The long term objective of this project is to understand the role of ubiquilin in the pathogenesis of late-onset Alzheimer's disease (AD). AD, the most common age-related dementia, is associated with extracellular deposition and aggregation of proteolytic fragments of amyloid precursor protein (APP). It is believed that these changes are associated with progressive neuronal dysfunction, dementia, and death. Therefore, understanding the molecular events directing the proteolytic processing of APP is essential to understanding the pathogenesis of this disease. Although the mechanism(s) of altered APP processing is (are) beginning to be understood in cases of early-onset familial AD, an overwhelming majority of AD cases are sporadic, or late-onset, with unknown etiology. Recent studies have reported a genetic link between late-onset AD and polymorphisms in the gene encoding ubiquilin. Ubiquilin contains a ubiquitin-like domain, which directly interacts with the proteasome, and it has been implicated in the regulation of the degradation of several proteins. Preliminary studies indicate that ubiquilin binds APP in a yeast-two-hybrid screen. The central hypothesis of this proposal is that ubiquilin contributes to AD by interacting with APP and altering the processing and/or turnover of its potentially toxic cleavage products.
The specific aims of this project are as follows: 1) Determine the requirements for ubiquilin to bind APP. Deletion and point-mutation constructs will be generated, and subcellular fractionation and co-immunoprecipitation experiments will be performed to determine the residues necessary for binding and the localization of the interaction. 2) Test whether ubiquilin affects APP processing and/or turnover. Western blotting and ELISA will be used to examine proteolytic fragment production in the presence and absence of ubiquilin. APP turnover will be examined using cycloheximide chase experiments in cultured cell lines and immunocytochemistry/fluorescence tracking in primary neurons. The role of the proteasome will be investigated using proteasome inhibitors. 3) Determine whether ubiquilin can protect cells from APP-induced cytotoxicity. APP-induced cell death in cultured cell lines and primary neurons will be determined in the presence and absence of ubiquilin. Apoptosis assays and fluorescence microscopy will be used. The role of the proteasome will be investigated using proteasome inhibitors. The purpose of this proposal is to examine a novel regulatory role for ubiquilin in the processing of APP. The results of these studies will contribute to our understanding of the pathogenesis of AD and may lead to the development of new therapeutics. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Individual Predoctoral NRSA for M.D./Ph.D. Fellowships (ADAMHA) (F30)
Project #
5F30AG030878-02
Application #
7452300
Study Section
Special Emphasis Panel (ZRG1-F03A-M (20))
Program Officer
Snyder, Stephen D
Project Start
2007-06-03
Project End
2013-06-02
Budget Start
2008-06-03
Budget End
2009-06-02
Support Year
2
Fiscal Year
2008
Total Cost
$27,131
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Neurosciences
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555
El Ayadi, Amina; Stieren, Emily S; Barral, José M et al. (2013) Ubiquilin-1 and protein quality control in Alzheimer disease. Prion 7:164-9
El Ayadi, Amina; Stieren, Emily S; Barral, José M et al. (2012) Ubiquilin-1 regulates amyloid precursor protein maturation and degradation by stimulating K63-linked polyubiquitination of lysine 688. Proc Natl Acad Sci U S A 109:13416-21
Stieren, Emily S; El Ayadi, Amina; Xiao, Yao et al. (2011) Ubiquilin-1 is a molecular chaperone for the amyloid precursor protein. J Biol Chem 286:35689-98
Stieren, Emily; Werchan, Walter P; El Ayadi, Amina et al. (2010) FAD mutations in amyloid precursor protein do not directly perturb intracellular calcium homeostasis. PLoS One 5:e11992