Unravelling the role of the TGN export machinery for basolateral proteins in Amyloid precursor proteins (APP) processing and transport

The secretory pathway is responsible for the synthesis, modification, and transport to specific cellular compartments of approximately 30% of human proteins. Within the secretory pathway, the Trans-Golgi Network (TGN) plays a central role as a sorting hub, directing proteins and lipids to various subcellular destinations, including apical and basolateral plasma membranes. The transport of basolateral proteins from the TGN is regulated by a complex molecular machinery involving GPRC5A, the G protein ⍺i3 subunit, PLCβ3, specific isoforms of Protein Kinase C, and Protein Kinase D. Alterations in this sorting process are associated with several human pathologies, including neurodegenerative disorders. This study investigates the role of the basolateral sorting machinery in the transport and processing of the amyloid precursor protein (APP), which undergoes cleavage by ⍺-, β-, and ɣ-secretases. In neurons, β-secretase cleavage generates the β-amyloid peptide, a key player in the pathogenesis of Alzheimer’s disease. Using non-neuronal cell models, it was observed that depletion of GPRC5A (GPRC5A-KD), in combination with secretase inhibitors, leads to increased secretion of the N-terminal APP fragment in an ⍺-secretase-dependent manner, accompanied by a reduction in intracellular levels of the C-terminal fragment. These findings suggest enhanced ⍺-secretase activity, potentially linked to defects in the transport or localization of APP or ⍺-secretase itself. Further analyses using immunofluorescence targeting the C-terminal epitope of APP in GPRC5A-KD cells revealed alterations in the intracellular localization of the protein. To investigate the dynamics of APP processing in more detail, we used HeLa cells stably transfected with APP dual-tag RUSH system to synchronize APP trafficking. It is known that GPRC5A expression is induced by all-trans retinoic acid (ATRA); indeed, combined treatment with ATRA and GPRC5A-KD produced an additive effect on the secretion of the N-terminal APP fragment. Finally, to further validate the experimental system, the endogenous processing of APP is currently being studied in a polarized cell model. The overarching aim of this research is to identify the mechanisms linking TGN regulation to APP transport and maturation, with the goal of reducing the production of amyloidogenic peptides and promoting non-amyloidogenic processing, thereby contributing to the understanding of the molecular processes underlying neurodegeneration.