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(12) Patent Application Publication (10) Pub. No.: US 2004/0194158A1 Botas Et Al US 2004O1941.58A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0194158A1 Botas et al. (43) Pub. Date: Sep. 30, 2004 (54) MODEL FOR NEURODEGENERATIVE Publication Classification DSORDERS (51) Int. Cl." ....................... A01K 67/00; AO1K 67/033; (75) Inventors: Juan Botas, Houston, TX (US); Diego AO1K 67/027 Rincon-Limas, Houston, TX (US); (52) U.S. Cl. ................................................................ 800/13 Pedro Fernandez-Funez, Houston, TX (US); Ismael Al-Ramahi, Houston, TX (US) (57) ABSTRACT Correspondence Address: The present invention discloses a double transgenic fly that PALMER & DODGE, LLP expresses both human Tau protein and the human AB42 KATHLEEN M. WILLIAMS peptide of human amyloid-fi precursor protein (APP). The 111 HUNTINGTONAVENUE double transgenic flies of the present invention display a Synergistic altered phenotype as compared to the altered BOSTON, MA 02199 (US) phenotype displayed by transgenic flies expressing either (73) Assignee: Baylor College of Medicine human Tau or human AB42 alone. Thus, the flies provide for models of neurodegenerative disorders, Such as Alzheimer's (21) Appl. No.: 10/402,420 disease. The invention further discloses methods for identi fying therapeutic compounds to treat neurodegenerative (22) Filed: Mar. 28, 2003 disorders using the double transgenic flies. Patent Application Publication Sep. 30, 2004 Sheet 1 of 10 US 2004/01941.58A1 FIGURE 1A A342 Amino acid Sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQID NO:1) Patent Application Publication Sep. 30, 2004 Sheet 2 of 10 US 2004/01941.58A1 FIGURE 1B A342 Nucleic acid Sequence gatgcagaatticcgacatgacticaggatatgaagttcatcatcaaaaattggtgttctgcagaagatgtgg gttcaaacaaaggtgcaatcattggacticatggtggg.cggtgttgtcatagogtga (SEQ ID NO:2) Patent Application Publication Sep. 30, 2004 Sheet 3 of 10 US 2004/01941.58A1 FIGURE 2A MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLOT PTEDGSEEPGSETSDAKSTPTAEDVTAPLVDEGAPGKQAAAQPHTEIPEG TTAEEAGIGDTPSLEDEAAGHVTOARMVSKSKDGTGSDDKKAKGADGKTK IATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSP GSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVPM PDLKNVKSKIGSTENLKHQPGGGKVQIINKKLDLSNVQSKCGSKDNIKHV PGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRV QSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKTDHGAEIVYKSPVVS GDTSPRHLSNVSSTGSIDMVDSPQLATLADEVSASLAKQGL (SEQ ID NO:3) Patent Application Publication Sep. 30, 2004 Sheet 4 of 10 US 2004/01941.58A1 FIGURE 2B atggctgagcccc.gc.caggagttcgaagtgatggaagatcACGCTGGGAC GTACGGGTTGGGGGACAGGAA AGATCAGGGGGGCTACACCATGCACCAAG ACCAAGAGGGTGACACGGACGCTGGCCTGAAAGAATCTCCCCTGCAGACC CCCACTGAGGACGGATCTGAGGAACCGGGCTCTGAAACCTCTGATGCTAA GAGCACTCCAACAGCGGAAGATGTGACAGCACCCTTAGTGGATGAGGGAG CTCCCGGCAAGCAGGCTGCCGCGCAGCCCCACACGGAGATCCCAGAAGGA ACCACAGCTGAAGAAGCAGGCATTGGAGACACCCCCAGCCTGGAAGACGA AGCTGCTGGTCACGTGACCCAAGCTCGCATGGTCAGTAAAAGCAAAGACG GGACTGGAAGCGATGACAAAAAAGCCAAGGGGGCTGATGGTAAAACGAAG ATCGCCACACCGCGGGGAGCAGCCCCTCCAGGCCAGAAGGGCCAGGCCAA CGCCACCAGGATTCCAGCAAAAACCCCGCCCGCTCCAAAGACACCACCCA GCTCTGGTGAACCTCCAAAATCAGGGGATCGCAGCGGCTACAGCAGCCCC GGCTCCCCAGGCACTCCCGGCAGCCGCTCCCGCACCCCGTCCCTTCCAAC CCCACCCACCCGGGAGCCCAAGAAGGTGGCAGTGGTCCGTACTCCACCCA AGTCGCCGTCTTCCGCCAAGAGCCGCCTGCAGACAGCCCCCGTGCCCATG CCAGACCTGAAGAATGTCAAGTCCAAGATCGGCTCCACTGAGAACCTGAA GCACCAGCCGGGAGGCGGGAAGGTGCAGATAATTAATAAGAAGCTGGATC TTAGCAACGTCCAGTCCAAGTGTGGCTCAAAGGATAATATCAAACACGTC CCGGGAGGCGGCAGTGTGCAAATAGTCTACA AACCAGTTGACCTGAGCAA GGTGACCTCCAAGTGTGGCTCATTAGGCA ACATCCATCATAAACCAGGAG GTGGCCAGGTGGAAGTAAAATCTGAGAAGCTTGACTTCAAGGACAGAGTC CAGTCGAAGATTGGGTCCCTGGACAATATCACCCACGTCCCTGGCGGAGG AAATAAAAAGATTGAAACCCACAAGCTGACCTTCCGCGAGA ACGCCAAAG CCAAGACAGACCACGGGGCGGAGATCGTGTACAAGTCGCCAGTGGTGTCT GGGGACACGTCTCCACGGCATCTCAGCAATGTCTCCTCCACCGGCAGCAT CGACATGGTAGACTCGCCCCAGCTCGCCACGCTAGCTGACGAGGTGTCTG CCTCCCTGGCCAAGCAGGGTTTGTGATCAGGCCCCTGGGGCGGTCAATAA TTGTGGAGAGGAGAGAATGAGAGAGTGTGGAAAAAAAAAGAATAATGACC CGGCCCCCGCCCTCTGCCCCC (SEQID NO: 4) Patent Application Publication Sep. 30, 2004 Sheet 5 of 10 US 2004/0194158A1 FIGURE 3 Hutton, M. et al., Nature 393: 702-708 (1998) Hutton, M. et al., Nature 393: 702-708 (1998) Tatebayashi, Y. et al., Proc. Nat. Acad. Sci. 99. - - - 13896-13901 (2002) 13103-13107 (1998) Murrell, J.R. et al., J. Neuropath. Exp. Neurol. 58: 1207-1226 (1999) Yasuda, M. et al., Neurology 55: 1224-1227 (2000) Spillantini, M.G. et al. Ann. Neurol. 48: 939 943 (2000) Lippa, C.F. et al., Ann. Neurol. 48: 850-858 (2000) Pickering-Brown, S. et al., Ann. Neurol. 48: 859-867 (2000 Neumann, M., Ann. Neurol. 50: 503–513 (2001) Iijima, M. et al., Nueroreport 10: 497-501, (1999) R5H Hayashi, S. et al., Ann Neurol. 51: 525-530 (2002 S.M. van Herpen, et al., Ann Nuerol. 51: 373 376 (2002) R5L Poorkaj, P. et al, Ann Nuerol. 52: 511-516 (2002) Patent Application Publication Sep. 30, 2004 Sheet 6 of 10 US 2004/01941.58A1 FIGURE 4 Signal Peptide Sequences Dint (wingless) Signal peptide Amino acid sequence MDISYIFVICLMALSGGS (SEQ ID NO:5) Dint (wingless) Signal peptide plus linker Nucleic acid sequence atggataticagotatatottcgtCatctgcCtgatggcc.cgtgcagogg cggcagcagottcgcgatg (SEQ ID NO: 6) Argos Signal Peptide Amino acid sequence MPTTLMLLPCMLLLLLTAAAVAVGG (SEQ ID NO: 7) Argos Signal Peptide Nucleic acid sequence atgcctacgacattgatgttgctg.ccgtgcatgctgctgttgctgcigac cgcc.gctg.ccgttgctgtcggcggc (SEQ ID NO: 8) Patent Application Publication Sep. 30, 2004 Sheet 7 of 10 US 2004/01941.58A1 FIGURE 5A UAS E10 R. R. R. R. Patent Application Publication Sep. 30, 2004 Sheet 8 of 10 US 2004/0194158A1 AAli arlal Patent Application Publication Sep. 30, 2004 Sheet 9 of 10 US 2004/01941.58A1 afiy(sKep) (%) fuquio (%) fuquio FIGURE 5 Patent Application Publication Sep. 30, 2004 Sheet 10 of 10 US 2004/01941.58A1 3: O f Co l Co Of 'ueig Jie Iod 1. SI3) oA) ISO S-HILJO JeCUUIn N FIGURE 7 US 2004/O1941.58A1 Sep. 30, 2004 MODEL FOR NEURODEGENERATIVE (2001); Jackson et al., Neuron 34:509-519 (2002)). In flies, DISORDERS expression of human Tau can lead to shortened life-span, loss of cholinergic neurons (Wittman et al., Science GOVERNMENT SUPPORT 293:711-714 (2001)) and eye phenotypes (Jackson et al., Neuron 34:309-519 (2002)). However, these wild type and 0001. The invention was supported, in whole or in part, mutant transgenic Tau fly models do not develop, on their by a grant number NS42179 from the National Institute of own, neurofibrillary tangles characteristic of human AD. Health The Government has certain rights in the invention. Neurofibrillary pathology was only observed when com bined with other alterations in genes of the Wint Signaling BACKGROUND pathway (Jackson et al., Neuron 34:309-519 (2002)). 0002 Alzheimer's disease (AD) is the most common 0006 Thus, despite significant advances in the field, there neurodegenerative disorder in humans. The disease is char is still a need in the art for improved non-mammalian animal acterized by a progressive impairment in cognition and models of Alzheimer's disease that can be easily and inex memory. The hallmark of AD at the neuropathological level pensively generated for Screening potential therapeutic is the extracellular accumulation of the amyloid-fi peptide agents. (AB) in “senile' plaques, and the intracellular deposition of neurofibrillary tangles made of the microtubule-associated SUMMARY OF THE INVENTION protein Tau. In neuronal tissue of AD patients, Tau is hyperphosphorylated and adopts pathological conforma 0007. The present invention discloses a double transgenic tions evident with conformation-dependent antibodies. The fly that expresses both the human Tau protein and the human amyloid-3 peptide is a cleavage product of the amyloid Af342 peptide of APP. The double transgenic flies of the precursor protein (APP). In normal individuals, most of AB present invention display a Synergistic altered phenotype as is in a 40-amino acid form, but there are also minor amounts compared to the altered phenotype displayed by transgenic of A? that are 42 amino acids in length (A?8). In patients flies expressing either human Tau or human AB42 alone. with AD, there is an overabundance of AB that is thought Thus, the flies provide for models of neurodegenerative to be the main toxic A? form. disorders, Such as Alzheimer's disease. Accordingly, the 0003) A number of transgenic mouse models have been invention further discloses methods for identifying thera generated that express wild-type or mutant human APP. The peutic compounds useful for treating neurodegenerative mutant form of APP is differentially cleaved to result in disorders, Such as Alzheimer's disease. increased amounts of AB42 deposited within AB plaques. 0008. The present invention provides a transgenic fly These transgenic mice present with neurological Symptoms whose Somatic and germ cells comprise two transgenes of Alzheimer's disease, Such as impaired memory and motor operatively linked to a promoter, wherein the transgenes function (Janus C. et al., Curr. Neurol. Neurosci. Rep 1 (5): encode human Tau and human AB42, and wherein the 451-457 (2001)). A transgenic mouse that expresses both expression of the transgenes in the nervous System results in mutant human APP and mutant human Tau has also been the fly having a predisposition to, or resulting in, progressive generated (Jada, et. al., Science, (5534) 293: 1487-1491 neural degeneration. (2001)). This double transgenic mouse is a rodent model for 0009. In one embodiment, the transgenic fly is transgenic AD that shows enhanced neurofibrillary degeneration indi Drosophila. cating that either APP or AB influences the formation of neurofibrillary tangles. 0010. In preferred embodiments
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