Tracking de novo Aβ generation in live cells.
The amyloid-beta peptide (Aβ), the principal component of amyloid plaques in Alzheimer’s disease (AD) is derived from the serial proteolytic cleavage of the amyloid precursor protein (APP), a type 1 transmembrane protein. Most APP undergoes proteolytic cleavage of the N-terminal extracellular domain at the plasma membrane through the activity of α-secretase, producing non-amyloidogenic fragments (soluble APP-α and α -C-terminal fragment); however, a small fraction of APP escapes α-cleavage and instead is endocytosed where it can subsequently undergo further proteolytic cleavages via β-secretase and γ-secretase, resulting in the liberation of Aβ (amyloidogenic pathway). The importance of intracellular trafficking in AD pathogenesis has been underscored by the identification of multiple genes involved in endocytosis and trafficking in several AD genome-wide association studies. However, the trafficking itinerary of APP following endocytosis and the subcellular site of Aβ generation are still unknown. One reason for this gap in knowledge is the lack of probes that can interrogate the de novo proteolytic cleavage of Aβ from endogenously synthesized APP. I have developed a method to track de novo Aβ generation in live cells using fluorogenic click chemistry. This labeling technique produces a biofluorescent APP construct that permits labeling of cell surface APP and visualization of subsequent trafficking and processing events.