Identifying positional candidate genes on chromosome 10 that are involved in APP processing

Abstract

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. 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. 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.Genes that increase AD risk are not fully accounted for and genome scans suggest large genomic regions contain AD positional candidate genes. Our goal is to identify these genes that function to modulate APP processing and Aβ generation. Here we are using RNA interference to silence expression of positional candidate genes and monitor changes in APP processing, using an AICD-mediated reporter gene expression assay in human neuroblastoma cell lines. The validity and sensitivity of this assay system was tested by pharmacologically and genetically targeting the α-, β-, and γ- secretases. To identify modulators of APP metabolism, genes on chromosomes 10 were screened for their ability to modulate APP metabolism. Here we identify 16 putative regulators of APP metabolism and characterize them to elucidate their mechanism of action. We further determine the mechanism by which two putative regulators; PPP3CB, the catalytic subunit of calcium dependent serein/threonine phosphatase and SUFU, a component of the hedgehog pathway modulate APP metabolism.%%%%Ph.D., Biological Sciences – Drexel University, 2008