Visualizing APP and BACE-1 approximation in neurons yields insight into the amyloidogenic pathway

Abstract

Cleavage of APP (amyloid precursor protein) by BACE-1 (β-site APP cleaving enzyme-1) is the rate-limiting step in amyloid-beta (Aβ) production and a neuropathologic hallmark of Alzheimer's disease (AD); thus physical approximation of this substrate-enzyme pair is a critical event with broad biological and therapeutic implications. Despite much research, neuronal locales of APP/BACE-1 convergence and APP-cleavage remain unclear. Here we report an optical assay – based on fluorescence complementation – to visualize in-cellulo APP/BACE-1 interactions as a simple on/off signal. Combined with other assays tracking the fate of internalized APP in hippocampal neurons, we found that APP/BACE-1 interact in both biosynthetic and endocytic compartments; particularly along recycling-microdomains such as dendritic spines and presynaptic boutons. In axons, APP and BACE-1 are co-transported, and also interact during transit. Finally, our assay reveals that the AD-protective “Icelandic” mutation greatly attenuates APP/BACE-1 interactions, suggesting a mechanistic basis for protection. Collectively, the data challenge canonical models and provide concrete insights into long-standing controversies in the field.

Increased localization of APP‐C99 in mitochondria‐associated ER membranes causes mitochondrial dysfunction in Alzheimer disease

“…Consistent with the data of others (Das et al , 2016), FL‐APP and BACE1 co‐migrated partially with a marker for endosomes (Rab7), but not with lysosomal, ER‐intermediate, or MAM markers (Fig 2C). Similarly, the APP‐CTFs C83 and C99 co‐migrated with endosomal and lysosomal markers (Rab5, Rab7, and LAMP‐2) (Haass et al , 2012; Das et al , 2016), whereas PS1 co‐migrated with MAM markers, such as FACL4 (Area‐Gomez et al , 2009; Newman et al , 2014; Schreiner et al , 2015). We reasoned that the difficulty in seeing APP‐CTFs and PS1 together was probably due to the rapid cleavage of the CTFs by γ‐secretase once both are in the same compartment.…”

“…Confocal microscopy analysis revealed that C99 (in red) was present mainly in the cytosol and in ER membranes (in green), as shown previously by others (Das et al , 2016). In addition to those sites, C99 (in red) also colocalized with regions where both ER (in green) and mitochondria (in blue) were present (white arrows in Fig 2E and Appendix Fig S2D).…”

“…Many reports have shown that C99 is localized mainly in endosomes (Haass et al , 2012; Das et al , 2016). Therefore, it is possible that the presence of C99 in MAM regions was the result of a cross‐contamination of MAM samples with endosomes during the process of subcellular fractionation.…”

Extracellular Tau Oligomers Induce Disruption of Endogenous Tau Distribution, Invasion of Tau into the Somatodendritic Compartment and Axonal Transport Dysfunction

“…Similar results were obtained for neurons transfected with BACE1-mRFP1 (Fig. 3-15, B), a protease that helps produce amyloid-β (Αβ) from the amyloid precursor protein (AβPP), and has been shown to be trafficked along with APP by kinesin-1 [252]. BACE1-mRFP1 expressing neurons treated with oligomeric tau showed an ~2-fold increase in anterograde run length and a ~40% increase in anterograde instantaneous velocity, suggesting that oligomeric 2N4R tau treatments affect multiple kinesin-1 cargoes in the same general manner.…”

“…To analyze the effect, if any, that the mislocalization of tau we describe here has on axonal transport, primary neurons were transfected with fluorescently-tagged proteins to label vesicles undergoing microtubule-directed fast axonal transport. Specifically, BDNF-mRFP1 and BACE1-mRFP1 were utilized to analyze effects on kinesin-1-dependent transport [103,252], and NeuropeptideY-mCherry to observe kinesin-3-dependent transport [103] (see Supplementary movie 1). Because 2N4R tau oligomers produced a robust response in previous experiments and data analysis for these experiments was extremely time consuming, we focused only on 2N4R tau oligomers.…”