AI-BASED ANALYSIS OF AN EXTRACELLULAR MOTIF OF APP REQUIRED FOR ITS ENDOCYTOSIS IN NEURONAL AND NON-NEURONAL CELL TYPES

The intracellular sorting and internalization of type I transmembrane proteins such as APP is typically mediated by short cytoplasmic motifs (e.g., NPTY, YTSI). APP localization within specific compartments of the secretory pathway critically influences its processing, as β-secretase (BACE1) activity is highest in endosomes. Endocytosis therefore plays a central role in amyloid-β (Aβ) production.
To gain a deeper understanding of APP endocytosis, we established an antibody uptake assay combined with an AI-based image analysis pipeline, enabling unbiased, high-throughput, and highly reproducible analysis across large datasets. Using this approach we observed that perturbation of the extracellular motif located between the β- and α-cleavage sites of APP significantly reduces APP internalization, to a similar extend as deletion of the intracellular NPTY motif. Notably, this effect is conserved across multiple neuronal and non-neuronal cell lines. These findings indicate that, in addition to the well-established intracellular motifs, extracellular domains can critically influence APP endocytosis. We therefore propose that this extracellular region modulates endocytic trafficking indirectly, potentially by engaging extracellular binding partners or co-receptors that couple to the intracellular endocytic machinery. To further validate our hypothesis we will systematically combine the uptake assay with targeted knockdowns of putative co-receptors to identify extracellular interaction partners required for APP internalization.
Together, this work suggests a previously unappreciated role of extracellular determinants in the regulation of APP endocytosis and provides a framework to explore how extracellular interactions may influence amyloidogenic processing.