Duggan, Claire

High, Stephen

English

Alzheimer???s disease (AD) is the most common cause of a progressive decline ofcognitive function in aged humans. It is thought to be a result of the formation ofamyloid plaques in the brain that are largely composed of ??-amyloid (A??), which isone of the cleavage products of the amyloid precursor protein (APP). Theamyloidogenic processing of APP to produce the A?? peptides requires sequentialproteolytic cleavages by the ??- and ??-secretases. APP is first cleaved by the ??-secretase to produce APP-C99, and this product is a substrate for furtherprocessing by the ??-secretase that cleaves within its transmembrane domain toproduce N-terminal A?? peptides and the C-terminal APP intracellular domain(AICD). On the basis of similarities to the Notch processing pathway, it has beenpostulated that the AICD may play a role in gene regulation following its release inresponse to some form of extracellular signal.In order to better understand the production and fate of the AICD, I haveinvestigated the potential for exploiting a cell-free system to study its generationand properties. Having generated a number of model APP-derived fragments andshown them to be efficiently membrane integrated in vitro, I went on to study AICDproduction. I discovered that AICD-like fragments are extremely labile whensynthesised in a rabbit reticulocyte lysate system and are rapidly degraded via ametalloproteinase, most likely the insulin degrading enzyme (IDE). The in vitrostability of these model AICD-like fragments was dependent upon the precisechain length of the polypeptide and N-terminal processing may preface the activityof IDE in vitro. The rapid degradation of the AICD in vitro is in close agreementwith previous in vivo studies, and taken together such data are consistent with arole for the AICD in a signalling pathway of some form.A variety of approaches were also taken to try to generate the AICD by the ??-secretase mediated cleavage of the APP-C99 fragment, a biologically relevantsubstrate. In no case was any evidence of such cleavage observed in vitro andhence I conclude that the endoplasmic reticulum does not possess an active formof the ??-secretase. Preliminary in vivo-based studies did provide evidence for the??-secretase cleavage of APP-C99 fragments, consistent with current modelsimplying that such processing takes place at the cell surface and/or in endosomesand not at the endoplasmic reticulum

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The generation of the amyloid precursor protein intracellular domain

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The generation of the amyloid precursor protein intracellular domain

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