Objectives

Amyloid-β (Aβ) accumulates in the Alzheimer brain and impairs neuronal function. Aβ is produced by the sequential cleavage of amyloid precursor protein (APP) by secretases. However, APP itself can be trafficked to the late endosome/lysosome where it is destroyed by powerful lysosomal hydrolases. We hypothesised that lysosomal trafficking of APP could provide treatments for Alzheimer’s disease. We therefore aimed to create a tool that could be used to quantitatively measure the trafficking of APP to the lysosome.

Methods

APP695 was fused to mCherry and green fluorescent protein (GFP) to create tandem fluorescent-APP (tf-APP). Flow cytometry was used to quantify the ratio of red fluorescence/green fluorescence (R/G) in a monoclonal HeLa tf-APP expressing cell line. When trafficked to an acidic environment (late endosome/lysosome), GFP quenches while mCherry does not, resulting in increased R/G.

Results

Flow cytometric measurement of R/G revealed that starvation induces autophagic flux (measured by tf-LC3) and also traffics APP to the lysosome. Inhibiting mTOR activity using AZD8055 had the same effect as starvation, inducing lysosomal trafficking of APP. Interestingly, whereas activation of mTOR by RHEB over-expression dramatically inhibited autophagic flux, RHEB expression did not affect lysosomal trafficking of APP.

Conclusions

We demonstrate trafficking of APP to the lysosome can be measured using tf-APP. Whereas APP usually cycles between the plasma membrane, the early endosome, retromer and the Golgi apparatus, we show the effect of starvation, or inhibition of mTOR signalling, can divert APP to the lysosome for hydrolysis by lysosomal proteases.