In-Depth Characterization of Endo-Lysosomal Aβ in Intact Neurons

Amyloid-beta (A beta) peptides are produced within neurons. Some peptides are released into the brain parenchyma, while others are retained inside the neurons. However, the detection of intracellular A beta remains a challenge since antibodies against A beta capture A beta and its precursor proteins (i.e., APP and C99). To overcome this drawback, we recently developed 1) the C99 720-670 biosensor for recording gamma-secretase activity and 2) a unique multiplexed immunostaining platform that enables the selective detection of intracellular A beta with subcellular resolution. Using these new assays, we showed that C99 is predominantly processed by gamma-secretase in late endosomes and lysosomes, and intracellular A beta is enriched in the same subcellular loci in intact neurons. However, the detailed properties of A beta in the acidic compartments remain unclear. Here, we report using fluorescent lifetime imaging microscopy (FLIM) that intracellular A beta includes both long A beta intermediates bound to gamma-secretase and short peptides dissociated from the protease complex. Surprisingly, our results also suggest that the dissociated A beta is bound to the glycoproteins on the inner membrane of lysosomes. Furthermore, we show striking cell-to-cell heterogeneity in intracellular A beta levels in primary neurons and APP transgenic mouse brains. These findings provide a basis for the further investigation of the role(s) of intracellular A beta and its relevance to Alzheimer's disease (AD).