Development of biochemical tools to unravel proteins trafficking in the endolysosomal system in Alzheimer’s Disease

Details

Supervisors

Annaert ; Kienlen-Campard, Pascal

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

Alzheimer's Disease (AD) is a neurodegenerative disease in which the Amyloid Precursor Protein (APP) is involved in the generation of one of AD hallmarks Amyloid β (Aβ) plaques. The enzyme γ-secretase is implicated in the processing of APP, leading to the generation of Aβ peptides. This enzyme is composed of four distinct subunits. The catalytic subunit, Presenilin (PSEN) has two homologs, PSEN1 and PSEN2, resulting in the formation of either the PSEN1-associated or PSEN2-associated γ-secretase complex. PSEN is also engaged in the regulation of the subcellular localisation of the complex. Indeed, PSEN1/γ-secretase is broadly distributed throughout the cell including the Plasma Membrane (PM). In contrary, PSEN2/γ-secretase is mainly observed in Late Endosomes and lysosomes (LE/Lys). γ-secretase traffics through the endolysosomal system, which has been found to play a key role along with the autophagic system in the generation of AD when impaired. Aim: Over the past years, several studies have focused on investigating APP and BACE1 intracellular trafficking. When it comes to γ-secretase, a lot of questions remain unanswered, and studies regarding its intracellular trafficking are expanding. The first aim of the project was to set up three new techniques in the laboratory that will be used to unravel protein trafficking in the endolysosomal system. These techniques are: 1) Lysosomal immunoprecipitation (LysoIP) allowing the intact isolation of LE/Lys; 2) proximity-dependent labelling using the combination of an ascorbate peroxidase enzyme and a chemical click reaction to assess the environment of a protein of interest 3) knock-in using a pOrange approach to endogenously tag a protein of interest. The second aim of the project was to apply these techniques in investigating the endosomal sorting and environment of PSEN1/γ-secretase.