Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Supervisor

Dr. Stephen Pasternak

Abstract

One of the hallmarks of Alzheimer’s disease (AD) is the pathological accumulation of β-amyloid (Aβ) in the brains of AD patients. Oligomeric and fibrillar aggregates of Aβ have been shown to be neurotoxic to neurons and hippocampal slices. Therefore, limiting Aβ production is an important area of research in order to delay or stop AD progression. Aβ is produced by amyloidogenic cleavage of amyloid precursor protein (APP). Amyloidogenic cleavage requires ectodomain removal by β-secretase and intramembrane γ-cleavage by γ-secretase to release Aβ products ranging from 38-43 residues. Work from our lab has shown that APP and γ-secretase are resident proteins of the lysosome. Furthermore, the acidic environment of lysosomes that promotes the aggregation of Aβ. While many lines of evidence demonstrate that APP internalization is important to the Aβ production, the intracellular itinerary of APP, from production to cleavage, is unclear.

In order to follow the intracellular trafficking of APP and Aβ, we have applied various microscopy techniques, in combination with fluorescently-tagged proteins. Using a photoactivatable mutant of GFP (paGFP), we accurately photoactivated nascent APP and followed its trafficking to lysosomes. To our surprise, we found that APP was delivered to lysosomes, where it is cleaved by γ-secretase, through an entirely intracellular pathway. This intracellular pathway was dependent upon an interaction between APP and adaptor protein 3. We found that the interaction between APP and AP-3 is dependent on the 709YTSI712 tyrosine motif. Furthermore, phosphorylation of the serine within this motif, by PKCε, can disrupt this interaction. By decreasing APP trafficking to lysosomes, through disrupting the APP/AP-3 interaction we decreased the production of Aβ. While lysosomes have traditionally been thought to be responsible for cellular waste disposal, they also have a secretory role in a number of cell types; including neurons. We demonstrate that lysosomes are not only responsible for the production of Aβ, but may also be responsible for the secretion of lysosomal Aβ into the extracellular space. This research may provide new therapeutic targets to limit the production and release of Aβ.

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Available for download on Friday, November 01, 2019


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