blocks and ages. Analysis of Trial 1reveals an effect of Ll~lsgcllic status (Group effect: p=0.018), suggesting a dirrclellce in pclrollllance before le~rning has occurred. However, an analysis of covariance, with trial 1 as the covariate, still yields a .cignifir~nt deficit in TG mice (p=0.00051).
It was also possible that some physical dirrelc,~ces between TG and nTG mice, rather than cognitive dirrclcllces, could have been responsible for some of the ~clrollll~lce differences seen in the water maze tasks. However, no cignifir~nt dirrclcllce in strength or activity was observed (see above).
Another possibility considered was the effect of ~willllllillg speed on pclrollllallce since a slower swimmer with equivalent cognitive ability would take longer to reach the platform. To test this, video tracking was used in the hidden platform task to measure the ~lict~nre travelled to reach the platform (a measure of the amount of sealchillg done by the mice which is related to cognitive ability), the swimming speed (a measure of physical ability unrelated to cognitive ability), and the amount of time need to find theplatform (a measure of the combination of both the ~ t~n~e travelled and the ~willllllillg speed). This was done in older and younger mice than reported above, using three trials per block and no pretraining. The time needed to find the platform was cignifir~ntly dirrclcllL in TG and nTG mice (Group effect: p<0.0005), with the TG mice taking longer. However, the swimming speed was not cignifir~ntly dirrclcllL between TG and nTG mice (Group effect: p=0.879). Thus, the dirr~lcnce in time needed to find the W O 96/40896 PCT~US9G~

platform is likely to be due to a cognitive dirrel~nce beLw~ell TG and nTG
mice. This is confirmPd by measures of the ~ t~nre travelled to find the platform. The TG mice travelled .~i~nifir~ntly further than the nTG mice before reaching the platform (Group effect: p<O.OOOS). These results imliratP, that the dirrelcllces seen between TG and nTG mice in the time to reach the platform in the water maze tasks are due to ~lirr.,lel.ces in cognitive ability.
To test whether nTG mice retain a better memory of the platform location than TG mice, a probe trial was given immP~ tPly following hidden o platform training in which the platform was removed. Video tracking was used to dcl~ P the number of crossings of the former platform location made by the mice relative to crossings of non-platform locations. There was a signifir~nt dirre~cllce seen between the relative crossings of TG and nTG
mice (Group effect: p=0.006). This is evidence that the nTG mice S remember the former location of the platform better than TG mice.
It was also possible that the dirre~ ce observed between TG and nTG
mice in the time needed to reach the platform could have been infl-nPnre~l by dirr~r~lces in p~lct;~lion of the cues or motivational dirÇele-lces. To test this, TG and nTG mice were subjected to visible platform tasks in the water maze. For these tasks, a platform was placed in the pool so that it was visible above the water. Three different platforms were tested, a dark platform 25 mm above the surface (most visible), a gray platform 25 mm above the surface, and a dark platform 5 mm above the surface (both less visible). The results show no difference in the time to find the most visible platform between TG and nTG mice (Group effect: p=0.403). There was not any greater decrease in performance in TG mice when less visible platforms were used, suggesting that their vision was as good as nTG mice.
These results indicate that ~el.;epLual and motivational differences do not infln~Pnre the time to reach the platform in the water maze tasks described above.
P~lrollllance dirre.~nces between TG and nTG mice were shown for RAM, Y maze, and water maze cognitive tasks in mice aged 4 to 8 months -WO ~G/1a~9~ PCTrUS96/09857 (2 to 12 months for the water maze). All of these dirr~ ces in~lir~tr, and are concictrnt with, cognitive deficits in the transgenic mice as a group. The various tasks combined to test working memory and lc;r~l~nce memory, both of which are implicated in cognitive impainnrnt observed in ~l~ l's s victims.
~nr'~ 10: Detecti-n and Mea~u,~ l of ~l~hrimrr~s Dise~se Markers.
A. Dete~tion and Mea~u~ lll of GFAP.
Glial fibrillary acidic protein (GFAP), a marker which increases in o AD brain tissue, was measured in the following manner. Tissue extracts were p~ d from hippocampi of control and PDAPP transgenic mice, as described in Example 6, aged 14 months. Tissue was sonicated in 10 volumes (v/w) of 10 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.1 mM PMSF, 10 ~g/ml lt:u~liul, 5 ~ug/ml calpain inhibitor 1. Protein 1~ ionc were made on the extract and SDS-PAGE sample buffer added before boiling the samples for S mimltec. SDS-PAGE was pc~lroluled using 12.5 ~g of protein of each sample loaded onto 10% Tris-glycine gels (Novex). The ~lu~eins were ~-dl~r~ d to Pro-Blot PVDF membranes by standard methods. GFAP immlmt reactive proteins were fletecte~l using an anti-GFAP antibody from Sigma (G9269) used at a dilution of 1:2,000. An increase in immnn-)reactivity in general was observed, and a smaller anti-GFAP reactive species was also found to increase subst~nti~lly, in the transgenic animals. In non-transgenic animals, this approximately 40 kD
fragment gave a mean densitometer signal of 142.47, while in the transgenic ~nim~lc, it gave a mean densitometer signal of 591.51. This difference was .cignffilr~nt with a P value of 0.0286.
B. Dete~tioll of Gliosis.
Gliosis is one of the changes that is associated with the neuropathology of Alzheimer's disease. The isoquinoline carboxamide PK
11195 has been shown to be a ~lefelellLial marker of the peripheral benzodiazepine sites associated with gliosis. These sites have been shown to be enh~nre~l in several ~lice~ces and animal models associated with nt:uiul~al WO 9C/1-~6 PCT~US~G/u5~3/

damage and activated necroglia including stroke (Stephenson et al., J.
Neurc!sci~onre 15:5263-5274 (1991)) and ~ ;lllel~s disease (Diorio et al., Neurobiology of Aging 12:255-258 (1991)). In particular Diorio and coll~ s have shown an a~pr~ ;""3le 200% h~ ase in [3H] PK 11195 binding in some brain regions of AD patie,l~, such as the ~ l cortex cc,lll~d with age-matched controls. In this example, the brains from the PDAPP mouse, described in Example 6, were ~x~ i for qll~lit~tive and q~ livt; changes in the binding of [3H] PK 11195 in order to correlate with the previously described AD disease pathology. Two ~iirr~le~
0 approaches were lltili7~cl; radioreceptor binding to homogenates of dirrtlc.ll brain regions and receptor autoradiography.
1. l~th".l~.
For the homogenate binding studies, PDAPP mice were e~lth~ni~e~l by cervical dislocation and the brains rapidly ~ sected on ice. Homogenates (10 lS mg/ml wet weight) of cerebral cortex, hippocampus and cerebellum were prepared in 50 mM Tris HCl, pH 7.4. 0.3, 1.0 and 3.0 nM [3H] PK 11195 was inrnb~ttorl with these brain regions for 60 mimltec at 23~C followed by rapid filtration over Whatman GF/B filters using a Brandell cell h~ ./e~ r.
Non-specific binding was ~l~tcrmin.o-l using 1 ,uM unlabelled PK 11195.
20 Qll~ntit~tion was performed by liquid scintillation spectrometry.
In the autoradiographic studies, PDAPP mice were ellth~ni~ed using carbon dioxide, the brains removed and snap frozen in methyl butane/dry ice.
The brains were sectioned in the coronal plane through the hippocampus.
Twenty micron thick sections were mounted on glass slides and stored at 2s -20~C. Sections were incubated at 1 hour at 23~C in 170 mM Tris-HCl, pH
7.4 cont~inin~ 1 nM [3H] PK 11195. Non-specific binding was determined using 1 ,uM unlabelled PK 11195. Tnrllbations were termin~tr~l by rinsing sections twice for 5 mimltes in ice-cold inrub~tinn buffer followed by a brief wash in ice-cold distilled water. Following rapid drying, sections were 30 exposed to tritium Hyperfilm (Amersham International) for up to 5 weeks.
2. R~e--lt.~.

CA 02222174 1997-ll-24 W O9G/~C8~6 PCT~US96/09857 ~ our hc~clvzy~,uus transgenic mice 34 months of age were evaluated in the homogenate binding studies and co~llpalcd with litter-mate controls.
No .ci~nifir~nt differences were observed between any of the brain regions of the transgenic animals and their respective controls. t3H] PK 1119 autoradiography was performed to compare binding in a 12-month old heterozygotic tr~ncgenic mouse with an aged-m~trhrd non-L.cu,cgellic control.
Prel;.,.;..,..y results from an autoradiogram exposed for five weeks in~lirs~tr-l that several plaque-like structures were labeled in the retrosplenial cortex of the L-~sgenic mouse, a region that invariably contains A,B deposits. The o pattern of labeling corresponded to microglial cell or astrocytic clumps associated with plaques, rather than the more widespread pattern of astrocytosis or microgliosis in the hippocampal and cortical parellcllyula.
The non-transgenic mouse did not show this labeling pattern.
No changes were observed in the 3-4 month animals but some 15 evidence for an increase in [3H] PK 11195 binding was seen in the 12-month animal.
C. Det~cti~n and Mea~u~ of Cholinergic Nerve Termin~
A population of cholinergic neurones projecting to the folcbldill have been shown to be selectively decreased in the postmortem brains of patients tii~gnosed with Alzheimer's disease. ~omirhnlinium-3 is a potent inhibitor of high affinity choline uptake and has been shown to be a good marker of cholinergic nerve terminals (Pascual et al., J Neurochem 54:792-800 (1990)).
The total number of high affinity choline uptake sites in PDAPP l~nsge~c animals, which are described in Example 6, has been measured using both crude whole-brain preparationc and homogenates from selective brain regions using the selective ligand [3H]-Wemichnlinium-3 ([3H]HCh-3).
[3H]-~emichnlinium-3 binding was determinP-l using a modification of the methods described in Pascual et al. Mice were ellth~nice-l by asphyxiation with carbon dioxide and the brains rapidly removed and tiicsect~d on ice. The cortex, cerebellum, striatum and hippocampus were homogenized in 5 ml of 10 mM phosphate buffer without NaCl. Samples were spun at 17,000 x g for 10 mimlt~?c, and the pellets washed twice in 5 ml WO 96/40896 PCTAJS96/098~77 10 mM PO4 buffer. The final pellet was le~us~cllded in 5 ml lX phosphate buffered saline (PBS) to produce a protein collcellLlaLion of 0.5 mg/ml.
Brain regions were assayed in triplicate for high affinity choline uptake sites by the addition of [3H]HCh-3 (3 nM final collcellLlalion). Following a 20 5 minute inf~lb~tion, assays were t~~ l by rapid filtration through Whatman GF/B filters using a Brandell cell hal~ 7~ and w~sl~illg with PBS.
Filters were Lldl~rwlcd into scintillation vials, and specific binding estim~t~dby liquid scintillation spectrometry.
D. Det~ti-n and Mea~ulc~ L of Sodium-r~ ... ATPase.
Ouabain has been shown to bind specifically to high affinity sites in m~mm~ n brain and that these sites correspond to a neuronal form of sodium-potassium ATPase (Na/K-ATPase; Hauger et al., J Neurochem 44:1709-1715 (1985)). These sites have been shown to decrease in animal models of neurodegenerative ~ e~es. Al_heimer's disease is char~cteri7e~1 15 by massive neurodegeneration (DeLacoste and White, Neurobiology of Aging 4(1):1-16 (1993)).
In order to estim~te the extent of neurode~ Lion in the PDAPP
mouse the binding of ouabain was tit~termin~(l in mouse brain homogenates.
Methods were adapted from tnose described by Hauger et al. Brain tissue 20 was homogenized in 100 mM Tris HCl, pH 7.4 cont~inin~ 200 mM NaCI
and 10 mM MgCl2 and resuspended in assay buffer to produce a final concentration of 100~ g protein per assay. Specific binding was determined with 1 to 200 nM [3H] ouabain in a solution of 5 mM ATP, 100 mM Tris HCl, pH 7.4, 10 mM MgCl2, and 200 mM NaCl, inr~lbatPcl for 30 mimlt~s at 25 37~C. Non specific binding was determined in the presence of 100 mM
ouabain and the absence of ATP. Assays were termin~te~l by rapid filtration over Whatman GF/B filters. Tubes were washed with ice cold 50 mM Tris HCl, pH 7.4, 15 mM KCl, 5 mM MgCl2. Filters were Lldl~r~ ,d into scintillation vials, and specific binding estim~tecl by liquid scintillation 30 spectrometry.
Measurements of Na/K-ATPase and Mg-ATPase activity in brain tissue of PDAPP transgenic mice of various ages and in non-L.ansg~l~ic W O9G/4~896 PCT~US96/09857 control brain tissue. These results show some ~ignifil~nt dirr~lcllces in activity between transgenic and non~ ns~,t;nic samples in older mice. Mouse brain homogenates from 4, 8, and 12 month old PDAPP transgenic (TG) mice, and from non-transgenic (nTG) mice, were prepared and assayed generally as described above. The activity of Mg-ATPase was also The results are shown in Tables 7 and 8.
Table 7. Na/K-ATPase Activity in PDAPP Tr~,~ lic Mouse Brain.
Age Tissue Na/K-ATPase o rate % of nTG
(pmole Pi/mg protein/min) 4 TG hippocampus2.57iO.62 88il4 4 nTG hippocampus2.53iO.51 4 TG cortex 0.57iO.13 72i9 4 nTG cortex 0.77iO.17 4 TG cerebellum1.39iO.17 85i 14 4 nTG cerebellum2.14iO.53 8 TG hippocampus4.63il.72 153i53 8 nTG hippocampus2.87iO.41 20 8 TG cortex 1.16iO.08 121i23 8 nTG cortex 1.15iO.20 8 TG cerebellum 2.74iO.81 183i53 8 nTG cerebellum1.47iO.13 12 TG hippocampus1.66+0.36 58i9 25 12 nTG hippocampus 3.11iO.94 12 TG cortex 1.45+0.40 lO9il7 12 nTG cortex 1.60iO.46 -12 TG cerebellum1.43iO.32 74i7 12 nTG cerebellum2.04iO.64 CA 02222174 1997-ll-24 W O ~6/~ 6 PCT/U~,GI'03 Table 8. Mg-ATPase Activity in PDAPP T~ Mouse Brain.
Age Tissue Mg-ATPase rate % of nTG
(pmole Pi/mg protein/min) 4 TG hippocampus 2.83~0.38 110+14 4 nTG hippocampus 2.44+0.54 4 TG cortex1.74+0.14 92:~5 4 nTG cortex1.87+0.18 o 4 TG cerebellum 2.58 ~0.44 100 + 12 4 nTG cerebellum 2.59+0.35 8 TG hippocampus 3.28+0.69 99+22 8 nTG hippocampus 3.32+0.39 8 TG cortex1.72~0.11 73+6 15 8 nTG cortex2.40iO.31 8 TG cerebellum 3.19 +0.49 113 + 15 8 nTG cerebellum 2.77+0.23 12 TG hippocampus 1.48+0.21 65+7 12 nTG hippocampus 2.33 ~0.46 20 12 TG cortex1.60+0.30 76+7 12 nTG cortex2 06 +0.40 12 TG cerebellum 1.61 +0.26 78 +8 12 nTG cerebellum 1.93+0.99 The difference in Na/K-ATPase activity between Ll~ulSgel~lC and non-25 transgenic tissue is ~ignific~nt (p < 0.05) in the case of 12 month old cerebellum, and is highly ~ignifir~nt (p < 0.01) in the case of 12 month old hippocampus The difference in Mg-ATPase activity between transgenic and non-transgenic tissue is ~ignifir~nt (p < 0.05) in the case of 8 and 12 month old cortex, and is highly ~ignifi/~nt (p ~ 0.01) in the case of 12 month old ~ 30 hippocampus E. In situ Hybri~ tion with Probes to Neur~ ol,hic Factors.

CA 02222l74 l997-ll-24 WO ~6/10896 PCT~US96/09857 The use of in situ hybridization to detect and localize mRNAs for specific gene products is well ~locnm~ont~l in the liL~la~ulc (Lewis et al., Molecular Imaging in Neuroscience: A Practical Approach (New York, Oxford U.~ ..iLy Press. 1st ed., 1-21, 1993), Lu and Gillett, Cell Vision 1(2): 169-176 (1994), Si~ hji and Dunnett, Molecular Imaging in Neuroscience: A Practical Approach (New York, Oxford Ulli~ y Press.
lst., ed. 43-67, 1993), Lawerence and Singer, Nuc. Acids Res. 13:1777-1799 (1985), Zeller and Rogers, Current Protocols in Molecular Biology (New York, John Wiley and Sons. 14.3.1-14.5.5, 1995)). For illu~dlive 0 purposes, the specific example described below utilizes 35S-radiolabeled oligodeoxyribonucleotide probes to detect BDNF mRNA in cryostat sectioned mouse brain from the PDAPP tr~n~geni~ mouse described in Example 6 and non-transgenic control mice. However, 33P-labeled r~ rtive DNA probes, as well as in vitro transcribed complemF~nt~ry RNA probes, could be used as well. Non radioactive probe labeling methods may also be used (Knoll, Current Protocols in Molecular Biology (New York, John Wiley and Sons.
14.7.1-14.7.14, 1995)). Additionally, the choice of tissue pre-tre~tm~nt for hybridization with probe (for example, p~rr~l embedded sections) and post-hybridization washes depend on the method used, examples of which are described in the references cited above. Known and a~luL,liate precautions against RNase co..~ ion should be employed and are also tli~c~lssed in the above references.
1. Tissue Preparation.
Freshly fli~sect~(1 whole brains, or sub-regions of interest, from 2s transgenic or control mice at various developmental stages, or post-natal ages, are snap frozen in isopentane pre-equilibrated to -70~C. If desired, the animals may be perfused with PBS to elimin~te circlll,.ting cells from brain prior to dissection. The brains are removed following 15 to 20 seconds immersion in isopentane, wrapped in al..,..i..",.. foil, labeled a~lu~liately, 30 and stored at -80~C for sectioning. It should be noted that although the signal from in situ hybridization to cryostat sectioned tissues is more sensitive than to ~drr"l embedded sections, it is dependent upon the time from WO 96/40896 PCTAJS~610~3 ection to hybridi~Lioll. Frozen tissue is preferably analyzed by hybridization with probe within six weeks. l~NA hlL~ liLy in tissues declin~s beyond this time. Thus, if longer time periods between ~ ction and analysis are antirirat~ the tissue should be fixed (see, for example, Lu and Gillett) before long term storage at -80~C.
Prior to sectioning, Probe-On-Plus glass slides (Fisher Scientific, PiLL~bul~ PA) can be made RNAase free by overnight soaking in absolute ethanol, air dried briefly in a dust free en~dlol~llellL, and baked at 180~C fora Illillillllllll of 4 hours. After cooling to room ~ ldlUlC, the slides are l0 coated with 0.01% poly-lysine (prepared in DEPC treated H2O) for a~ro~ lately S seconds, and air dried in a dust free area. The coated slides can be stored for up to one month before use in a slide box with silica gel or drierite pellets.
For sectioning, the frozen brain stored at -80~C is L.dl~Çell.,d to a cryostat at -20~C, mounted onto a sectioning block, embedded in OCT.~, and allowed to equilibrate. The tissue is then cut into 7 to 14 ,~Lm thick sections using a sterilized microtome knife (treated with 70% EtOH in DEPC H2O), and thaw mounted onto poly-lysine coated slides. The slides are kept at -20~C until the sectioning is complete. The sections are fixed and dehydrated 20 by illllel~hlg the slides sequentially in the solutions noted below.
1. once in 4% paraform~ yde, lX PBS, pH 7.4, at 0~C for S
minutes (this solution should be made fresh, and can be stored for up to 1 week at 4~C);
2. twice in lX PBS, 2.5 minutes each time;
3. once in 50% EtOH in DEPC H2O for 5 minutes;
4. once in 70% EtOH in DEPC H2O for 5 ",i"~c;
5. once in 95% EtOH in DEPC H2O for 5 ,..i"~ s;
The fixed sections are stored immersed in the 95% EtOH/DEPC H2O
solution at 4~C until use. If the sections are not fixed imm~ tely, they may be stored at -80~C in the presence of drierite until use. In this case the sections are allowed to equilibrate to room temperature prior to the fixation/dehydration steps.

CA 02222174 1997-ll-24 W 0~6/~0~96 PCT~US96/09857 2. Probe Design and Pl~dLion.
The sequence of the mouse BDNF mRNA/cDNA (accession #55573) is available from the Genbank ~l~t~h~e of Nucleic acid seq~ rçs (NCBI, RethPs-l~, MD). Anti-sense oligodeoxynucleotide probes against BDNF were ~leci~nP~l using t_e primer select module of the DNAstar~ sor~wal~ package (Lasergene Inc., Madison, WI). Nulllcl~ us other software p~ gPS, such Oligo~ (NBI, Plymouth, MN), offer similar capabilities, and are also suitable. C~nr~ t~ probes of 45 to 55 nucleotides length, approximately 50% G+C content, and hybridi_ing to the pre-cursor or mature peptide lO encoding regions of the BDNF mRNA were synthP~i7Pd on an ABI 380B
DNA synthP~i7Pr. As specificity controls, sense oligonucleotides corresponding to each probe were also synthP~i7P~l. Using the convention that the first nucleotide in the BDNF coding region is position 1, the BDNF
probes syn~hPci7Pd correspond to BDNF nucleotide positions 47 to 94 (probes 15 2710 & 2711), 158 to 203 (probes 2712 & 2713), 576 to 624 (probes 2714 &
2715), and 644 to 692 (probes 2716 & 2717). The even numbered oligonucleotides are probes for the sense strand, and the odd numbered oligonucleotides are probes for the anti-sense strand.
For radiolabeling, the probes are gel purified on .lt~
20 acrylamide gels and reco~ l in H20 using standard protocols (Sambrook et al.). The probes (30 to 35 ng, 2 pmoles) are labeled by 3' homopolymeric tailing using termin~l deoxynucleotidyl Lldl~r~ldse (Promega, Madison WI) and 35S-dATP (1000 Ci/mmol, ~mPr~h~m Inc.) according to the en_yme m~mlf~ctllrer's recommpnrl~tion. The radiolabeled probes are purified by 25 column chromatography on si_e exclusion mini-spin columns (Biospin-6, Biorad Inc., Hercules, CA). The specific ac~ivity of the probes is qn~ntit~t~l by scintillation counting. Typical specific activities of the probes ranged from 1 X 109 to 5 X 109 cpm/,ug.
3. Tissue Hybri~i7~tion and Post Hybrilli7~tinn Washes.
In ~ L,aldLion for hybridi7ation, the desired number of slides are removed from storage under alcohol, and allowed to air dry thoroughly in the slide rack (approximately 1 hour). Meanwhile, the probe is heat denatured in CA 02222l74 l997-ll-24 W O 9''1S~96 PCT/U~,G1~5~57 a boiling H2O bath for 2 to S mimltes, quick chilled in an ice/H2O bath, and diluted in hybridi_ation buffer (10% dextran sulfate, 50% deioni7ed fo"--~-"i(11-, 4X SSC, SX Dehardts, 100 ~g/ml sheared salmon sperm DNA, 100 ,ug/ml polyadenylic acid) to a final concentration of 5 X 103 to 10 X 103 cpm/~4l. DTT is added to a 10 mM final concentration.
For hybridi7ation, 100 ~l of diluted probe in hybridization buffer (corresponding to O.S X 106 to 1.0 X 106 cpm probe) is carefully applied to each section being hybridized with probe. The solution is gently spread over the section with a pipet tip to cover the entire section(s) on each slide. The lO slides cont:~ining probe are then placed in hllmitlifi.-~l hybridization chambers at 42~C for hybri~li7~finn overnight with the probe. The hybridi7ation ch~mhers can be covered utility boxes, or acrylic boxes, wit'n raised platforms to accommodate slides. The boxes are lined with filter paper (or paper towels), saturated in 4X SSC, 50% form~mitle, and hllmi-lified by pre-inrllhating them with closed lids in a 42~C incubator for 1 to 3 hours before the slides are placed inside them. Although cover slips can be placed on the sections after the hybridi7ation buffer is applied, this is not nrcess~ry provided the hybritli7~tion rh~mhers are adequately hllmi~lifi-od during the procedure. If pre-hybridi_ation is used to obtain a lower background, the 20 sections may be inrllh~te-l at 42~C under 50 ~l hybridization buffer (minus probe) per section for 1 to 2 hours. After this time, an equal volume of hybridization buffer cont~ining probe at twice the concentration described above is applied to each section, and hybridization is carried out as described above.
For washes, the slides are Lldl~.rell~d from the hybridi_ation chamber to a slide holder. The slides can be placed in the slide holder every 4th or 5th slot so as to allow adequate flow of wash solution over the surface of each section. This placement can ~ignifir~ntly lowers background on the sections. A moderate flow rate of wash solution over the surface of the 30 sections promotes removal of unhybridized probe, and con~eq~lently reduces background. This is best accomplished during the 55~C wash steps by suspending the slides in the slide holder, above a m~gnrtir stir bar. The stir CA 02222174 1997-ll-24 WO ~ IS~96 PCT/US96/09857 bar is preferably placed approximately one inch under the slide holder. This can be done by h~nging the slide holder(s) from pipets straddling the wash chamber. A large beaker, or a 4 to 6 inch deep Pyrex b~king dish makes a suitable wash rh~mher. The wash chamber is placed on a hot-plate stirrer, s the temperature setting of which is precalibrated to ~ i" the wash solution at 55~C during the procedure. The changes of wash solution are made using pre-equilibrated solution. Washes and post wash dehydration of the sections are carried out as follows:
1. twice in lX SSC at room temperature for 5 ,i~es each time;
2. three times in lX SSC at 55~C for 30 .. i.. ~ s each time;
3. once in lX SSC at room Lt:lllpel~Lul~ for 1 min.
4. once in 0.1X SSC at room temperature for 15 seconds;
5. once in ultra pure H20 at room L~lllpc~ldLul~ for 15 seconds;
6. once in 50% EtOH for 15 seconds;
7. once in 70% EtOH for 15 seconds;
8. once in 95% EtOH for 15 seconds;
The sections are air dried thoroughly at room L~ aLul~ for 2 hours, followed by 30 minutes at 55~C. The dried sections on the slides are placed in autoradiographic cassette and exposed to X-ray film (Hyperfilm"B-max, 20 Amersham Inc., Arlington Heights, IL) at 4~C for 2 to 3 days to çstim~tt~ the exposure time required under emulsion. The X-ray film is developed according to the m~mlf~ rers .c:co.,.",~ntl~tit)n. The sections are coated with emulsion (Amersham LM-l, #RPN40) by dipping in emulsion at 42~C
under ap~ L~: safelight conditions. The emulsion coated slides are air 25 dried on a cooled surface for apprnxim~t~ly 30 mimlt~c, and ~ldl~r~ ..ed to aplastic slide box cont~ining a drying agent (drierite pellets). The seams of the box can be sealed with black tape, and the box wrapped in several layers of ~ ..lill..lll foil to ensure a light-tight enclosure. Following 2 to 4 hours at room telll~.dture to finish the drying, the boxes are transferred to 4~C for 30 autoradiographic exposure for 2 to 6 weeks. Prior to developing the emulsion coated slides, the box is removed from the refrigerator and allowed to equilibrate to room Lellll~eldlule for approximately 1 hour. The slides are W O 96/~16 PCT~US96/09857 then developed according to the m~mlf~rtnrers instructions, air dried for 1 to 2 hours, and if desired, co~ inP(l with the a~ro~lia~ cou,lle,~i,l.
Morlifi-~tinn~ and variations of the making and testing of L~ l~iC
animal models for testing of Alzheimer's disease will be obvious to those 5 skilled in the art from the foregoing (1et~ description. Such mo(1ifi~tion~
and variations are int~n-le-l to come within the scope of the following claims.

CA 02222l74 l997-ll-24 W0~6/~8~6 PCT~US96/09857 ~ U~N~ LISTING
( 1 ) ~.~N~ ~T- INFORMATION:
(i) APPLICANT: Athena Neurosciences, Inc.
(ii) TITLE OF lNv~NllON: Method For Identifying Al~h~;~s Disease Therapeutics Using Transgenic Animal Models (iii) NUMBER OF ~U~N~S: 10 (iv) COR~Nu~N~ ~nnRR~s (A) ~nDT~RCs~ Patrea L. Pabst (B) STREET: 2800 One ~tl Ant; c Center 1201 West Peachtree Street (C'~ CITY: AtlAntA
D~ STATE: GA
~E~ COUN'1'~Y: USA
~,F,~ ZIP: 30309-3450 (v) COMPUTER READABLE FORM:
'A' MEDIUM TYPE: Floppy disk 'B~ COMPUTER: IBM PC compatible C~ OPERATING SYSTEM: PC-DOS/MS-DOS
,D,~ SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) ~UK~N'l' APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vi) PRIOR APPLICATION DATA:
(A) APPLICATION N~MBER: US 08/480,653 (B) FILING DATE: June 7, 1995 (C) CLASSIFICATION:
(viii) AllOKN~Y/AGENT INFORMATION:
(A) NAME: Pabst, Patrea L.
(B) REGISTRATION NUMBER: 31,284 (C) REFERENCE/DOCKET N~MBER: ANSlOlCIP
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (404)-873-8794 (B) TELEFAX: (404)-873-8795 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2085 base pairs (B) TYPE: nucleic acid (C) sTRANn~nN~s: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1-2085 (D) OTHER INFORMATION: /function= "coding region ~or APP695."
(Xi ) ~U~N~ DESCRIPTION: SEQ ID NO:l:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala GlU Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 = 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu -CA 02222l74 l997-ll-24 W 096/40896 PCT~Us96~583-/

Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu GTT CCT GAC AAG TGC A~A TTC TTA CAC CAG GAG AGG ATG GAT GTT TGC 432 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys GAA ACT CAT CTT CAC TGG CAC ACC GTC GCC A~A GAG ACA TGC AGT GAG 480 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu GAG ACA CCT GGG GAT GAG AAT GAA CAT GCC CAT TTC CAG A~A GCC AAA 960 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg = 325 330 335 GAA TGG GAA GAG GCA GAA CGT CAA GCA AAG AAC TTG CCT A~A GCT GAT 1056 WO 9C/1~995 PCT~US96/09857 Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp AAG AAG GCA GTT ATC CAG CAT TTC QG GAG A~A GTG GAA TCT TTG GAA 1104Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu CAG GAA G Q GCC AAC GAG AGA CAG CAG CTG GTG GAG ACA QC ATG GCC 1152Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala 370 375 3~0 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His ACC CTA AAG CAT TTC GAG CAT GTG CGC ATG GTG GAT CCC AAG A~A GCC 1344 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu CGC ATG AAT QG TCT CTC TCC CTG CTC TAC AAC GTG CCT G Q GTG GCC 1440Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala GAG GAG ATT CAG GAT GAA GTT GAT GAG CTG CTT CAG A~A GAG CAA AAC 1488 Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 ~550 555 - 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val CA 02222l74 l997-ll-24 W O ~G~4W9C PCT~US9G/'~3Z

CAT CAT CAA A~A TTG GTG TTC TTT GCA GAA GAT GTG GGT TCA AAC A~A 1872 His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val ATC GTC ATC ACC TTG GTG ATG CTG AAG AAG A~A CAG TAC ACA TCC ATT 1968 Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn =690 695 ~2) INFORMATION FOR SEQ ID NO:2:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 695 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu CA 02222l74 l997-ll-24 W O 96/40896 PCT~US96/09857 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu ~lu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu ~lu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys ~lu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg ~lu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 . 400 ~yr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe ~sn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 . 480 ~lu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn ~yr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn CA 02222l74 l997-ll-24 W 0 96/40896 PCTrUS96/09857 ~lu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr ~hr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val ~le Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile ~is His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg ~is Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2253 base pairs (B) TYPE: nucleic acid (C) STR~n~n~R~S: dou~le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1-2253 (D) OTHER INFORMATION: /~unction= "coding region ~or APP751."
(Xi) S~Qu~Nc~ DESCRIPTION: SEQ ID NO:3:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly A~n Ala Gly Leu Leu Ala Glu Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln AAT GGG AAG TGG GAT TCA GAT CCA TCA GGG ACC A~A ACC TGC ATT GAT 192 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn CA 02222l74 l997-ll-24 W O9G/~U96 ~ PCT~US96/09857 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cy5 Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys GAA ACT CAT CTT CAC TGG CAC ACC GTC GCC A~A GAG ACA TGC AGT GAG 480 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu GIu Glu Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr 340 345 . 350 Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu CA 02222l74 l997-ll-24 W O~G/4~6 PCTAJS~G

His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp GAG CTG CTT CAG A~A GAG CAA AAC TAT TCA GAT GAC GTC TTG GCC AAC 1680 Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr CA 02222l74 l997-ll-24 W 096/40896 PCT~US96/09857 Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe GCA GAA GAT GTG GGT TCA AAC A~A GGT GCA ATC ATT GGA CTC ATG GTG 2064 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn (2) INFORMATION FOR SEQ ID NO:4:
( i ) S~U~N~'~: CHARACTERISTICS:
(A) LENGTH: 751 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg ~la Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu ~ln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln~Asn ~rp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Va Ile Pro Tyr Arg Cys ~eu Val Gly Glu Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 :160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile wo ~ r~3c PCT~US96,'0~3;~

Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu ~lu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu ~lu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe ~yr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr ~ys Met Ala Val Cys Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala Glu Arg Gln ~la Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe ~ln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val 450 g55 460 Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg ~la Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val ~rg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met ~hr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu CA 02222l74 l997-ll-24 WO9G/4~~96 PCT~US96~03 Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn ~et Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro ~er Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu'Thr Thr Arg Pro Gly Ser Gly Leu Thr ~sn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe ~rg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp ~la Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn ~ly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2310 base pairs (B) TYPE: nucleic acid (C) STR~Nn~nN~-~S: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1-2310 '' (D) OTHER INFORMATION: /function= "coding region ~or APP770."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln WO 96/40896 PCT~US961~33~7 AAT GGG AAG TGG GAT TCA GAT CCA TCA GGG ACC A~A ACC TGC ATT GAT 192 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu GTT CCT GAC AAG TGC A~A TTC TTA CAC CAG GAG AGG ATG GAT GTT TGC 432 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys GAA ACT CAT CTT CAC TGG CAC ACC GTC GCC A~A GAG ACA TGC AGT GAG 480 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val TGG TGG GGC GGA GCA GAC ACA GAC TAT GCA GAT GGG AGT GAA GAC A~A 672 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile CA 02222l74 l997-ll-24 WO 9~ 96 PCT~US9 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala GAA CGT CAA GCA AAG AAC TTG CCT A~A GCT GAT AAG AAG GCA GTT ATC 1296 Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile CAG CAT TTC CAG GAG A~A GTG GAA TCT TTG GAA CAG GAA GCA GCC AAC 1344 Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val . --CA 02222l74 l997-ll-24 WO 96/4 396 PCT~US96/~9~/

Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met Gln Asn (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 770 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) S~QU~N~'~ DESCRIPTION: SEQ ID NO:6:
~et Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg ~la Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro ~ln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln CA 02222l74 l997-ll-24 W 096/40896 PCT~US96/05 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu ~ln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn ~rp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 ; 160 ~ys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile ~sp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu GlU Glu ~lu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu ~lu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile Ser Arg Trp Tyr Phe Asp Val Thr Çlu Gly Lys Cys Ala Pro Phe Phe 305 310 315 : 320 ~yr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr ~y8 Met Ala Val Cys Gly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr Thr Gln Glu Pro Leu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala WO ~G/1C~6 PCT~Us96/09857 11~

Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val ~25 630 635 640 Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met = 755 760 765 CA 02222l74 l997-ll-24 WO96/40896 PCT~US96/09857 Gln Asn (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 20 base pairs B~ TYPE: nucleic acid ~C STRPNn~nN~-~S: single ~ ,D, TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) EYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CCGATGATGA CGAGGACGAT ~ 20 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CEARACTERISTICS:
~'A LENGTH: 20 base pairs B~ TYPE: nucleic acid 'C,~ STRPNn~nN~-~S: single ~ D:~ TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(iii) ~Y~O~ CAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

(2) INFORMATION FOR SEQ ID NO:9:
(i) ~h'~U~N~'~ CHARACTERISTICS:
A) LENGTH: 5 amino acids B) TYPE: a~ino acid ,D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) ~U~N~ DESCRIPTION: SEQ ID NO:9:
Phe Arg Val Gly Ser (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 a~ino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Asp Ala Glu Phe Arg Gly Gly Cys

Claims (27)

We claim:

1. A method for testing compounds for an effect on an Alzheimer's disease marker comprising a) administering the compound to be tested to a non-human transgenic mammal, or mammalian cells derived from the transgenic mammal, wherein the transgenic mammal has a nucleic acid construct stably incorporated into the genome, wherein the construct comprises a promoter for expression of the construct in a mammalian cell and a region encoding an A.beta.-containing protein, wherein the promoter is operatively linked to the region, wherein the region comprises DNA encoding the A.beta.-containing protein, wherein the A.beta.-containing protein consists of all or a contiguous portion of a protein selected from the group consisting of APP770, APP770 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, APP751, APP751 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, APP695, and APP695 bearing a mutation in one or more of the amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, and 717, wherein the A.beta.-containing protein includes amino acids 672 to 714 of human APP, wherein the promoter mediates expression of the construct such that A.beta.tot is expressed at a level of at least 30 nanograms per gram of brain tissue of themammal when it is two to four months old, A.beta.1-42 is expressed at a level of at least 8.5 nanograms per gram of brain tissue of the mammal when it is two to four months old, APP and APP.alpha. combined are expressed at a level of at least 150 picomoles per gram of brain tissue of the mammal when it is two to four months old, APP.beta. is expressed at a level of at least 40 picomoles per gram of brain tissue of the mammal when it is two to four months old, and/or mRNA
encoding the A.beta.-containing protein is expressed to a level at least twice that of mRNA encoding the endogenous APP of the transgenic mammal in brain tissue of the mammal when it is two to four months old; and detecting or measuring the Alzheimer's disease marker such that any difference between the marker in the transgenic mammal, or by mammalian cells derived from the transgenic mammal, and the marker in a transgenic mammal, or by mammalian cells derived therefrom, to which the compound has not been administered, is observed, wherein an observed difference in the marker indicates that the compound has an effect on the marker.

2. The method of claim 1 wherein the A.beta.-containing protein is selected from the group consisting of APP770; APP770 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; APP751; APP751 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; APP695; APP695 bearing a mutation in the codon encoding one or more amino acids selected from the group consisting of amino acid 669, 670, 671, 690, 692, 717; a protein consisting of amino acids 646 to 770 of APP; a protein consisting of amino acids 670 to 770 ofAPP; a protein consisting of amino acids 672 to 770 of APP; and a protein consisting of amino acids 672 to 714 of APP.

3. The method of claim 2 wherein the DNA encoding the A.beta.-containing protein is cDNA or a cDNA/genomic DNA hybrid, wherein the cDNA/genomic DNA hybrid includes at least one APP intron sequence wherein the intron sequence is sufficient for splicing.

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

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

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

7. The method of claim 1 wherein the Alzheimer's disease marker is a protein and the observed difference is an increase or decrease in the amount of the protein present in the transgenic mammal, or in mammalian cells derived therefrom, to which the compound has been administered.

8. The method of claim 7 wherein the protein is selected from the group consisting of Cat D,B, Neuronal Thread Protein, nicotine receptors, 5-HT2 receptor, NMDA receptor, .alpha.2-adrenergic receptor, synaptophysin, p65, glutamine synthetase, glucose transporter, PPI kinase, GAP43, cytochrome oxidase, heme oxygenase, calbindin, adenosine A1 receptors, choline acetyltransferase, acetylcholinesterase, glial fibrillary acidic protein (GFAP), .alpha.1-antitrypsin, C-reactive protein, .alpha.2-macroglobulin, IL-1.alpha., IL-1.beta., TNF.alpha., IL-6, HLA-DR, HLA-A, D,C, CR3 receptor, MHC I, MHC II, CD 31, CR4, CD45, CD64, CD4, spectrin, tau, ubiquitin, MAP-2, apolipoprotein E, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), advanced glycosylation end products, receptor for advanced glycosylation end products, 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, beta-3 integrin,Apo J, clusterin, type 2 plasminogen activator inhibitor, midkine, macrophage colony stimulating factor receptor, MRP14, 27E10, interferon-alpha, S100.beta., cPLA2, c-jun, c-fos, HSP27, HSP70, MAP5, membrane lipid peroxidase, protein carbonyl formation, junB, junD, fosB, fral, cyclin D1, pS3, NGFI-A, NGFI-B, IKB, NFKB, IL-8, MCP-1, MIP-1.alpha., matrix metaloproteinases, 4-hydroxynonenal-protein conjugates, amyloid P component, laminin, and collagen type IV.

9. The method of claim 1 wherein the Alzheimer's disease marker is a protein and the observed difference is a reduction or absence of the protein in plaques or neuritic tissue present in the transgenic mammal to which the compound has been administered.

10. The method of claim 9 wherein the protein is selected from the group consisting of Cat D,B, protein kinase C, NADPH, C3d, C1q, C5, C4bp, C5a-C9, tau, ubiquitin, MAP-2, neurofilaments, heparin sulfate, chrondroitin sulphate, apolipoprotein E, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glycosylation end products, amyloid P component, laminin, and collagen type IV.

11. The method of claim 1 wherein the Alzheimer's disease marker is a protein and the observed difference is an increase or decrease in the enzymatic or biochemical activity of the protein in the transgenic mammal, or in mammalian cells derived therefrom, to which the compound has been administered.

12. The method of claim 11 wherein the protein is selected from the group consisting of nicotine receptors, 5-HT2 receptor, NMDA receptor, .alpha.2-adrenergic receptor, glutamine synthetase, glucose transporter, PPI kinase, cytochrome oxidase, heme oxygenase, calbindin, adenosine A1 receptors, choline acetyltransferase, acetylcholinesterase, glial fibrillary acidic protein (GFAP),.alpha.1-antitrypsin, C-reactive protein, .alpha.2-macroglobulin, IL-1, TNF.alpha., IL-6, HLA-DR, HLA-A, D,C, CR3 receptor, MHC I, MHC II, CD 31, CR4, CD45, CD64, CD4, spectrin, ubiquitin, and apolipoprotein E.

13. The method of claim 1 wherein the Alzheimer's disease marker is a nucleic acid encoding a protein and the observed difference is an increase or decrease in the amount of the nucleic acid present in the transgenic mammal, or in mammalian cells derived therefrom, to which the compound has been administered.

14. The method of claim 13 wherein the encoded protein is selected from the group consisting of growth inhibitory factor, Cat D,B, Neuronal Thread Protein, nicotine receptors, 5-HT2 receptor, NMDA receptor, .alpha.2-adrenergic receptor, synaptophysin, p65, glutamine synthetase, glucose transporter, PPI
kinase, GAP43, cytochrome oxidase, heme oxygenase, calbindin, adenosine A1 receptors, choline acetyltransferase, acetylcholinesterase, glial fibrillary acidic protein (GFAP), .alpha.1-antitrypsin, C-reactive protein, .alpha.2-macroglobulin, IL-1, TNF.alpha., IL-6, HLA-DR, HLA-A, D,C, CR3 receptor, MHC I, MHC II, CD 31, CR4, CD45, CD64, CD4, spectrin, tau, ubiquitin, MAP-2, apolipoprotein E, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), advanced glycosylation end products, receptor for advanced glycosylation end products, 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, beta-3 integrin, Apo J, clusterin, type 2 plasminogen activator inhibitor, midkine, macrophage colony stimulating factor receptor, MRP14, 27E10, interferon-alpha, S100.beta., cPLA2, c-jun, c-fos, HSP27, HSP70, MAP5, membrane lipid peroxidase, protein carbonyl formation, junB, junD, fosB, fra1, cyclin D1, p53, NGFI-A, NGFI-B, IKB, NFKB, IL-8, MCP-1, MIP-1.alpha., matrix metaloproteinases, 4-hydroxynonenal-protein conjugates, amyloid P
component, laminin, and collagen type IV.

15. The method of claim 1 wherein the Alzheimer's disease marker is a behavior and the observed difference is a change in the behavior observed in thetransgenic mammal to which the compound has been administered.

16. The method of claim 15 wherein the behavior is selected from the group consisting of behavior using working memory, behavior using reference memory, locomotor activity, emotional reactivity to a novel environment or to novel objects, and object recognition.

17. The method of claim 1 wherein the Alzheimer's disease marker is a histopathology and the observed difference is a decrease in the extent or severity of the histopathology present in the transgenic mammal to which the compound has been administered.

18. The method of claim 17 wherein the histopathology marker is selected from the group consisting of compacted plaques, neuritic dystrophy, gliosis, A.beta.
deposits, decreased synaptic density, and neuropil abnormalities.

19. The method of claim 1 wherein the Alzheimer's disease marker is cognition and the observed difference is a change in the cognition of the transgenic mammal to which the compound has been administered.

20. The method of claim 1 wherein the marker is detected or measured using RT-PCR, RNase protection, Northern analysis, R-dot analysis, ELISA, antibody staining, laser scanning confocal imaging, and immunoelectron micrography.

21. The method of claim 1 wherein the mammals are rodents.

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

23. The method of claim 22 wherein the codon encoding amino acid 717 is mutated to encode Phe.

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

25. The method of claim 24 wherein the codon encoding amino acid 670 is mutated to encode Asn, and/or the codon encoding amino acid 671 is mutated to encode Leu or Tyr.

26. The method of claim 1 wherein the promoter mediates expression of the construct such that A.beta.tot is expressed at a level of at least 30 nanograms per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, A.beta.1-42 is expressed at a level of at least 8.5 nanograms per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, APP and APP.alpha. combined are expressed at a level of at least 150picomoles per gram of hippocampal or cortical brain tissue of the mammal when it is two to four months old, APP.beta. is expressed at a level of at least 40 picomoles per gram of hippocampal or cortical brain tissue of the mammal when it is two tofour months old, and/or mRNA encoding the A.beta.-containing protein is expressed to a level at least twice that of mRNA encoding the endogenous APP of the transgenic mammal in hippocampal or cortical brain tissue of the mammal when it is two to four months old.

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