Presenilin/c-Secretase Activity Is Located in Acidic Compartments of Live Neurons

Abstract

Presenilin (PSEN)/c-secretase is a protease complex responsible for the proteolytic processing of numerous substrates. These substrates include the amyloid precursor protein (APP), the cleavage of which by c-secretase results in the production of b-amyloid (Ab) peptides. However, exactly where within the neuron c-secretase processes APP C99 to generate Ab and APP intracellular domain (AICD) is still not fully understood. Here, we employ novel Förster resonance energy transfer (FRET)based multiplexed imaging assays to directly “visualize” the subcellular compartment(s) in which c-secretase primarily cleaves C99 in mouse cortex primary neurons (from both male and female embryos). Our results demonstrate that c-secretase processes C99 mainly in LysoTracker-positive low-pH compartments. Using a new immunostaining protocol which distinguishes Ab from C99, we also show that intracellular Ab is significantly accumulated in the same subcellular loci. Furthermore, we found functional correlation between the endo-lysosomal pH and cellular c-secretase activity. Taken together, our findings are consistent with Ab being produced from C99 by c-secretase within acidic compartments such as lysosomes and late endosomes in living neurons.