Quantification of gamma-secretase activity in an endogenous context reveals biphasic GSI-mediated Notch/APP selectivity switch and the novel detection of potential proteolytic cleavage fragments of the Notch Intracellular Domain

Abstract

Gamma-secretase is a promiscuous intra-membrane protease implicated in the proteolytic processing of two notable substrates: amyloid-precursor-protein (APP), in which gamma-secretase will irreversibly cleave to produce Amyloid-beta (Aβ) in Alzheimer’s disease; and the Notch receptor, where gamma-secretase is essential for liberating the Notch intracellular domain (NICD) to activate Notch-mediated transcriptional regulation in the nucleus. Gamma-secretase inhibitors such as DAPT and Avagacestat have been tested as therapies to prevent the formation of amyloid plaques, however, off-target interference with the Notch signalling pathway leading to Notch-related malignant side effects in clinical trials makes these pharmaceuticals unsuitable. The high-throughput search for selective drugs that block the production of Aβ but don’t interfere with the Notch signalling pathway has been hindered by a lack of reliability in detecting Notch signal inhibition in pre-clinical, cell-based assays with ectopic substrate expression. Therefore, the development of a highly-sensitive, high-throughput cell-based assay to quantify the level of proteolytic processing of APP and Notch by gamma-secretase is a promising addition to the gamma-secretase inhibitor drug discovery pipeline. This thesis presents the combination of immunofluorescence staining, western blotting, and the bromo-deoxyuridine (BrdU) cell proliferation assay as three orthogonal methods to sensitively quantify the gamma-secretase cleavage of Notch and APP in SH-SY5Y human neuroblastoma cells, which rely on active Notch signalling to maintain proliferation. Using our assay, we found that the selectivity for gamma-secretase to cleave APP versus Notch was dependent on the time the GSI was replenished before harvesting, which may directly reflect a GSI concentration-dependent selective potency. Using this high-throughput cell-based assay, cleavage of APP and Notch activation by gamma-secretase was sensitively quantified; while a novel profile of cell-type specific proteolytic fragments of the Notch ICD have been identified that may have biological implications in normal development and the pathological proliferation and metastasis of some cancers.