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An AICD-based functional screen to identify APP metabolism regulators

An AICD-based functional screen to identify APP metabolism regulators

Authors :
Lee Jeremy C
O'Connor Michael
Cosentino Christopher P
Patel Shyam A
Sarangi Srikant
Cuellar Trinna L
Chakraborty Ranjita
Khandelwal Preeti J
Zhang Can
Tanzi Rudolph E
Saunders Aleister J
Source :
Molecular Neurodegeneration, Vol 2, Iss 1, p 15 (2007)
Publication Year :
2007
Publisher :
BMC, 2007.

Abstract

Abstract Background A central event in Alzheimer's disease (AD) is the regulated intramembraneous proteolysis of the β-amyloid precursor protein (APP), to generate the β-amyloid (Aβ) peptide and the APP intracellular domain (AICD). Aβ is the major component of amyloid plaques and AICD displays transcriptional activation properties. We have taken advantage of AICD transactivation properties to develop a genetic screen to identify regulators of APP metabolism. This screen relies on an APP-Gal4 fusion protein, which upon normal proteolysis, produces AICD-Gal4. Production of AICD-Gal4 induces Gal4-UAS driven luciferase expression. Therefore, when regulators of APP metabolism are modulated, luciferase expression is altered. Results To validate this experimental approach we modulated α-, β-, and γ-secretase levels and activities. Changes in AICD-Gal4 levels as measured by Western blot analysis were strongly and significantly correlated to the observed changes in AICD-Gal4 mediated luciferase activity. To determine if a known regulator of APP trafficking/maturation and Presenilin1 endoproteolysis could be detected using the AICD-Gal4 mediated luciferase assay, we knocked-down Ubiquilin 1 and observed decreased luciferase activity. We confirmed that Ubiquilin 1 modulated AICD-Gal4 levels by Western blot analysis and also observed that Ubiquilin 1 modulated total APP levels, the ratio of mature to immature APP, as well as PS1 endoproteolysis. Conclusion Taken together, we have shown that this screen can identify known APP metabolism regulators that control proteolysis, intracellular trafficking, maturation and levels of APP and its proteolytic products. We demonstrate for the first time that Ubiquilin 1 regulates APP metabolism in the human neuroblastoma cell line, SH-SY5Y.

Details

Language :
English
ISSN :
17501326
Volume :
2
Issue :
1
Database :
Directory of Open Access Journals
Journal :
Molecular Neurodegeneration
Publication Type :
Academic Journal
Accession number :
edsdoj.6173579d786e4fa1aeb3b62a74ea96ae
Document Type :
article
Full Text :
https://doi.org/10.1186/1750-1326-2-15