Investigating novel interaction partners of amyloid precursor protein: the mechanistic target of rapamycin and pikyve complex

Abstract

Previous Work Although the amyloid precursor protein (APP) is known to have a central role in Alzheimer's disease, its cellular function is poorly characterised. To better understand the cellular functions of APP, an interactome of APP’s intracellular domain (AICD) was generated using a proteo-lipososome based assay, which enabled interactions to be identified within a membrane context. In addition to proteins known to bind AICD, novel interactors were identified, including the mechanistic target of rapamycin complex 1 (mTORC1) and the phosphoinositide kinase PIKfyve complex. Binding of AICD to the two complexes was confirmed by Western blotting of treated AICDproteoliposomes and pulldowns of purified protein by AICD. Project Aims This project aimed to investigate the biological relevance of the APP/mTOR and APP/PIKfyve complex interactions. Results Investigation of the APP/mTOR interaction showed mTOR signalling increased in mammalian cells overexpressing APP/AICD, while loss of function studies determined C. elegans APP (APL-1) is involved in mTOR ortholog function. The APP/PIKfyve interaction was investigated with APP family knockdown, and TAT-AICD: a new molecular tool to allow acute AICD overexposure within the cell. Knockdown decreased PIKfyve function, while TAT-AICD exposure increased PIKfyve function in mammalian tissue culture. mTOR and PIKfyve are important to degradative pathway progression, and results suggested APP modulates the activity of these proteins. Protein degradation is important in human disease, including Alzheimer's disease. Experiments elaborating APP relevance in the lysosome demonstrated that APP degradation is dependent on sorting, endosomal acidification and the inhibition of mTOR. Further experiments linked PIKfyve inhibition to these degradative processes, in particular, to lower organelle acidification and altered late endosome morphology. Summary These results suggest an interdependence between APP, mTOR and PIKfyve, where APP appears to impact lysosomal function, while also being dependent upon it for down-regulation.