George R. Jackson - US grants

Huntington's disease (HD) is caused by expansion of a polymorphic CAG repeat within exon 1 of huntingtin, a gene of unknwon function. In HD and mouse and cell culture models, huntingtin fragments containing the polyglutamine tract undergo progressive nuclear aggregation. We have established a Drosophila model of HD that shares important features of the human phenotype, including age- and repeat-length-dependent neuronal degeneration and death, as well progressive nuclear localization of expanded repeat-containing protein. The similarities between the polyglutamine-expanded phenotype in humans and flies suggest that the molecular mechanisms underlying polyglutamine-induced cell death are, at least in part, conserved from Drosophila to man. The long-term objective of the proposed research is to utilize our Drosophila model of HD to unravel the molecular mechanisms of polyglutamine-induced cell death in an effort to identify therapeutic targets. A three-pronged approach will be used: 1. The role of nuclear aggregation of polygluytamine-containing protein on cytotoxicity in vivo will be assessed by examining the distribution of various epitope-tagged constructs over time and by expressing these constructs in the presence of a nuclear export signal. We will also examine the interaction of polyglutamine-expanded fragments with both pathologic and wild type repeat lengths presented as truncated fragments or within the full length protein. 2. The role of identified genes in Drosophila that may modify polyglutamine-induced neuronal cell death will be examined by expressing the Q120 transgene in a background homozygous for mutations in these genes. We will assess potential disease-modifying genes by expressing the Q120 construct in a genetic mosaic background, including patches homozygous for the mutation of interest. 3. A large-scale genetic screen for mutations that alter the photoreceptor degeneration associated with Q120 expression will be used to identify enhancers and suppressors of polyglutamine-induced cell death. Mutagenized males will be crossed to Q120-bearing females. Mutations affecting degeneration will be scored by examining the pseudopupil pattern and by scoring reversion to the wild type response in a UV choice test. Mutations will be localized and cloned using the technizue of "local hopping." Suppression or enhancement will be verified by co-expressing such mutations with Q120 lines and in inducible cell culture systems.

ABSTRACT NOT AVAILABLE

DESCRIPTION (provided by applicant): Mutations in the microtubule-associated protein tau occur in some cases of inherited frontotemporal dementia (FTD), demonstrating that tau abnormalities can cause neurodegeneration. Many FTD mutations occur in regulatory elements that alter splicing and thereby expression of tau isoforms, rather than tau coding sequence. One of the hallmark neuropathologic features of Alzheimer's disease (AD) is the neurofibrillary tangle (NFT), which contains hyperphosphorylated tau. Characterization of the events that modify tau-associated neurodegenerative processes is critical for understanding the pathophysiology of AD, as well as FTD and related diseases, such as progressive supranuclear palsy and corticobasal degeneration, and for the development of therapeutics. In order to test the hypothesis that neurodegeneration can be caused by aberrant expression of wild-type tau, the longest isoform of human tau was overexpressed in the fruit fly, producing degeneration of the eye and underlying brain, but failing to produce neurofibrillary tangles. However, tau phosphorylation by co-expression of shaggy, the Drosophila homologue of glycogen synthase kinase (GSK)-3beta, an important tau kinase in vitro, produced a more severely degenerated eye, as well as lesions resembling NFT (Jackson, G.R., et al. (2002): Human wild-type tau interacts with wingless pathway components and produces neurofibrillary pathology in Drosophila. Neuron 34: 509-519). Here, we will examine whether Shaggy is incorporated into NFT-like lesions in double tau + Shaggy transgenics. We will examine the role of puromycin-sensitive aminopeptidase, which was identified in a pilot screen, as a tau modifier. Finally, we will perform loss of function and gain of function genetic screens in order to identify novel modifiers of the abnormal Drosophila phenotype associated with expression of hyperphosphorylated tau. Modifier genes will be characterized and evaluated as targets for the development of new therapies aimed at alleviating neurofibrillary pathology and neuronal cell death in AD and other neurodegenerative disorders.

Mutations in the microtubule-associated protein tau occur in some cases of inherited frontotemporal dementia (FTD), demonstrating that tau abnormalities can cause neurodegeneration. Many FTD mutations occur in regulatory elements that alter splicing and thereby expression of tau isoforms, rather than tau coding sequence. One of the hallmark neuropathologic features of AIzheimer's disease (AD) is the neurofibrillary tangle (NFT), which contains hyperphosphorylated tau. Characterization of the events that modify tau-associated neurodegenerative processes is critical for understanding the pathophysiology of AD, as well as FTD and related diseases, such as progressive supranuclear palsy and corticobasal degeneration, and for the development of therapeutics. In order to test the hypothesis that neurodegeneration can be caused by aberrant expression of wild-type tau, the longest isoform of human tau was overexpressed in the fruit fly, producing degeneration of the eye and underlying brain, but failing to produce neurofibrillary tangles. However, tau phosphorylation by co-expression of shaggy, the Drosophila homologue of glycogen synthase kinase (GSK)-3beta, an important tau kinase in vitro, produced a more severely degenerated eye, as well as lesions resembling NFT (Jackson, G.R., et al. (2002): Human wild-type tau interacts with wingless pathway components and produces neurofibrillary pathology in Drosophila. Neuron 34: 509-519). Here, we propose to test selected kinase inhibitors, caspase inhibitors, and antioxidants for their ability to suppress the dual tau + shaggy bioassay phenotype. We also will test a library of commercially available, FDA-approved compounds (Microsource Discovery Systems) for their ability to suppress the external eye phenotype of tau + shaggy flies. Subsequently, we will determine the effects of identified compounds on cell death and conformation and solubility of tau. This project contributes to the commitment of the UCLA Alzheimer's Disease Research Center (ADRC) to foster research that will lead to identification of disease-modifying therapies for AD and related conditions. The Project will interact with Project 2 assessing similar compounds in transgenic mice.