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Title: Analysis of Gamma-Secretase and Amyloid Precursor Protein in Bone
Author: McLeod, Jane
ISNI:       0000 0001 3625 6890
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2007
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y-secretase IS an intramembrane protease responsible for the cleavage of numerous molecules which regulate osteoblast activity, such as Notch and EphrinB2. Cleavage of its substrates results in release of a fragment into the extracellular space, and a Cterminal peptide into the cytoplasm. Amyloid precursor protein (APP) is also processed by y-secretase, though its role in bone is unknown. Amyloia-p (AP) peptides are secreted into the extracellular space as a result ofy-secretase activity on APP, and in the brain can aggregate to form the plaques characteristic of -Alzheimer's disease (AD). The amyloid intracellular domain (AICD) binds to the nuclear adaptor protein Fe65 in the , . cytoplasm, and translocates to the nucleus to form a transcriptionally active AFT complex with Tip60. In this study the expression, cleavage and potential function of APP were investigated during osteogenesis. Expression of all y-secretase subunits (pS 1, PS2, APH-la, APH-1P, Net, PEN-2) was confirmed throughout osteogenesis and a significant increase in enzyme activity was observed during osteogenic differentiation by use of a specific fluorimetric assay. Characterisation ofAPP in osteoblasts identified expression and processing of a longer isoform of APP in osteoblasts compared to neuronal APP, which contained an additional Kunitz protease inhibitory domain. Application of y-secretase inhibitors confirmed that this enzyme specifically cleaves APP within differentiating mesenchymal stem cells (MSCs). A specific chemiluminescent immunoassay demonstrated secretion of Ap by osteoblasts. MSCs showed a significant increase in adhesion to extracellular matrices containing aged Ap plaques compared to non-aged Ap peptide controls and Ap plaques were localized to the endosteal and periosteal surfaces in sections of adult rat ulna. Expression of the AFT complex components, Fe65 and Tip60, was confirmed throughout in vitro osteogenesis and over-expression of both AICD and Fe65 in C3HlOT~ stromal cells resulted in colocalisation within discrete domains of the nucleus. JlCT analysis (15Jlm resolution) of vertebrae from a 12 month old AD mouse model, Tg2576, which over-expresses a mutant APP resulting in increased y-secretase cleavage, showed' a decrease in bone volume, surface area and thickness compared to wild type controls. These findings indicate that APP may function as a novel regulator of osteoblastic activity, with functions ranging from adhesion to regulation of gene transcription.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available