Huntington's disease (HD) is a dominant hereditary neurodegenerative disease that is caused by the expansion of a stretch of CAG repeats within the HD gene that encodes a large protein (huntingtin; htt) of unknown function. HD is thought to be the consequence of a deleterious gain-of-function that is conferred by the expanded stretch of polyglutamine encoded by the CAG repeats. The role of the normal function of htt in the disease process is unknown, but our recent work and that of others suggests that loss of normal htt function may also contribute to pathogenesis. Our long-term objective is to use genetic approaches to understand the role of htt's normal functions in HD pathogenesis using both cell culture and mouse models. To accomplish this objective, we propose three complementary specific aims that are designed to test the potential contribution of different loss-of-function mechanisms in HD.
A fourth aim i s designed to test a potential therapeutic strategy based on restoring normal htt function in HD mouse models. (1) To test the hypothesis that loss-of-function in HD may occur through mutant htt's ability to sequester wild-type htt via the polyglutamine stretch, we will generate an epitope-tagged allele of the mouse HD gene homologue (Hdh-deltaQ) that lacks precisely the polyglutamine stretch. The ability of this modified version of htt to resist sequestration by mutant htt will be assessed in cell culture. In addition, in order to test if htt is capable of participating in potential dominant-negative interactions by interacting with itself, an ES cell line with targeted insertion of different epitope tags in each Hdh allele will be generated for use in immunoprecipitation pull-down assays. (2) To test if htt loss-of-function may occur through mutant htt's ability to activate caspase-mediated proteolysis, and if proteolytic cleavage of htt is a rate-limiting step in HD pathogenesis, we will compare the onset and progression of phenotypes exhibited by two knockin HD mouse models: the first expressing a full-length mutant htt, and the second expressing a truncated version of mutant htt. Both mutant proteins are expressed under the control of the endogenous Hdh promoter, enabling a direct comparison between the two models. (3) Htt loss-of-function may also occur via dominant-negative interference of wild-type htt interactions with protein partners. To test this hypothesis in vivo, we will characterize the impact of losing htt interactions with the postsynaptic density 95 protein that could lead to altered N-methyl-D-aspartate (NMDA) receptor function in an Hdh conditional knockout mouse model. (4) Finally, we will attempt to rescue phenotypes in an HD mouse model by over-expressing a temporally regulated dominant-negative resistant form of htt in the forebrain.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Molecular, Cellular and Developmental Neurosciences 2 (MDCN)
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Sutherland, Margaret L
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University of Virginia
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André, Emily A; Braatz, Elise M; Liu, Jeh-Ping et al. (2017) Generation and Characterization of Knock-in Mouse Models Expressing Versions of Huntingtin with Either an N17 or a Combined PolyQ and Proline-Rich Region Deletion. J Huntingtons Dis 6:47-62
Ochaba, Joseph; Lukacsovich, Tamás; Csikos, George et al. (2014) Potential function for the Huntingtin protein as a scaffold for selective autophagy. Proc Natl Acad Sci U S A 111:16889-94
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Culver, Brady P; Savas, Jeffrey N; Park, Sung K et al. (2012) Proteomic analysis of wild-type and mutant huntingtin-associated proteins in mouse brains identifies unique interactions and involvement in protein synthesis. J Biol Chem 287:21599-614
Fang, C; Bolivar, V J; Gu, J et al. (2012) Neurobehavioral abnormalities in a brain-specific NADPH-cytochrome P450 reductase knockout mouse model. Neuroscience 218:170-80
Neveklovska, Michelle; Clabough, Erin B D; Steffan, Joan S et al. (2012) Deletion of the huntingtin proline-rich region does not significantly affect normal huntingtin function in mice. J Huntingtons Dis 1:71-87
Klionsky, Daniel J (see original citation for additional authors) (2012) Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8:445-544
Liu, Jeh-Ping; Zeitlin, Scott O (2011) The long and the short of aberrant ciliogenesis in Huntington disease. J Clin Invest 121:4237-41
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