WO2005041650A1 - TRANSGENIC FLIES EXPRESSING MUTANT Aβ42 - Google Patents
TRANSGENIC FLIES EXPRESSING MUTANT Aβ42 Download PDFInfo
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- WO2005041650A1 WO2005041650A1 PCT/US2004/034838 US2004034838W WO2005041650A1 WO 2005041650 A1 WO2005041650 A1 WO 2005041650A1 US 2004034838 W US2004034838 W US 2004034838W WO 2005041650 A1 WO2005041650 A1 WO 2005041650A1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
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- C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
Definitions
- AD Alzheimer's disease
- a ⁇ amyloid- ⁇ peptide
- Tau is hyperphosphorylated and adopts pathological conformations evident with conformation-dependent antibodies.
- the amyloid - ⁇ peptide is a cleavage product of the amyloid precursor protein (APP).
- a ⁇ 42 A ⁇ 42 amino acids in length
- a number of pathogenic mutations have been found within APP which are associated with hereditary forms of AD, several of which are located within the A ⁇ sequences. These mutations result in a phenotype different from AD, with massive amyloid accumulation in cerebral blood vessel walls.
- Two mutations namely the Dutch (Glu22Gln) and the Flemish (Ala21Gly) mutations, have been reported (Levy, et al., Science 248, 1124-1126 (1990)), (van Broeckhoven et al. (1990)), (Hendriks,-et al., Nature Genet 1, 218- 221 (1992)). Patients having these mutations suffer from cerebral hemorrhage and vascular symptoms.
- a third pathogenic intra-A ⁇ ; mutation was recently discovered in an Italian family (Glu22Lys), with clinical findings similar to the Dutch patients (Tagliavini, et al, Alz Report 2, S28 (1999)).
- Glu22Gly another pathogenic AD mutation within APP, named the "Arctic mutation” (Glu22Gly)
- Carriers of this mutation develop progressive dementia with clinical features typical of AD without symptoms of cerebrovascular disease.
- AD is distinctly characterized by accelerated formation of protofibrils comprising mutated A ⁇ peptides (A ⁇ 4 ⁇ ARc and/or A ⁇ 42ARc) compared to protofibril formation of wild type A ⁇ peptides.
- carriers of the "Iowa” mutation, carrying a Asp23Asn mutation within A ⁇ exhibit severe cerebral amyloid angiopathy, widespread neurofibrillary tangles, and unusually extensive distribution of A ⁇ 40 in plaques. (Grabowski et al, Ann. Neurol. 49: 691-693 (2001))
- transgenic mouse models have been generated that express wild-type or mutant human APP.
- the mutant form of APP is differentially cleaved to result in increased amounts of A ⁇ 42 deposited within A ⁇ plaques.
- These transgenic mice present with neurological symptoms of Alzheimer's disease, such as impaired memory and motor function (Janus C. et al., Curr. Neurol. Neurosci. Rep 1 (5): 451-457 (2001)).
- a transgenic mouse that expresses both mutant human APP and mutant human Tau has also been generated (Jada, et. al, Science, (5534) 293:1487-1491 (2001)).
- This double transgenic mouse is a rodent model for AD that shows enhanced neurofibrillary degeneration indicating that either APP or A ⁇ influences the formation of neurofibrillary tangles.
- mice have proven very useful for testing potential AD therapeutics. However, the use of mice for testing therapeutics is both expensive and time consuming. Thus, it would be beneficial to find alternative models which are less expensive and that can be efficiently used to screen for therapeutic agents for Alzheimer's disease.
- non-mammalian animal models such as Caenorhabditis elegans or Drosophila melanogaster.
- Drosophila as a model organism has proven to be an important tool in the elucidation of human neurodegenerative pathways (reviewed in Fortini, M. and Bonini, N. Trends Genet. 16: 161-167 (2000)), as the Drosophila genome contains many relevant human orthologs that are extremely well conserved in function (Rubin, G. M., et al., Science 287: 2204-2215 (2000)).
- Drosophila melanogaster carries a gene that is homologous to human APP which is involved in nervous system function.
- the gene, APP-like (Appl) is ' approximately 40% identical to the neurogenic isoform (Rosen et al., Proc. Natl. Acad. Sci.
- Drosophila models of polyglutamine repeat diseases Jackson, G. R., et al (1998). Neuron 21: 633-642; Kazemi-Esfarani, P. and Benzer, S. (2000). Science 287: 1837- 1840; Femandez-Funez et al. (2000) Nature 408 (6808): 101-6), Parkinson's disease (Feany, M. B. and Bender, W. W. (2000). Nature 404: 394-398) and others have been established which closely mimic the disease state in humans at the cellular and physiological levels, and have been successfully employed in identifying other genes that may be involved in these diseases.
- This invention generally relates to a method to identify compounds and genes acting on the APP pathway in transgenic Drosophila melanogaster that ectopically express genes related to AD. Expression of these transgenes can induce visible phenotypes and it is contemplated herein that genetic screens disclosed herein may be used to identify genes involved in the APP pathway by the identification of mutations that modify the induced visible phenotypes. The genes affected by these mutations will be called herein "genetic modifiers”. It is contemplated herein that human homologs of such genetic modifiers would be useful targets in the development of therapeutics to treat conditions associated with, but not limited to, Alzheimer Disease.
- the present invention discloses transgenic flies that express the human A ⁇ 42 peptide of APP containing a pathogenic mutation selected from the group consisting of the 'Iowa mutation' (D23N) within the A ⁇ 42 (A ⁇ 42 ⁇ owa ) peptide of SEQ ID NO: 1, the 'Dutch mutation' (E22Q) within the A ⁇ 42 (A ⁇ 42 Dutch ) peptide of SEQ ID NO: 2, the 'Flemish mutation' (A21G) within the A ⁇ 42 (A ⁇ 42 F iemish ) peptide of SEQ ID NO: 3, and the 'Italian mutation' (E22K) within the A ⁇ 42 (A ⁇ 42 Ita ⁇ ian ) peptide of SEQ ID NO: 4.
- a pathogenic mutation selected from the group consisting of the 'Iowa mutation' (D23N) within the A ⁇ 42 (A ⁇ 42 ⁇ owa ) peptide of SEQ ID NO: 1, the 'Dutch mutation' (
- the present invention discloses transgenic flies that express the human A ⁇ 42 peptide of APP containing a pathogenic 'Arctic mutation' (E22G) within the A ⁇ 42 (A ⁇ 42Arctic ) peptide of SEQ ID NO: 5 and the human Tau protein.
- the present invention provides transgenic flies whose somatic and germ cells comprise a transgene encoding the human A ⁇ 42 ⁇ OWa containing the Iowa mutation, the human A ⁇ 42 DutC containing the Dutch mutation, the human A ⁇ 42 F iemish containing the Flemish mutation, and the human A ⁇ 42 lta ii an containing the Italian mutation and wherein expression of the transgene results in the fly having a predisposition to, or resulting in, progressive neural degeneration.
- the present invention provides transgenic flies whose somatic and germ cells comprise transgenes encoding the Tau and human A ⁇ 42Arctic containing the Arctic mutation and wherein expression of the transgene results in the fly having a predisposition to, or resulting in, progressive neural degeneration.
- the transgenic fly is transgenic Drosophila.
- the transgenic fly comprising the Iowa, Dutch, Flemish, or Italian A ⁇ 42 mutation comprises a second transgene, encoding the Tau protein.
- the double transgenic fly of this embodiment as well as the double transgenic fly comprising the Arctic A ⁇ 42 mutation and Tau displays a synergistic altered phenotype as compared to the altered phenotype displayed by transgenic flies expressing mutant human A ⁇ 42 protein alone.
- the Tau and human A ⁇ 42 ⁇ owa , A ⁇ 42Dutch. A ⁇ 42Fiemish. A ⁇ 42 lta iian. or A ⁇ 42Arctic mutant transgenes are operatively linked to an expression control sequence and expression of the transgenes results in an observable phenotype.
- the transgene is temporally regulated by the expression control sequence.
- the transgene is spatially regulated by the expression control sequence.
- the expression control sequence is a heat shock promoter.
- the heat shock promoter is derived from the hsp70 or hsp83 genes.
- the Tau and human A ⁇ 42 Iowa , A ⁇ 42 Du tch. A ⁇ 42 Fle mish. A ⁇ 42 Ita iian. or A ⁇ 42A-ctic transgenes are operatively linked to a GAL4 Upstream Activating Sequence ("UAS").
- UAS Upstream Activating Sequence
- the transgenic Drosophila comprising Tau and human A ⁇ 42 lowa , A ⁇ 42D U tc .
- a ⁇ 42 ⁇ taiian . or A ⁇ 42Arctic mutant transgenes further comprise a GAL4 gene.
- the GAL4 gene is linked to a tissue specific expression control sequence, h a preferred mode of the embodiment, the tissue specific expression control sequence is derived from the sevenless, eyeless, gmr/glass or any of the rhodopsin genes, hi another preferred mode of the embodiment, the tissue specific expression control sequence is derived from the dpp, vestigial, or apterous genes. In another preferred mode of the embodiment, the tissue specific expression control sequence is derived from neural-specific genes like elav, nirvana or D42 genes. In yet other embodiments, the expression control sequence is derived from ubiquitously expressed genes like tubulin, actin, or ubiquitin.
- the expression control sequence comprises a tetracycline-controlled transcriptional activator (tTA) responsive regulatory element.
- tTA tetracycline-controlled transcriptional activator
- the transgenic Drosophila comprising the Tau and mutant human A ⁇ 42 ⁇ owa> A ⁇ 42 Dutch , A ⁇ 42 F iemish, A ⁇ 42 ⁇ tal i an , or A ⁇ 42 Arct i c transgenes further comprise a tTA gene.
- the DNA sequence encoding the mutant human A ⁇ 42 l0Wa , A ⁇ 42 Dutcll , A ⁇ 42 F ⁇ em j Sh , A ⁇ 42 Ita iian. or A ⁇ 42Arctic may be fused to a signal peptide, e.g., via an amino acid linker.
- the signal peptide may be a wingless (wg) signal peptide, such as the peptide represented by SEQ ID NO: 11, or an Argos (aos) signal peptide, such as the sequence of SEQ ID NO: 12.
- the transgenic fly may exhibit an altered phenotype, such as a rough eye phenotype, a concave wing phenotype, a locomotor dysfunction (e.g., reduced climbing ability, reduced walking ability, reduced flying ability, decreased speed, abnormal trajectories, and abnormal turnings), abnormal grooming, other abnormal behaviors, or reduced life span.
- a locomotor dysfunction e.g., reduced climbing ability, reduced walking ability, reduced flying ability, decreased speed, abnormal trajectories, and abnormal turnings
- abnormal grooming other abnormal behaviors, or reduced life span.
- the invention in another aspect, relates to a method for identifying an agent active in neurodegenerative disease.
- the method comprises the steps of: (a) providing a transgenic fly whose genome comprises DNA sequences that encode the mutant human A ⁇ 42 ⁇ owa , A ⁇ 42Dutch. A ⁇ 42 Flem ish. or A ⁇ 42 ⁇ taiian alone, or in combination with the Tau protein, or that encode the A ⁇ 42 Arc t i c in combination with Tau protein; (b) providing a candidate agent to the transgenic fly; and (c) observing the phenotype of the transgenic fly of step (b) relative to the control fly that has not been administered an agent.
- the invention relates to a method for identifying an agent active in neurodegenerative disease.
- the method comprises the steps of: (a) providing a transgenic fly and a control wild-type fly; (b) providing a candidate agent to the transgenic fly and to the control fly; and (c) observing a difference in phenotype between the transgenic fly and the control fly, wherein a difference in phenotype is indicative of an agent active in neurodegenerative disease.
- the invention relates to a method to identify genetic modifiers of the APP pathway, comprising: providing a transgenic fly whose genome comprises a DNA sequence encoding a polypeptide comprising the A ⁇ 42 ⁇ OW a, A ⁇ 42 Du tch. A ⁇ 42 F ⁇ em ish. or A ⁇ 42 Ita ii an (SEQ.
- the present invention discloses transgenic flies that express human A ⁇ 42 lowa , containing a D23N mutation, human A ⁇ 42Dutch. containing a E22Q, human A ⁇ 42 F ⁇ en -ish. containing a A21G mutation, or human A ⁇ 42 ⁇ ta ii a n, containing a E22K mutation either alone or in combination with the Tau protein, or human A ⁇ 42 Arct i c. containing a E22G mutation in combination with the Tau protein.
- the transgenic flies exhibit progressive neurodegeneration which can lead to a variety of altered phenotypes including locomotor phenotypes, behavioral phenotypes (e.g., appetite, mating behavior, and/or life span), and morphological phenotypes (e.g., shape, size, or location of a cell, organ, or appendage; or size, shape, or growth rate of the fly).
- locomotor phenotypes e.g., appetite, mating behavior, and/or life span
- morphological phenotypes e.g., shape, size, or location of a cell, organ, or appendage; or size, shape, or growth rate of the fly.
- transgenic fly refers to a fly whose somatic and germ cells comprise a transgene operatively linked to a promoter, wherein the transgene encodes the human A ⁇ 42 ⁇ owa , A ⁇ 42 Du tc » A ⁇ 42 F ⁇ em ish. or A ⁇ 42 ⁇ ta iian. or A ⁇ 42A-ctic in combination with Tau, and wherein the expression of said transgenes in the nervous system results in said Drosophila having a predisposition to, or resulting in, progressive neural degeneration.
- double transgenic fly refers to a transgenic fly whose somatic and germ cells comprise at least two transgenes, wherein the transgenes encode the Tau and human A ⁇ 42 ⁇ OW a, A ⁇ 42 Dute h, A ⁇ 42 F iemish. A ⁇ 42 ⁇ t aiian. or A ⁇ 42Arctic. It will be readily apparent to one of skill in the art that a "transgenic fly” comprising the human A ⁇ 42A-c t i c mutation also expresses Tau, and will thus necessarily be a "double transgenic fly”.
- transgenic fly and “double transgenic fly” include all developmental stages of the fly, i.e., embryonic, larval, pupal, and adult stages.
- the development of Drosophila is temperature dependent.
- the Drosophila egg is about half a millimeter long. It takes about one day after fertilization for the embryo to develop and hatch into a worm-like larva. The larva eats and grows continuously, molting one day, two days, and four days after hatching (first, second and third instars).
- Drosophila refers to any member of the Drosophilidae family, which include without limitation, Drosophila funebris, Drosophila multispina, Drosophila subfunebris, guttifera species group, Drosophila guttifera, Drosophila albomicans, Drosophila annulipes, Drosophila curviceps, Drosophila formosana, Drosophila hypocausta, Drosophila immigrans, Drosophila keplauana, Drosophila kohkoa, Drosophila nasuta, Drosophila neohypocausta, Drosophila niveifrons, Drosophila pallidiftons, Drosophila pulaua, Drosophila quadrilineata, Drosophila siamana, Drosophila
- a ⁇ 42 ⁇ owa A ⁇ 42 Du tch.
- a ⁇ 42 F ⁇ em ish.
- a ⁇ 42 lta iian. and A ⁇ 42Arctic is used to refer to a mutant form of the 42-amino acid polypeptide that is produced in nature through the proteolytic cleavage of human amyloid precursor protein (APP) by beta and gamma secretases.
- APP amyloid precursor protein
- a ⁇ 42 Flem i_h, A ⁇ 42 ⁇ ta ii an , or A ⁇ 42A ⁇ ctic differs from wildtype A ⁇ 42 in that it contains a Asp23Asn mutation (SEQ ID NO: 1), a Glu22Gln mutation (SEQ ID NO: 2), a Ala21Gly mutation (SEQ ID NO: 3), a Glu22Lys mutation (SEQ ID NO: 4), or a Glu22Gly mutation (SEQ ID NO: 5), respectively.
- a ⁇ 42 is a major component of extracellular amyloid plaque depositions found in neuronal tissue of Alzheimer's disease patients.
- a ⁇ 42 ltal i an , or A ⁇ 42A- c tic includes a peptide encoded by a recombinant DNA wherein a nucleotide sequence encoding A ⁇ 42iowa, A ⁇ 42 Du tch.
- a ⁇ 42 F ⁇ emis h, A ⁇ 42 Ital ia--. or A ⁇ 42A-ctic is operatively linked to an expression control sequence such that the A ⁇ 42 l0W a, A ⁇ 42 Du tch.
- a ⁇ 42 Arct i c peptide is produced in the absence of cleavage of APP by beta and gamma secretase. It is noted that, because of the degeneracy of the genetic code, different nucleotide sequences can encode the same polypeptide sequence.
- amyloid plaque depositions refers to insoluble protein aggregates that are formed extracellularly by the accumulation of amyloid peptides, such as A ⁇ 42.
- signal peptide refers to a short amino acid sequence, typically less than 20 amino acids in length, which directs proteins to or through the endoplasmic reticulum secretory pathway of Drosophila.
- signal peptides include, but are not limited to, the Drosophila signal peptides of Dint protein synonymous to "wingless (wg) signal peptide” (SEQ ID NO: 11) and the “Argos (aos) signal peptide” (SEQ ID NO: 12), the Drosophila Appl (SEQ ID NO: 13), presenilin (SEQ ID NO: 14), or wind milk (SEQ ID NO: 15).
- Any conventional signal sequence that directs proteins through the endoplasmic reticulum secretory pathway can be used in the present invention.
- amino acid linker refers to a short amino acid sequence from about 2 to 10 amino acids in length that is flanked by two individual peptides.
- tau protein refers to the microtubule-associated protein Tau that is involved in microtubule assembly and stabilization, hi neuronal tissues of Alzheimer's disease patients, Tau is found in intracellular depositions of neurofibrillary tangles.
- the human gene that encodes the human Tau protein contains 11 exons, and is described by Andreadis, A. et al., Biochemistry, 31 (43):10626-10633 (1992), herein incorporated by reference. In adult human brain, six tau isoforms are produced from a single gene by alternative mRNA splicing.
- Tau protein includes various Tau isoforms produced by alternative mRNA splicing as well as mutant forms of a sequence encoding human Tau proteins as described in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID 21.
- the Tau protein used to generate the double transgenic fly is represented by SEQ ID NO: 16 (amino acid sequence) and SEQ ID NO: 17 (nucleotide sequence).
- SEQ ID NO: 16 amino acid sequence
- SEQ ID NO: 17 nucleotide sequence
- Tau protein includes Tau protein recognized by these conformation specific-antibodies.
- Tau protein also includes Tau proteins containing mutations and variants. These mutations include but are not limited to: Exon 10 +12 "Kumamoto pedigree” (Yasuda et al., (2000) Ann Neurol. 47: 422-9); I260V (Grover et al., Exp Neurol. 2003 Nov ; 184(l):131-40); G272V (Hutton et al., 1998 Nature 393:702-5; Heutink et al., (1997) Ann Neurol.
- Tau gene sequences exist in adult human brain, six tau isoforms are produced from a single gene by alternative mRNA splicing (Goedert et al., Neuron. 1989 3:519-26). It is noted that, because of the degeneracy of the genetic code, different nucleotide sequences can encode the same polypeptide sequence.
- the invention further contemplates the use of Tau genes containing sequence polymorphisms (See, for example, Table 1).
- the invention also contemplates the use of Tau proteins or genes from other animals, including but not limited to mice (Lee et al., (1988) Science 239, 285-8), rats (Goedert et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89 (5), 1983-1987), Bos taurus (Himmler et al, (1989) Mol. Cell. Biol. 9 (4), 1381-1388), Drosophila melanogaster (Heidary & Fortini, (2001) Mech. Dev. 108 (1-2), 171-178) andXenopus l ⁇ evis (Olesen et al., (2002) Gene 283 (1-2), 299-309).
- the Tau genes from other animals may additionally contain mutations equivalent to those previously described. Equivalent positions can be identified by sequence alignment, and equivalent mutations can be introduced by means of site-directed mutagenesis or other means known in the art.
- neuroofibrillary tangles refers to insoluble twisted fibers that form intracellularly and that are composed mainly of Tau protein.
- operatively linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
- An expression control sequence "operatively linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the activity of the control sequences.
- the term "expression control sequence” refers to promoters, enhancer elements, and other nucleic acid sequences that contribute to the regulated expression of a given nucleic acid sequence.
- promoter refers to DNA sequences recognized by RNA polymerase during initiation of transcription and can include enhancer elements.
- the term “enhancer element” refers to a cz-?-acting nucleic acid element, which controls transcription initiation from homologous as well as heterologous promoters independent of distance and orientation.
- an “enhancer element” also controls the tissue and temporal specification of transcription initiation.
- enhancer elements include, but are not limited to, the UAS control element.
- UAS refers to an Upstream Activating Sequence recognized and bound by the Gal4 transcriptional activator.
- a "tissue specific” expression control sequence refers to expression control sequences that drive expression in one tissue or a subset of tissues, while being essentially inactive in at least one other tissue. "Essentially inactive” means that the expression of a sequence operatively linked to a tissue specific expression control sequence is less than 5% of the level of expression of that sequence in that tissue where the expression control sequence is most active.
- tissue specific expression control sequences include those that are specific for organs such as the eye, wing, notum, brain, as well as tissues of the central and peripheral nervous systems. Examples of tissue specific control sequences include, but are not limited to, the sevenless promoter/enhancer (Bowtell et al., Genes Dev. 2(6):620-34 (1988)); the eyeless promoter/enhancer (Bowtell et al., Proc. Natl. Acad. Sci. U.S.A.
- expression control sequences include, but are not limited to the heat shock promoters/enhancers from the hsp70 and hsp83 genes, useful for temperature induced expression; and promoters/enhancers derived from ubiquitously expressed genes, such as tubulin, actin, or Ubiquitin.
- phenotype refers to an observable and/or measurable physical, behavioral, or biochemical characteristic of a fly.
- altered phenotype refers to a phenotype that has changed relative to the phenotype of a wild-type fly. Examples of altered phenotypes include a behavioral phenotype, such as appetite, mating behavior, and/or life span, that has changed by a measurable amount, e.g. by at least 10%, 20%, 30%, 40%, or more preferably 50%, relative to the phenotype of a control fly; or a morphological phenotype that has changed in an observable way, e.g.
- a synergistic altered phenotype refers to a phenotype wherein a measurable and/or observable physical, behavioral, or biochemical characteristic of a fly is more than the sum of its components.
- a “change in phenotype” or “change in altered phenotype,” as used herein, means a measurable and/or observable change in a phenotype relative to the phenotype of a control fly.
- the "rough eye” phenotype is characterized by irregular ommatidial packing, occasional ommatidial fusions, and missing bristles that can be caused by degeneration of neuronal cells.
- the eye becomes rough in texture relative to its appearance in wild type flies, and can be easily observed by microscope.
- the "concave wing" phenotype is characterized by abnormal folding of the fly wing such that wings are bent upwards along their long margins.
- locomotor dysfunction refers to a phenotype where flies have a deficit in motor activity or movement (e.g., at least a 10% difference in a measurable parameter) as compared to control flies.
- Motor activities include flying, climbing, crawling, and turning.
- movement traits where a deficit can be measured include, but are not limited to: i) average total distance traveled over a defined period of time; ii) average distance traveled in one direction over a defined period of time; iii) average speed (average total distance moved per time unit); iv) distance moved in one direction per time unit; v) acceleration (the rate of change of velocity with respect to time; vi) turning; vii) stumbling; viii) spatial position of a fly to a particular defined area or point; ix) path shape of the moving fly; and x) undulations during larval movement; xi) rearing or raising of larval head; and xii) larval tail flick.
- Examples of movement traits characterized by spatial position include, without limitation: (1) average time spent within a zone of interest (e.g., time spent in bottom, center, or top of a container; number of visits to a defined zone within container); and (2) average distance between a fly and apoint of interest (e.g., the center of a zone).
- Examples of path shape traits include the following: (1) angular velocity (average speed of change in direction of movement); (2) turning (angle between the movement vectors of two consecutive sample intervals); (3) frequency of turning (average amount of turning per unit of time); and (4) stumbling or meander (change in direction of movement relative to the distance).
- Turning parameters can include smooth movements in turning (as defined by small degrees rotated) and/or rough movements in turning (as defined by large degrees rotated).
- control fly refers to a larval or adult fly of the same genotype of the transgenic fly as to which it is compared, except that the control fly either i) does not comprise one or both of the transgenes present in the transgenic fly, or ii) has not been administered a candidate agent.
- the term “candidate agent” refers to a biological or chemical compound that when administered to a transgenic fly has the potential to modify the phenotype of the fly, e.g. partial or complete reversion of the altered phenotype towards the phenotype of a wild type fly.
- Agents as used herein can include any recombinant, modified or natural nucleic acid molecule, library of recombinant, modified or natural nucleic acid molecules, synthetic, modified or natural peptide, library of synthetic, modified or natural peptides; and any organic or inorganic compound, including small molecules, or library of organic or inorganic compounds, including small molecules.
- small molecule refers to compounds having a molecular mass of less than 3000 Daltons, preferably less than 2000 or 1500, more preferably less than 1000, and most preferably less than 600 Daltons.
- a small molecule is a compound other than an oligopeptide.
- a “therapeutic agent” refers to an agent that ameliorates one or more of the symptoms of a neurodegenerative disorder such as Alzheimer's disease in mammals, particularly humans.
- a therapeutic agent can reduce one or more symptoms of the disorder, delay onset of one or more symptoms, or prevent or cure the disease.
- a ⁇ 42 lta iian. or A ⁇ 42Arctic (phis Tau) as well as a double transgenic fly carrying both the Tau protein and the mutant human A ⁇ 42 ⁇ owa>
- a ⁇ 42 ⁇ ta ii an. or A ⁇ 42Arctic peptide are disclosed.
- the transgenic flies provide a model for neurodegenerative disorders such as Alzheimer's disease, which is characterized by an extracellular accumulation of A ⁇ 42 ⁇ owa peptide and an intracellular deposition of a hyperphosphorylated form of microtubule-associated protein Tau.
- the transgenic flies of the present invention can be used to screen for therapeutic agents effective in the treatment of Alzheimer's disease.
- transgenic flies of the present invention can be generated by any means known to those skilled in the art. Methods for production and analysis of transgenic Drosophila strains are well established and described in Brand et al., Methods in Cell Biology 44:635-654 (1994); Hay et al., Proc. Natl. Acad. Sci. USA 94(10):5195-200 (1997); and in Robert D.B. Drosophila: A Practical Approach, Washington D.C. (1986), herein incorporated by reference in their entireties.
- a transgene of interest is stably incorporated into a fly genome.
- Any fly can be used, however a preferred fly of the present invention is a member of the Drosophilidae family.
- An exemplary fly is Drosophila Melanogaster.
- transformation vectors are useful for the generation of the transgenic flies of the present invention, and include, but are not limited to, vectors that contain transposon sequences, which mediate random integration of transgene into the genome, as well as vectors that use homologous recombination (Rong and Golic, Science 288: 2013-2018 (2000)).
- a preferred vector of the present invention is pUAST (Brand and Perrimon, Development 118:401-415 (1993)) that contains sequences from the transposable P-element which mediate insertion of a transgene of interest into the fly genome.
- Another preferred vector is PdL that is able to yield doxycycline-dependent overexpression (Nandis, Bhole and Tower, Genome Biology 4 (R8):l-14, (2003)).
- P-element transposon mediated transformation is a commonly used technology for the generation of transgenic flies and is described in detail in Spradling, P element mediated transformation, In Drosophila: A Practical Approach (ed. D. B. Roberts), pp#175-197, IRL Press, Oxford, UK (1986), herein incorporated by reference.
- Other transformation vectors based on transposable elements include for example, the hobo element (Blackman et al., Embo J.
- the terminal repeat sequences of the transposon that are required for transposition are incorporated into a transformation vector and arranged such that the terminal repeat sequences flank the transgene of interest.
- the transformation vector contains a marker gene used to identify transgenic animals.
- marker genes affect the eye color of Drosophila, such as derivatives of the Drosophila white gene (Pirrotta V., & C. Brockl, EMBO J. 3(3):563-8 (1984)) or the Drosophila rosy gene (Doyle W. et al., Eur. J Biochem. 239(3):782-95 (1996)) genes.
- Any gene that results in a reliable and easily measured phenotypic change in transgenic animals can be used as a marker.
- marker genes used for transformation include the yellow gene (Wittkopp P. et al., Curr Biol. 12(18):1547-56 (2002)) that alters bristle and cuticle pigmentation; the forked gene (McLachlan A., Mol Cell Biol. 6(l).T-6 (1986)) that alters bristle morphology; the Adh+ gene used as a selectable marker for the transformation o ⁇ Adh- strains (McNabb S.
- Plasmid constructs for introduction of the desired transgene are coinjected into Drosophila embryos having an appropriate genetic background, along with a helper plasmid that expresses the specific transposase needed to mobilize the transgene into the genomic DNA.
- Animals arising from the injected embryos (GO adults) are selected, or screened manually, for transgenic mosaic animals based on expression of the marker gene phenotype and are subsequently crossed to generate fully transgenic animals (Gl and subsequent generations) that will stably carry one or more copies of the transgene of interest.
- Binary systems are commonly used for the generation of transgenic flies, such as the UAS/GAL4 system.
- transgenic Drosophila termed "target" lines, are generated where the gene of interest (e.g. A ⁇ 42 Iowa> A ⁇ 42 Du tch. A ⁇ 42 F ⁇ emish , A ⁇ 42 ⁇ ta iian. or A ⁇ 42A ⁇ ctic and/or TAU)) is operatively linked to an appropriate promoter controlled by UAS.
- the gene of interest e.g. A ⁇ 42 Iowa> A ⁇ 42 Du tch.
- driver lines Other transgenic Drosophila strains, termed “driver” lines, are generated where the GAL4 coding region is operatively linked to promoters/enhancers that direct the expression of the GAL4 activator protein in specific tissues, such as the eye, antenna, wing, or nervous system.
- the gene of interest is not expressed in the "target” lines for lack of a transcriptional activator to "drive” transcription from the promoter joined to the gene of interest.
- the UAS- target line is crossed with a GAL4 driver line, the gene of interest is induced.
- the resultant progeny display a specific pattern of expression that is characteristic for the GAL4 line.
- the present invention discloses transgenic flies that have incorporated into their genome a DNA sequence that encodes a mutant human A ⁇ 42 ⁇ O a, A ⁇ 42outch. A ⁇ 42 F ⁇ em ish. A ⁇ 42 lta iia n . or A ⁇ 42 Arct i c fused to a signal peptide, as well as double transgenic flies which comprise a DNA sequence that encodes the Tau protein as well as a DNA sequence encoding the mutant human A ⁇ 42 ⁇ owa , A ⁇ 42 DutC h, A ⁇ 42 F ⁇ em i-- ⁇ , A ⁇ 42 ⁇ ta ii a n, or A ⁇ 42A- c tic fused to a signal peptide.
- transgenic Drosophila that express either the A ⁇ 42 l0 w a> A ⁇ 42 Dut c h , A ⁇ 42 F ⁇ emish , or A ⁇ 42 ⁇ ta ii an , or the Tau protein, or A ⁇ 42 rc t ic and the Tau protein are independently made and then crossed to generate a Drosophila that expresses both proteins.
- transgenic Drosophila are produced using the UAS/GAL4 control system.
- a DNA sequence encoding Tau is cloned into a vector such that the sequence is operatively linked to the GAL4 responsive element UAS.
- Vectors containing UAS elements are commercially available, such as the pUAST vector (Brand and Perrimon, Development 118:401-415 (1993)), which places the UAS sequence element upstream of the transcribed region.
- the DNA is cloned using standard methods (Sambrook et al., Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y.
- the vector After cloning the DNA into appropriate vector, such as pUAST, the vector is injected into Drosophila embryos (e.g. yw embryos) by standard procedures (Brand et al., Methods in Cell Biology 44:635-654 (1994)); Hay et al., Proc. Natl. Acad. Sci. USA 94(10):5195-200 (1997) to generate transgenic Drosophila.
- Drosophila embryos e.g. yw embryos
- standard procedures Brand et al., Methods in Cell Biology 44:635-654 (1994)
- Hay et al. Proc. Natl. Acad. Sci. USA 94(10):5195-200 (1997) to generate transgenic Drosophila.
- the transgenic progeny can be crossed with Drosophila driver strains to assess the presence of an altered phenotype.
- a preferred Drosophila comprises the eye specific driver strain g r-GAL4, which enables identification and classification of transgenics flies based on the severity of the rough eye phenotype.
- Expression of Tau in Drosophila eye results in the rough eye phenotype (characterized by an eye with irregular ommatidial packing, occasional ommatidial fusions, and missing bristles), which can be easily observed by microscope.
- the severity of the rough eye phenotype exhibited by a transgenic line can be classified as strong, medium, or weak.
- the weak or mild lines have a rough, disorganized appearance covering the ventral portion of the eye.
- the medium severity lines show greater roughness over the entire eye, while in strong severity lines the entire eye seems to have lost/fused many of the ommatidia and interommatidial bristles, and the entire eye has a smooth, glossy appearance.
- a ⁇ 42 lta iian, or A ⁇ 42Arctic is ligated in frame to a DNA sequence encoding a signal peptide such that the A ⁇ 42 ⁇ owa , A ⁇ 42D tch.
- a ⁇ 42 F ⁇ emis h, A ⁇ 42 ⁇ tal i an , or A ⁇ 42Arctic peptide can be exported across cell membranes.
- the signal sequence is directly linked to the A ⁇ 42 Iowa , A ⁇ 42 D utch, A ⁇ 42 F ⁇ emish , A ⁇ 42 ⁇ ta iia n , or A ⁇ 42Arctic coding sequence or indirectly linked by using a DNA linker sequence, for example of 3, 6, 9, 12, or 15 nucleotides.
- a signal peptide that directs proteins to or through the endoplasmic reticulum - secretory pathway of Drosophila is used.
- Preferred signal peptides of the present invention are the Argos (aos) signal peptide (SEQ ID NO: 12), the wingless (wg) signal peptide (SEQ ID NO: 11) the Drosophila Appl (SEQ ID NO: 13), presenilin (SEQ ID NO: 14), and wind zucchini (SEQ ID NO: 15).
- a ⁇ 42 Ifa ⁇ ian , or A ⁇ 42 rctic peptide is linked to a signal sequence by standard ligation techniques and is then cloned into a vector such that the sequence is operatively linked to the GAL4 responsive element UAS.
- a ⁇ 42 lta iian . or A ⁇ 42Arctic transgenic flies is the pUAST vector (Brand and Perrimon, Development 118:401-415 (1993)).
- the vector is injected into Drosophila embryos (e.g. yw embryos) by standard procedures (Brand et al., Meth. in Cell Biol. 44:635-654 (1994)); Hay et al., Proc. Natl. Acad. Sci. USA 94(10):5195-200 (1997)) and progeny are then selected and crossed based on the phenotype of the selected marker gene.
- Drosophila driver strains are gmr-GAL4 (eye) and e ⁇ v-GAL4 (CNS).
- neurodegeneration in the eye is believed to give rise to the rough eye phenotype; neurodegeneration in the wing (i.e., neuromuscular degeneration) is believed to give rise to morphological wing abnormalities such as the concave wing; and neurodegeneration in the CNS or PNS is believed to give rise to numerous locomotive and behavioral phenotypes.
- Abberant overexpression of the A ⁇ 42 and/or Tau sequences may be evaluated by screening flies for phenotypic changes which are commensurate with the tissue-specific expression of the sequence as dictated by a particular expression control sequence. For example, were the gmr, sevenless, eyeless, or rhodopsin- derived eye-specific promoter/enhancer is used to direct expression in the eye, a phenotype such as the rough-eye phenotype is expected to be observed. Where an enhancer/promoter derived from the dpp or vestigial genes is used to direct expression in the wing, a phenotype such as the concave wing is expected to be obaerved.
- a promoter/enhancer derived from elav, Appl, or nirvana is used to direct expression in the central nervous system, or a promoter/enhancer derived from neural specific D42 genes is used
- neurological, locomotor, and/or behavioral phenotypes can be expected to be obeserved.
- the converse approach is also contemplated.
- an eye phenotype e.g., rough eye phenotype
- a gmr-GKL driver strain is used in the cross. Ectopic overexpression of mutant A ⁇ 42 lowa , A ⁇ 42Dutch. A ⁇ 42Fie m ish.
- a ⁇ 42 ⁇ ta ii an , or A ⁇ 42Arctic in Drosophila eye is believed to disrupt the regular trapezoidal arrangement of the photoreceptor cells of the ommatidia (identical single units, forming the Drosophila compound eye), the severity of which is believed to depend on transgene copy number and expression levels.
- a locomotor phenotype e.g., climbing assay
- an elav (or other neural specific promoter)-Gal4 driver strain is used in the cross. Ectopic overexpression of mutant A ⁇ 42 Iowa , A ⁇ 42Dutch, A ⁇ 42 F ⁇ e mish.
- a ⁇ 42it a ii an , or A ⁇ 42 rct i c in Drosophila central nervous system (CNS) is believed to result in locomotor deficiencies, such as impaired movement, climbing and flying.
- CNS Drosophila central nervous system
- a wing phenotype e.g., concave wing
- a dpp- or ve-?t-gt-. -Gal4 driver strain is used in the cross.
- a ⁇ 42 ⁇ ta tian, or A ⁇ 42A-ctic in Drosophila wing is believed to result in a concave wing phenotype, evidenced by abnormal folding of the fly wing such that wings are bent upwards along their long margins.
- transgenic flies can be crossed with each other by mating. Flies are crossed according to conventional methods. When the binary UAS/GAL4 system is used, the fly is crossed with an appropriate driver strain and the altered phenotype assessed, as described above, transgenic flies are classified by assessing phenotypic severity. For example, as disclosed herein, the combination of Tau and mutant A ⁇ 42 lowa , A ⁇ 42 Du tch. A ⁇ 42 F i em i s h . A ⁇ 42 ⁇ ta ii a n . or A ⁇ 42 A rctic transgenes is believed to produce a synergistic effect on the eye.
- a ⁇ 42 ⁇ taii an , or A ⁇ 42Ar C tic proteins in transgenic flies is confirmed by standard techniques, such as Western blot analysis or by immunostaining of Drosophila tissue cross-sections, both of which are described below. a. Western Blot analysis
- Western blot analysis is performed by standard methods. Briefly, as means of example, to detect expression of the A ⁇ 42 peptide or Tau by western blot analysis, whole flies, or Drosophila heads (e.g. 80-90 heads) are collected and placed in an eppendorf tube on dry ice containing 100 ⁇ l of 2% SDS, 30% sucrose, 0.718 M Bistris, 0. 318 M Bicine, with "Complete" protease inhibitors (Boehringer Mannheim), then ground using a mechanical homogenizer. Samples are heated for 5 min at 95° C, spun down for 5 min at 12,000 rpm, and supernatants are transferred into a fresh eppendorf tube.
- 6E10 (Senetek PLC Napa, CA.) are hybridized, generally at a concentration of 1:2000, in 5% non-fat milk, l ⁇ PBS containing 0.1% Tween 20, for 90 min at room temperature. Samples are washed 3 times for 5 min., 15 min. and 15 min. each, in 1 x PBS-0.1% Tween-20. Labeled secondary antibody, (for example, anti-mouse-HRP from Amersham Pharmacia Biotech, NA 931) is prepared, typically at a concentration of 1:2000, in 5% non- fat milk, lx PBS containing 0.1% Tween 20, for 90 min at room temperature. Samples are then washed 3 times for 5 min., 15 min. and 15 min.
- Labeled secondary antibody for example, anti-mouse-HRP from Amersham Pharmacia Biotech, NA 931
- ECL ECL Western Blotting Detection Reagents, Amersham Pharmacia Biotech, # RPN 2209
- Drosophila organ cross sections As a manner of confirming protein expression in transgenic flies, immunostaining of Drosophila organ cross sections is performed. Such a method is of particular use to confirm the presence of hyperphosphorylated Tau, which is a modified form of the Tau protein that is present in non-diseased tissue. Hyperphosphorylated Tau exhibits altered pathological conformations as compared to Tau protein and is present in diseased tissue from patients with certain neurodegenerative disorders, such as Alzheimer's disease.
- Cross sections of Drosophila organs can be made by any conventional cryosectioning, such as the method described in Wolff, Drosophila Protocols, CSHL Press (2000), herein incorporated by reference.
- Cryosections can then be immunostained for detection of Tau and A ⁇ 42 peptides using methods well known in the art.
- the Vectastain ABC Kit which comprises biotinylated anti-mouse IgG secondary antibody, and avidin/biotin conjugated to the enzyme Horseradish peroxidase H (Vector Laboratories) is used to identify the protein.
- the secondary antibody is conjugated to a fluorophore.
- cryosections are blocked using normal horse serum, according to the Vectastain ABC Kit protocol.
- the primary antibody recognizing the human A ⁇ 42 peptide or Tau, is typically used at a dilution of 1 :3000 and incubation with the secondary antibody is done in PBS/1%BSA containing 1-2% normal horse serum, also according to the Vectastain ABC Kit protocol. The procedure for the ABC Kit is followed; incubations with the ABC reagent are done in PBS/0.1% saponin, followed by 4x10 minute washes in PBS/0.1% saponin.
- Sections are then incubated in 0.5 ml per slide of the Horseradish Peroxidase H substrate solution, 400 ug/ml 3,3'-diaminobenzidene (DAB), 0.006% H 202 in PBS/0.1% saponin, and the reaction is stopped after 3 min. with 0.02% sodium azide in PBS. Sections are rinsed several times in PBS and dehydrated through an ethanol series before mounting in DPX (Fluka).
- DAB 3,3'-diaminobenzidene
- Exemplary antibodies that can be used to immunostain cross sections include but are not limited to, the monoclonal antibody 6E10 (Senetek PLC Napa, CA.) that recognizes A ⁇ 42 peptide and anti-Tau antibodies ALZ50 and MCI (Jicha GA, et al., J. of Neurosci. Res. 48:128-132 (1997)).
- antibodies for use in the present invention that recognize A ⁇ 42 and Tau can be made using standard protocols known in the art (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
- a mammal, such as a mouse, hamster, or rabbit can be immunized with an immunogenic form of the protein (e.g., a A ⁇ 42 or Tau polypeptide or an antigenic fragment which is capable of eliciting an antibody response).
- Immunogens for raising antibodies are prepared by mixing the polypeptides (e.g., isolated recombinant polypeptides or synthetic peptides) with adjuvants.
- a ⁇ 42 or Tau polypeptides or peptides are made as fusion proteins to larger immunogenic proteins.
- Polypeptides can also be covalently linked to other larger immunogenic proteins, such as keyhole limpet hemocyanin.
- plasmid or viral vectors encoding A ⁇ 42 or Tau, or a fragment of these proteins can be used to express the polypeptides and generate an immune response in an animal as described in Costagliola et al., J. Clin. Invest. 105:803-811 (2000), which is incorporated herein by reference.
- immunogens are typically administered intradermally, subcutaneously, or intramuscularly to experimental animals such as rabbits, sheep, and mice.
- genetically engineered antibody derivatives can be made, such as single chain antibodies.
- the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
- Standard ELISA, flow cytometry or other immunoassays can also be used with the immunogen as antigen to assess the levels of antibodies.
- Antibody preparations can be simply serum from an immunized animal, or if desired, polyclonal antibodies can be isolated from the serum by, for example, affinity chromatography using immobilized immunogen.
- antibody-producing splenocytes can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
- immortalizing cells such as myeloma cells.
- Such techniques are well known in the art, and include, for example, the hybridoma technique (originally developed by Kohler and Milstein, Nature, 256: 495-497 (1975)), the human B cell hybridoma technique (Kozbar et al, Immunology Today, 4: 72 (1983)), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96(1985)).
- Hybridoma cells can be screened immunochemically for production of antibodies that are specifically reactive with A ⁇ 42 or Tau peptide, or polypeptide, and monoclonal antibodies isolated from the media of a culture comprising
- DNA sequences that encode Tau or human A ⁇ 42 l0W a, A ⁇ 42Dutch, A ⁇ 42Fi em is .
- a ⁇ 42 ⁇ ta ii an , or A ⁇ 42Arctic are cloned into transformation vectors suitable for the generation of transgenic flies.
- A. Generation of DNA sequences encoding Tau or human A ⁇ 42 DNA sequences encoding Tau and A ⁇ 42 ⁇ owa> A ⁇ 42 Du tc , A ⁇ 42 F iemis h. A ⁇ 42 ItaHan , or A ⁇ 42Arctic can be obtained from genomic DNA or be generated by synthetic means using methods well known in the art (Sambrook et al., Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y. (1989); Ausubel, et al., Current protocols in Molecular Biology, . Greene Publishing, Y, (1995)).
- human genomic DNA can be isolated from peripheral blood or mucosal scrapings by phenol extraction, or by extraction with kits such as the QIAa p Tissue kit (Qiagen, Chatsworth, Cal.), Wizard genomic DNA purification kit (Promega, Madison, Wis.), and the ASAP genomic DNA isolation kit (Boehringer Mannheim, Indianapolis, Ind.). DNA sequences encoding Tau and A ⁇ 42 lowa> A ⁇ 42 Dutch. A ⁇ 42 F iemish.
- a ⁇ 42 ⁇ ta ii an , or A ⁇ 42 Arctic can then be amplified from genomic DNA by polymerase chain reaction (PCR) (Mullis and Faloona Methods Enzymol, 155: 335 (1987)), herein incorporated by reference) and cloned into a suitable recombinant cloning vector.
- PCR polymerase chain reaction
- a ⁇ 42 ⁇ taKan . or A ⁇ 42 Arctic can be amplified from mRNA using RT-PCR and cloned into a suitable recombinant cloning vector.
- RNA may be prepared by any number of methods known in the art; the choice may depend on the source of the sample.
- sequences that encode Tau or A ⁇ 42 l0W a, A ⁇ 42 Du t C h.
- a ⁇ 42 F ⁇ emiS h, A ⁇ 42 ⁇ tal ian, or A ⁇ 42A-ctic by PCR or RT-PCR that the sequences are cloned into an appropriate sequencing vector in order that the sequence of the cloned fragment can be confirmed by nucleic acid sequencing in both directions.
- Suitable recombinant cloning vectors for use in the present invention contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells.
- this sequence is one that enables the vector to replicate independently of the host chromosomal DNA and includes origins of replication or autonomously replicating sequences.
- origins of replication or autonomously replicating sequences are well known for a variety of bacteria, yeast and viruses.
- the origin of replication from the plasmid pBR322 is suitable for most Gram- negative bacteria
- the 2 micron plasmid origin is suitable for yeast
- various viral origins e.g. SV40, adenovirus
- the origin of replication is not needed for mammalian expression vectors unless these are used in mammalian cells able to replicate high levels of DNA, such as COS cells.
- a cloning or expression vector may contain a selection gene also referred to as a selectable marker.
- This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will therefore not survive in the culture medium.
- Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.
- E. c ⁇ /z-selectable marker for example, the ⁇ -lactamase gene that confers resistance to the antibiotic ampicillin.
- E. coli plasmids such as pBR322 or a pUC plasmid such as pUC18 or pUC19.
- a ⁇ 42 F ⁇ e mish, A ⁇ 42 1 taii an . or A ⁇ 42 Arctic can also be directly cloned into a transformation vector suitable for generation of transgenic Drosophila such as vectors that allow for the insertion of sequences in between transposable elements, or insertion downstream of an UAS element, such as pUAST.
- Vectors suitable for the generation of transgenic flies preferably contain marker genes such that the transgenic fly can be identified such as, the white gene, the rosy gene, the yellow gene, the forked gene, and others mentioned previously.
- Suitable vectors can also contain tissue specific control sequences as described earlier, such as, the sevenless promoter/enhancer, the eyeless promoter/enhancer, ⁇ /ass-responsive promoters (gwr)/enhancers useful for expression in the eye; and enhancers/promoters derived from the dpp or vestigial genes useful for expression in the wing.
- tissue specific control sequences such as, the sevenless promoter/enhancer, the eyeless promoter/enhancer, ⁇ /ass-responsive promoters (gwr)/enhancers useful for expression in the eye; and enhancers/promoters derived from the dpp or vestigial genes useful for expression in the wing.
- DNA sequences that encode Tau or human A ⁇ 42 Iowaj A ⁇ 42 Dutch , A ⁇ 42 F ⁇ em i Sh , A ⁇ 42 lt aii a n, or A ⁇ 42Arctic can be generated through the use of Polymerase chain reaction (PCR), or RT-PCR which uses RNA-directed DNA polymerase (e.g., reverse transcriptase) to synthesize cDNAs which is then used for PCR.
- PCR Polymerase chain reaction
- RT-PCR RNA-directed DNA polymerase
- a double transgenic fly according to the invention can exhibit an altered eye phenotype, of progressive neurodegeneration in the eye that leads to measurable morphological changes in the eye (Fernandez-Funez et al., Nature 408:101-106 (2000); Steffan et. al, Nature 413:739-743 (2001)).
- the Drosophila eye is composed of a regular trapezoidal arrangement of seven visible rhabdomeres produced by the photoreceptor neurons of each Drosophila ommatidium.
- a phenotypic eye mutant according to the invention leads to a progressive loss of rhabdomeres and subsequently a rough-textured eye.
- a rough textured eye phenotype is easily observed by microscope or video camera, hi a screening assay for compounds which alter this phenotype, one may observe slowing of the photoreceptor degeneration and improvement of the rough-eye phenotype (Steffan et. al, Nature 413:739-743 (2001)).
- a transgenic or double transgenic fly according to the invention can exhibit an altered wing phenotype, believed to be rooted in neuromuscular degeneration in the wing, leading to measurable morphological changes in the wing structure.
- a concave wing phenotype may be easily observed by microscope, video camera, or other suitable imaging means.
- Neuronal degeneration in the central nervous system will give rise to behavioral deficits, including but not limited to locomotor deficits, that can be assayed and quantitated in both larvae and adult Drosophila.
- behavioral deficits including but not limited to locomotor deficits
- Drosophila adult animals to climb in a standard climbing assay (see, e.g. Ganetzky and Flannagan, J. Exp. Gerontology 13:189-196 (1978); LeBourg and Lints, J. Gerontology 28:59-64 (1992)) is quantifiable, and indicative of the degree to which the animals have a motor deficit and neurodegeneration.
- Neurodegenerative phenotypes include, but are not limited to, progressive loss of neuromuscular control, e.g.
- Drosophila behavior that can be assayed include but are not limited to circadian behavioral rhythms, feeding behaviors, inhabituation to external stimuli, and odorant conditioning. All of these phenotypes are measured by one skilled in the art by standard visual observation of the fly.
- Another neural degeneration phenotype is a reduced life span, for example, the Drosophila life span can be reduced by 10-80%, e.g., approximately, 30%, 40%), 50%, 60%, or 70%.
- Any observable and/or measurable physical or biochemical characteristic of a fly is a phenotype that can be assessed according to the present invention.
- Transgenic flies can be produced " by identifying flies that exhibit an altered phenotype as compared to control (e.g., wild-type flies, or flies in which the transgene is not expressed).
- Therapeutic agents can be identified by screening for agents, that upon administration, result in a change in an altered phenotype of the transgenic fly as compared to a transgenic fly that has not been administered a candidate agent.
- a change in an altered phenotype includes either complete or partial reversion of the phenotype observed.
- Complete reversion is defined as the absence of the altered phenotype, or as 100% reversion of the phenotype to that phenotype observed in control flies.
- Partial reversion of an altered phenotype can be 5%, 10%, 20%, preferably 30%, more preferably 50%, and most preferably greater than 50% reversion to that phenotype observed in control flies.
- Example measurable parameters include, but are not limited to, size and shape of organs, such as the eye; distribution of tissues and organs; behavioral phenotypes (such as, appetite and mating); and locomotor ability, such as can be observed in a climbing assays.
- locomotor ability can be assessed by placing flies in a vial, knocking them to the bottom of the vial, then counting the number of flies that climb past a given mark on the vial during a defined period of time. 100% locomotor activity of control flies is represented by the number of flies that climb past the given mark, while flies with an altered locomotor activity can have 80%, 70%, 60%, 50%, preferably less than 50%, or more preferably less than 30% of the activity observed in a control fly population. Locomotor phenotypes also can be assessed as described in provisional application 60/396,339, Methods for Identifying Biologically Active Agents, herein incorporated by reference.
- locomotor dysfunction phenotypes which may be measured according to the invention include deficits in motor activity or movement (e.g., at least a 10% difference in a measurable parameter) as compared to control flies.
- Motor activities include flying, climbing, crawling, and turning.
- movement traits where a deficit can be measured include, but are not limited to: i) average total distance traveled over a defined period of time; ii) average distance traveled in one direction over a defined period of time; iii) average speed (average total distance moved per time unit); iv) distance moved in one direction per time unit; v) acceleration (the rate of change of velocity with respect to time; vi) turning; vii) stumbling; viii) spatial position of a fly to a particular defined area or point; ix) path shape of the moving fly; and x) undulations during larval movement; xi) rearing or raising of larval head; and xii) larval tail flick.
- Examples of movement traits characterized by spatial position include, without limitation: (1) average time spent within a zone of interest (e.g., time spent in bottom, center, or top of a container; number of visits to a defined zone within container); and (2) average distance between a fly and a point of interest (e.g., the center of a zone).
- Examples of path shape traits include the following: (1) angular velocity (average speed of change in direction of movement); (2) turning (angle between the movement vectors of two consecutive sample intervals); (3) frequency of turning (average amount of turning per unit of time); and (4) stumbling or meander (change in direction of movement relative to the distance).
- Turning parameters can include smooth movements in turning (as defined by small degrees rotated) and/or rough movements in turning (as defined by large degrees rotated).
- Locomoter defects in a fly may be measured using methods known in the art, or by taking measurements including, but not limited to: a) total distance (average total distance traveled over a defined period of time); b) X only distance (average distance traveled in X direction over a defined period of time; c) Y only distance (average distance traveled in Y direction over a defined period of time); d) average speed (average total distance moved per time unit); e) average X-only speed (distance moved in X direction per time unit); f) average Y-only speed (distance moved in Y direction per time unit); g) acceleration (the rate of change of velocity with respect to time); h) turning; i) stumbling; j) spatial position of one animal to a particular defined area or point (examples of spatial position traits include (1) average time spent within a zone of
- path shape of the moving animal i.e., a geometrical shape of the path traveled by the animal
- path shape traits include the following: (1) angular velocity (average speed of change in direction of movement); (2) turning (angle between the movement vectors of two consecutive sample intervals); (3) frequency of turning (average amount of turning per unit of time); (4) stumbling or meandering (change in direction of movement relative to the distance); and the like. This is different from stumbling as defined above.
- Turning parameters may include smooth movements in turning (as defined by small degrees rotated) and or rough movements in turning (as defined by large degrees rotated).
- the transgenic flies of the invention provide a model for neurodegeneration as is found in human neurological diseases such as Alzheimer's and tauopathies, such as Amyotrophic lateral sclerosis/ arkinsonism-dementia complex of Guam Argyrophilic grain dementia, Corticobasal degeneration, Dementia pugilistica, Diffuse neurofibrillary tangles with calcification, Frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP- 17), Pick's disease, Progressive subcortical gliosis, Progressive supranuclear palsy (PSP), Tangle only dementia, Creutzfeldt- Jakob disease, Down syndrome, Gerstmann-Straussler- Scheinker disease, Hallervorden-Spatz disease, Myotonic dystrophy, Age-related memory impairment, Alzheimer's disease , Amyotrophic lateral sclerosis, Amyotrophic lateral/parkinsonism-dementia complex of Guam, Auto-imm
- the present invention further provides a method for identifying a therapeutic agent for neurodegenerative disease using the transgenic flies disclosed herein.
- a therapeutic agent refers to an agent that ameliorates the symptoms of neurodegenerative disease as determined by a physician.
- a therapeutic agent can reduce one or more symptoms of neurodegenerative disease, delay onset of one or more symptoms, or prevent, or cure.
- a candidate agent is administered to a transgenic fly.
- the transgenic fly is then assayed for a change in the phenotype as compared to the phenotype displayed by a control transgenic fly that has not been administered a candidate agent.
- An observed change in phenotype is indicative of an agent that is useful for the treatment of disease.
- a candidate agent can be administered by a variety of means.
- an agent can be administered by applying the candidate agent to the Drosophila culture media, for example by mixing the agent in Drosophila food, such as a yeast paste that can be added to Drosophila cultures.
- the candidate agent can be prepared in a 1% sucrose solution, and the solution fed to Drosophila for a specified time, such as 10 hours, 12 hours, 24 hours, 48 hours, or 72 hours.
- the candidate agent is microinjected into Drosophila hemolymph, as described in WO 00/37938, published June 29, 2000.
- Other modes of administration include aerosol delivery, for example, by vaporization of the candidate agent.
- the candidate agent can be administered at any stage of Drosophila development including fertilized eggs, embryonic, larval and adult stages.
- the candidate agent is administered to an adult fly. More preferably, the candidate agent is administered during a larval stage, for example by adding the agent to the Drosophila culture at the third larval instar stage, which is the main larval stage in which eye development takes place.
- the agent can be administered in a single dose or multiple doses.
- Appropriate concentrations can be determined by one skilled in the art, and will depend upon the biological and chemical properties of the agent, as well as the method of administration.
- concentrations of candidate agents can range from 0.0001 ⁇ M to 20 mM when delivered orally or through injection, 0.1 ⁇ M to 20 mM, l ⁇ M-10 mM, or 10 ⁇ M to 5 mM.
- the candidate agents can be administered as a mixture or population of agents, for example a library of agents.
- a "library” of agents is characterized by a mixture more than 2O, 100, 10 3 , 10 4 , 10 5 , 10 6 , 10 8 , 10 12 , or 10 15 individual agents.
- a "population of agents” can be a library or a smaller population such as, a mixture less than 3, 5, 10, or 20 agents.
- a population of agents can be administered to the transgenic flies and the flies can be screened for complete or partial reversion of a phenotype exhibited by the transgenic flies. When a population of agents results in a change of the transgenic fly phenotype, individual agents of the population can then be assayed independently to identify the particular agent of interest.
- a high throughput screen of candidate agents is performed in which a large number of agents, at least 50 agents, 100 agents or more are tested individually in parallel on a plurality of fly populations.
- a fly population contains at least 2, 10, 20, 50, 100, or more adult flies or larvae.
- locomotor phenotypes, behavioral phenotypes (e.g. appetite, mating behavior, and/or life span), or morphological phenotypes (e.g., shape size, or location of a cell, or organ, or appendage; or size shape, or growth rate of the fly) are observed by creating a digitized movie of the flies in the population and the movie is analyzed for fly phenotype.
- Agents that are useful in the screening assays of the present inventions include biological or chemical compounds that when administered to a transgenic fly have the potential to modify an altered phenotype, e.g. partial or complete reversion of the phenotype.
- Agents include any recombinant, modified or natural nucleic acid molecule; library of recombinant, modified or natural nucleic acid molecules; synthetic, modified or natural peptides; library of synthetic, modified or natural peptides; organic or inorganic compounds; or library of organic or inorganic compounds, including small molecules. Agents can also be linked to a common or unique tag, which can facilitate recovery of the therapeutic agent.
- Example agent sources include, but are not limited to, random peptide libraries as well as combinatorial chemistry-derived molecular library made of D-and/or L- configuration amino acids; phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang et al., Cell 72:767- 778 (1993)); antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti- idiotypic, chimeric or single chain antibodies, and FAb, F(ab')2 and FAb expression library fragments, and epitope-binding fragments thereof); and small organic or inorganic molecules.
- phosphopeptides including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang et al., Cell 72:767- 778 (1993)
- antibodies including, but not limited to, polyclonal, monoclon
- libraries are known in the art that can be used, e.g. chemically synthesized libraries, recombinant libraries (e.g., produced by phage), and in vitro translation-based libraries.
- chemically synthesized libraries are described in Fodor et al., Science 251 :767- 773 (1991); Houghten et al., Nature 354:84-86 (1991); Lam et al., Nature 354:82-84 (1991); Medyuski, Bio/Technology 12:709-710 (1994); Gallop et al., J. Medicinal Chemistry 37(9):1233-1251 (1994); Ohlmeyer et al., Proc. Natl. Acad. Sci.
- a benzodiazopine library (see e.g., Bunin et al., Proc. Natl. Acad. Sci. USA 91 :4708-4712 (1994)) can be adapted for use.
- Peptoid libraries (Simon et al., Proc. Natl. Acad. Sci. USA 89:9367-9371 (1992)) can also be used.
- Examples of phage display libraries wherein peptide libraries can be produced are described in Scott & Smith, Science 249:386-390 (1990); Devlin et al., Science, 249:404-406 (1990); Christian et al., J. Mol. Biol.
- Agents that can be tested and identified by methods described herein can include, but are not limited to, compounds obtained from any commercial source, including Aldrich (Milwaukee, Wl 53233), Sigma Chemical (St. Louis, MO), Fluka Chemie AG (Buchs, Switzerland) Fluka Chemical Corp. (Ronkonkoma, NY;), Eastman Chemical Company, Fine Chemicals (Kingsport, TN), Boehringer Mannheim GmbH (Mannheim, 25 Germany), Takasago (Rockleigh, NJ), SST Corporation (Clifton, NJ), Ferro (Zachary, LA 70791), Riedel-deHaen Aktiengesellschaft (Seelze, Germany), PPG Industries Inc., Fine Chemicals (Pittsburgh, PA 15272). Further any kind of natural products may be screened using the methods described herein, including microbial, fungal, plant or animal extracts.
- libraries may be commercially obtained from Specs and BioSpecs B.V. (Rijswijk, The Netherlands), Chembridge Corporation (San Diego, CA), Contract Service Company (Dolgoprudoy, Moscow Region, Russia), Comgenex USA Inc. (Princeton, NJ), Maybridge Chemicals Ltd. (Cornwall PL34 OHW, United Kingdom), and Asinex (Moscow, Russia).
- combinatorial library methods known in the art, can be utilized, including, but not limited to: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one- compound” library method; and synthetic library methods using affinity chromatography selection.
- the biological library approach is limited to peptide libraries, while the other approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des.12: 145 (1997)).
- Combinatorial libraries of test compounds including small molecule test compounds, can be utilized, and may, for example, be generated as disclosed in Eichler & Houghten, Mol. Med. Today 1:174-180 (1995); DoUe, Mol. Divers. 2:223-236 (1997); and Lam, Anticancer Drug Des. 12:145-167 (1997).
- a library of agents can also be a library of nucleic acid molecules; DNA, RNA, or analogs thereof.
- a cDNA library can be constructed from mRNA collected from a cell, tissue, organ or organism of interest, or genomic DNA can be treated to produce appropriately sized fragments using restriction endonucleases or methods that randomly fragment genomic DNA.
- a library containing RNA molecules can be constructed, for example, by collecting RNA from cells or by synthesizing the RNA molecules chemically. Diverse libraries of nucleic acid molecules can be made using solid phase synthesis, which facilitates the production of randomized regions in the molecules. If desired, the randomization can be biased to produce a library of nucleic acid molecules containing particular percentages of one or more nucleotides at a position in the molecule (U.S. Pat. No. 5,270,163).
- a transgenic Drosophila melanogaster strain containing a transgene encoding Tau and a transgenic Drosophila melanogaster strain containing a transgene encoding human A ⁇ 42 ⁇ OWa , A ⁇ 42Duteh, A ⁇ 42 F ⁇ em i S , A ⁇ 42 ⁇ tal i an , or A ⁇ 42 Arc ic peptide are generated as described herein.
- the two transgenic fly strains are then crossed to obtain a double transgenic Drosophila melanogaster strain containing both Tau and human A ⁇ 42 ⁇ owa , A ⁇ 42r- Utcr ⁇ , A ⁇ 42 F ⁇ em i sh. A ⁇ 42 ⁇ ta iian. or A ⁇ 42Arctic genes.
- the UAS/GAL4 system are used to generate both the A ⁇ 42 lowa , A ⁇ 42 DutCh , A ⁇ 42Fie ⁇ msh.
- a ⁇ 42 ⁇ ta vi a r 1 , or A ⁇ 42Arctic and Tau transgenic flies.
- a cDNA encoding the longest human brain Tau isoform is cloned using standard ligation techniques (Sambrook et al., Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y. 1989) into vector pUAST (Brand and Perrimon, Development 118:401-415 (1993)) as an EcoRI fragment in order to generate transformation vector, pUAS: 2N4R Tauwt.
- the Tau isoform which is represented by SEQ ID NO: 17 (nucleic acid sequence), and SEQ ID NO: 16 (amino acid sequence) contains Tau exons 2 and 3 as well as four microtubule-binding repeats.
- Two pUAST transformation vectors carrying DNA sequences encoding the A ⁇ 42 lowa , A ⁇ 42 Dutch , A ⁇ 42 F ie m is , A ⁇ 42 Italian , or A ⁇ 42 A rc ic peptide (SEQ ID NO: 6, 7, 8, 9, or 10, respectively) are generated.
- One vector encodes A ⁇ 42 l0W a, A ⁇ 42 Du tch.
- a ⁇ 42 Ita ii an , or A ⁇ 42Arctic peptide is first fused, in frame, to a synthetic oligonucleotide encoding the wingless (wg) signal peptide using a 4 amino acid linker (SFAM).
- the resulting DNA sequence is then cloned as an EcoRI fragment into vector pUAST (Brand and Perrimon, Development 118:401-415 (1993).
- the Argos (aos) signal peptide (SEQ ID NO: 12) is PCR amplified from DNA encoding Argos and ligated in frame, to DNA encoding A ⁇ 42 ⁇ OW a, A ⁇ 42 DutC h, A ⁇ 42 F ⁇ emish , A ⁇ 42 Ital i an , or A ⁇ 42Arctic in the absence of a linker sequence.
- the DNA encoding Argos (aos) signal peptide fused in frame to A ⁇ 42 l0Wa , A ⁇ 42 Dutcr ⁇ , A ⁇ 42 F ⁇ em i s , A ⁇ 42 ⁇ ta ii ar ⁇ , or A ⁇ 42 A --tic is cloned iiito pUAST (Brand and Perrimon, Development 118:401- 415 (1993)) as an EcoRI fragment.
- a ⁇ 42 F ie m is .
- a ⁇ 42 lta Han. or A ⁇ 42 rctic the pUAST constructs described above, either 1 l i e pUAS: ⁇ s , -A ⁇ 42, or pUAS: 2N4 _ t Tauwt are injected into ay w Drosophila Melanogaster embryos as described in (Rubin and Spradling, Science 218:348-353, 1982).
- transgenic lines are generated and classified by visual inspection, as described herein, as strong, medium, and weak based on the severity of the eye phenotype observed after crossing with a g- r-GAL4 driver strain.
- pUASr ⁇ as- A ⁇ 42 ⁇ owa A ⁇ 42 Du t C h, A ⁇ 42Fiemish.
- a ⁇ 42 ⁇ tali a a consequence of transgenic lines.
- Transgenic Drosophila strains of moderate eye phenotype that carry the gmr-GAIA driver and pUAS :aos- A ⁇ 42 ⁇ owa> A ⁇ 42 Dut ch, A ⁇ 42 F ⁇ er m-h, A ⁇ 42 ⁇ ta ⁇ ian , or A ⁇ 42Arctic or pUAS: 2N4R.
- Tauwt are then crossed to generate a double transgenic Drosophila line that express both Tau and human A ⁇ 42 ⁇ owa , A ⁇ 42 Dutcr ⁇ , A ⁇ 42 F ⁇ em is--, A ⁇ 42 ⁇ t a iian, or A ⁇ 42Arctic peptide.
- transgenic lines are generated by injecting the construct into ay x w ⁇ Drosophila Melanogaster embryos as described in (Rubin and Spradling, Science 218:348-353, 1982) and screened for the insertion of transgene into genomic DNA by monitoring eye color.
- the pUAST vector carries the white gene marker.
- a ⁇ 42 ⁇ ta ij an , or A ⁇ 42Arctic transgene are then crossed with elav-Gal4 driver strains for expression of the transgene in the central nervous system. If the crosses do not result in a measurable phenotype, the transgene is mobilized for expansion of copy number by crossing Transgenic Drosophila carrying wg- A ⁇ 42iowa, A ⁇ 42 Du tch. A ⁇ 42 F iemish. A ⁇ 42 ⁇ taiian. or A ⁇ 42Arctic transgene with Drosophila that carry a source of P-element. Progeny from this cross are selected based on a change in eye color.
- Flies carrying higher copy numbers of wg- A ⁇ 42 lowa , A ⁇ 42 Dutch , A ⁇ 42 Fle mi Sh , A ⁇ 42 ⁇ t a ⁇ ian , or A ⁇ 42Arctic transgene are then crossed with elav-Ga ⁇ 4 driver strains and locomotor ability of the crossed flies is tested in climbing assays.
- Transgenic lines may exhibit a locomotor phenotype and the flies are classified as strong, medium, weak and very weak (28 lines) as compared among themselves and to el ⁇ v-Ga ⁇ 4 driver control flies.
- a double transgenic Drosophila carrying wg- A ⁇ 42 ⁇ owa , A ⁇ 42 DutC h, A ⁇ 42 F ⁇ e mi-h, A ⁇ 42 ⁇ t a ⁇ ian , or A ⁇ 42Arctic and Tauwt transgenes is then generated by crossing a Tauwt transgenic Drosophila carrying an e/ ⁇ v-Gal4 driver, with an wg- A ⁇ 42 ⁇ owa , A ⁇ 42 Dutch.
- Locomotor ability is assessed and classified as strong, medium, weak and very weak as compared to elav-Ga ⁇ 4 driver control flies.
- Climbing performance as a function of age is determined for populations of flies of various genotypes at 27°C. Climbing assays are performed in duplicate (two groups of 30 individuals of the same age.
- Drosophila brain is then cyrosectioned, and horizontal cross sections of e/ ⁇ v-GAL4; Tauwt/wg- A ⁇ 42 ⁇ owa , A ⁇ 42 DutC h, A ⁇ 42 F ⁇ emisr ⁇ , A ⁇ 42 It aiian, or A ⁇ 42Arctic flies are immunostained with anti-Tau conformation dependent antibodies ALZ50 and MCI. Positive staining of neurons may be observed with both MCI antibody (data not shown) and ALZ50 antibody. The result is expected to show that Tau protein, which is expressed in the brain of A ⁇ 42/Tau double transgenic Drosophila, exhibits protein conformations associated with Alzheimer's disease.
- Thioflavin-S staining is also performed on cells and neurites of the transgenic flies, described herein, to assess the presence of amyloid. Amyloids, when stained with Thioflavin-S, fluoresce an apple green color under a fluorescent microscope. The methods for Thioflavin-S staining are well known in the art. All flies are developed at 27°C. Thioflavin-S positive cells are not expected to be observed in flies expressing Tau only. Thioflavin-S positive cells are expected to be observed in flies expressing A ⁇ 42 l0W a, A ⁇ 42 Du tc , A ⁇ 42 F ⁇ ern i Sh , A ⁇ 42itaiian. or A ⁇ 42 Ar ctic only.
- the number of Thioflavin-S- positive cells is expected to be much greater in flies expressing both Tau and A ⁇ 42 lowa> A ⁇ 42 Dut ch, A ⁇ 42 F ⁇ ernish , A ⁇ 42 lta ⁇ ian , or A ⁇ 42 A rctic
- candidate agents are administered to a plurality of the A ⁇ 42 ⁇ owa> A ⁇ 42Du ch.
- Candidate agents are microinjected into third instar transgenic Drosophila melanogaster larvae (three to 5 day old larvae). Larvae are injected through the cuticle into the hemolymph with defined amounts of each compound using a hypodermic needle of 20 gm internal diameter. Following injection, the larvae are placed into glass vials for completion of their development. After eclosion, the adult flies are anesthetized with C0 2 and visually inspected utilizing a dissecting microscope to assess for the reversion of the Drosophila eye phenotype as compared to control flies in which a candidate agent was not administered. An observed reversion of the A ⁇ 42 ⁇ owa , A ⁇ 42Dutch.
- a ⁇ 42 lta ⁇ ia n, or A ⁇ 42 Arct i c /Tau transgenic fly eye phenotype towards the phenotype displayed by the control g r-GAL4 driver strain is indicative of an agent that is useful for the treatment of Alzheimer's disease.
- Flies are trained by exposure to electroshock (12 pulses at 60 V, duration of 1.5 seconds, interval of 5 seconds) paired with one odor (benzaldehyde (BA, 4%) or methylcyclohexanol (MCH, 10°) for 60 seconds) and subsequent exposure to a second odor without electroshock.
- the odor concentrations are adjusted to assume no preference for flies exposed simultaneously to the two odors before the training.
- learning is measured by allowing flies to choose between the two odors used during training. No preference between odors results in zero (no learning) performance index (PI). Avoidance of the odor previously paired with electroshock is expected to produce a 0 ⁇ PI ⁇ 1.00 (see Tully, T. and Quinn, W. G., J. Comp. Physiol. A Sens. Neural. Behav. Physiol ., 157:263- 277 (1985)).
Abstract
Description
Claims
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EP04795934A EP1684574A4 (en) | 2003-10-20 | 2004-10-21 | Transgenic flies expressing mutant alpfa42 |
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US10/853,593 US20040250302A1 (en) | 2003-10-21 | 2004-05-25 | Transgenic flies expressing Abeta42-dutch |
US10/852,973 | 2004-05-25 | ||
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US10/852,973 US20040255342A1 (en) | 2003-10-21 | 2004-05-25 | Transgenic flies expressing Abeta42-Iowa |
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US10/852,892 US20040244064A1 (en) | 2003-10-20 | 2004-05-25 | Transgenic flies expressing Abeta42-Flemish |
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US10/853,593 | 2004-05-25 | ||
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EP1684574A4 (en) | 2008-08-06 |
CA2543058A1 (en) | 2005-05-12 |
AU2004284937A1 (en) | 2005-05-12 |
EP1684574A1 (en) | 2006-08-02 |
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