Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
2000-08-14
2004-11-09
Falk, Anne-Marie (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S003000, C800S009000
Reexamination Certificate
active
06815575
ABSTRACT:
TECHNICAL FIELD
This invention relates to an animal model that exhibits polyglutamine toxicity, and more particularly to methods for identifying genes that modulate polyglutamine toxicity using Drosophila.
BACKGROUND
Expansion of polyCAG tracts is associated with human hereditary neurodegenerative disorders and neuronal toxicity (Kaytor et al.,
J Bio. Chem
., 274:37507-37510 (1999)). Huntington's disease and several other hereditary neurodegenerative disorders are characterized by expansion of a polyglutamine sequence (LaSpada et al.,
Nature
, 352:77-79 (1991); Koide et al.,
Nat. Genet
., 6:9-13 (1994); Kawaguchi et al.,
Nat. Genet
., 8:221-228 (1994); Orr et al.,
Nat. Genet
., 4:221-226 (1993); Sanpei et al.,
Nat. Genet
., 14:277-284 (1996); and Zhuchenko et al.,
Nat. Genet
., 15:62-69 (1997)). The expanded polyCAG tracts encode abnormally long polyglutamine sequences within specific proteins promoting their nuclear and/or cytoplasmic aggregation. The protein aggregation is believed to contribute to cellular toxicity including cell death or apoptosis (Trottier et al., Nature, 378:403-406 (1995); Davies et al., Cell, 90:537-548 (1997); and DiFiglia et al., Science, 277:1990-1993 (1997)).
The mechanism of toxicity and cell death by expanded polyglutamines is not yet fully understood. Peptides containing expanded polyglutamine tracts are prone to forming cytoplasmic (CIs) and/or nuclear inclusions (NIs). Two variables appear as major determinants of the aggregation propensity, subcellular localization or toxicity of polyglutamine-containing peptides. The relative length of the polyglutamine tract determines the aggregation propensity and cytotoxicity; the longer it is, the more likely it is to form inclusions and cause cell death. The overall size of the peptide determines subcellular localization as well as aggregation propensity and cytotoxicity; shorter, truncated gene products with expanded repeats are more likely to form inclusions, and these inclusions are more likely to be in the nucleus than in the cytoplasm. These inclusions occasionally recruit their full-length counterpart.
Perinuclear inclusions produced by truncated huntingtin peptides recruit endogenous huntingtin in transfected human kidney epithelial 293Tcells (HEK 293T). Cotransfection of truncated ataxin-3 (SCA3 gene product) with its full-length counterpart, containing either a normal or an expanded polyglutamine tract, resulted in the recruitment of either of the two full-length proteins into perinuclear inclusions formed by the truncated ataxin-3. However, this type of recruitment was not observed in HD brains. In another set of experiments, huntingtin was recruited to neuritic plaques, neurofibrillary tangles and dystrophic neurites in Alzheimer's disease, and to Pick bodies found in Pick disease. Heteromerous aggregates were also formed between co-expressed ataxin-1, with normal or expanded polyglutamine, and ataxin-3 with an expanded polyglutamine repeat in transfected HEK 293T.
Experiments in mouse striatal cell culture and transgenic mice suggested that nuclear localization was necessary for the pathogenic effects. On the other hand, experiments in a human embryonic kidney cell line suggested that polyglutamine can be equally cytotoxic in the cytoplasm or the nucleus. Furthermore, in cultured mouse clonal striatal cells or in SCA1 transgenic mice, aggregation of polyglutamines appeared to be neither sufficient nor necessary for pathogenesis. When NI formation was suppressed in neurons transfected with mutant huntingtin, cell death increased.
The molecular components of the pathways involved in neuronal degeneration and protein aggregation have been investigated. These include: components of protein folding (Cummings et al.,
Nat Genet
, 19:148-154 (1998); Wyttenbach et al.,
Proc. Natl. Acad Sci. USA
, 97:2898-2903 (2000); and Kobayashi et al.,
J. Biol. Chem
., 275:8772-8778 (2000)), protein degradation (Chai et al., Hum.
Mol. Genet
., 8:673-682 (1999)), gene expression (Boutell et al.,
Hum. Mol. Genet
., 8:1647-1655 (1999); Kazantsev et at,
Proc. Natl. Acad. Sci. USA
, 96:11404-11409 (1999); and Li et al.,
J. Neurosci
., 19:5159-5172 (1999)), and programmed cell death (Portera et al.,
J. Neurosci
., 3775-3787 (1995); Wellington et al.,
J Biol. Chem
., 273:9158-9167 (1998); and Ona et al.,
Nature
, 399:263-267 (1999)), as well as interacting proteins (Kaichman et al.,
Nat, Genet
, 16:44-53 (1997); Sittler. et al., Mol. Cell, 2:427-436 (1998); Waragai et al.,
Hum. Mol Genet
., 8:977-987 (1999)), neurotransmitters, and their receptors (Cha et al.,
Proc. Natl. Acad. Sci. USA
, 95:6480-6485 (1998); Chen et a.,
J. Neurosci
., 72:1890-1898 (1999); and Reynolds et al.,
J. Neurochem
., 72:1773-1776 (1999)). A Drosophila model has recapitulated abnormal protein aggregation and neuronal toxicity associated with polyglutamine disorders, and a candidate heat shock gene has been shown to have a suppressing effect (Warrick et a.,
Cell
, 93:939-949 (1998); Jackson et al.,
Neuron
, 21:633-642 (1998); Marsh et al.,
Hum. Mol Genet
., 9:13-25 (2000); and Kazemi-Esfarjani,
Science
, 287:1837-1840 (2000)). The present invention is based upon an alternative animal model that mimics polyglutamine and/or protein folding abnormalities observed in humans.
SUMMARY
The present invention relates to an animal model useful for identifying molecules that modulate expression or activity of proteins involved in polyglutamine toxicity, neuronal and other degenerative disorders, cancer and other proliferative disorders in humans. This animal model is also useful for identifying molecules that modulate disorders associated with undesirable or aberrant protein folding, aggregation, degradation or aberrant transport. Such molecules include genes and other compounds that modulate protein aggregation or folding and associated disorders, including polyglutamine toxicity and polyglutamine related disorders.
A genetic screen using a Drosophila animal model of the invention identified in vivo genetic modulators of polyglutamine toxicity. Three Drosophila genes, heat shock protein 40/HDJ1 (dHDJ1), tetratricopeptide repeat protein 2 (dTPR2) and myeloid leukemia factor 1 (dMLF), were capable of decreasing polyglutamine toxicity in affected flies. Thus, the Drosophila genes or their mammalian homologues and other compounds identified using an in vivo animal model of the invention can be used as therapeutics in treating polyglutamine toxicity and associated disorders in humans. A method of the invention, and the genes and compounds identified, are also applicable for the identification and treatment of disorders associated with other diseases that result from or are associated with intracellular or extracellular protein misfolding/aggregation. Particular examples include Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jacob's disease (CJD), bovine spongiform encephalopathy, Huntington's disease (HD), Machado-Joseph disease (MJD), Spinocerebellar ataxias (SCA), dentatorubropallidoluysian atophy (DRPLA), Kennedy's disease, stroke and head trauma. In addition, as the human homologues of dTPR2 and DMLF (TPR2 and MLF, respectively) are associated with tumorigenesis (neurofibromatosis 1) and leukemias (myelodysplastic syndrome and acute myeloid leukemias), respectively, these genes, and the flies carrying dTPR2 and dMLF P-element insertions or their transgenic versions, will be helpful in identifying cancer therapeutics.
In accordance with the present invention, there are provided methods of screening for genes or compounds that modulate polyglutamine toxicity. In one embodiment, a method of the invention includes providing a first animal expressing a polyglutamine sequence, wherein the sequence produces polyglutamine toxicity in the animal; breeding the first animal to a second animal, wherein the second animal has a marker sequence inserted into its germline, thereby producing progeny; screening the progeny for increased or decreased polyglutamine toxicity relative to the first animal thereb
Benzer Seymour
Kazemi-Esfarjani Parsa
California Institute of Technology
Falk Anne-Marie
Sullivan Daniel M.
LandOfFree
Animal model of polyglutamine toxicity, methods of use, and... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Animal model of polyglutamine toxicity, methods of use, and..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Animal model of polyglutamine toxicity, methods of use, and... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3298028