An Investigation into the Intracellular Trafficking and Processing Pathways of Amyloid Precursor Protein

Abstract

Amyloid precursor protein (APP) is a type-I membrane-spanning protein, believed to be central to Alzheimer’s disease (AD) pathology. It undergoes a complex intracellular trafficking and processing itinerary to produce toxic amyloid-β peptides that accumulate in extracellular plaques in the brains of AD patients. Its cleavage is dependent on several different secretase enzymes that are distributed throughout the secretory pathway and endolysosomal system. The co-localisation of APP with various enzymes determines whether it is processed through either the pathogenic amyloidogenic pathway, generating amyloid-β peptides, or through the protective non-amyloidogenic pathway. Mutations to APP itself, or to the enzymes that cleave APP, are causal for familial Alzheimer’s disease (FAD). Despite this, the molecular determinants of APP processing remain unclear. This thesis introduces novel experimental techniques to characterise the trafficking and processing of APP. The short cytosolic tail of APP contains several tyrosine-based sorting motifs that are believed to direct its trafficking. Using a kinetic trafficking assay, these motifs have been systematically mutated and quantitatively assessed for their impact on anterograde APP processing. The effects of FAD APP mutations were also quantified using this assay, where significant perturbations to APP processing were observed in the presence of several mutations, including the Arctic and Swedish APP mutations. Using an unbiased mass spectrometry approach, several novel interactors of the APP tail have been identified. This includes RABGAP1, a cytosolic small GTPase-activating protein (GAP). Work presented in this thesis demonstrates that the PTB domain of RABGAP1 can directly interact with a YENPTY motif in the APP tail. This interaction is essential for the physiological processing of endogenous APP in multiple neuronal models. Knockdown of RABGAP1 in i3 neurons reduces the amyloidogenic processing of APP, subsequently decreasing the production of the intermediate membrane-bound C99 peptide fragment. This study uncovers a new role for RABGAP1, that is likely to apply to other YENPTY-containing cargoes. Here, RABGAP1 can act as a rheostat to sense levels of YENPTY cargoes in the endosomes and direct their sorting.