A Super-Resolved View of the Alzheimer's Disease-Related Amyloidogenic Pathway in Hippocampal Neurons.

Background Processing of the amyloid-β protein precursor (AβPP) is neurophysiologically important due to the resulting fragments that regulate synapse biology, as well as potentially harmful due to generation of the 42 amino acid long amyloid β-peptide (Aβ 42), which is a key player in Alzheimer's disease. Objective Our aim was to clarify the subcellular locations of the amyloidogenic AβPP processing in primary neurons, including the intracellular pools of the immediate substrate, AβPP C-terminal fragment (APP-CTF) and the product (Aβ 42). To overcome the difficulties of resolving these compartments due to their small size, we used super-resolution microscopy. Methods Mouse primary hippocampal neurons were immunolabelled and imaged by stimulated emission depletion (STED) microscopy, including three-dimensional, three-channel imaging and image analyses. Results The first (β-secretase) and second (γ-secretase) cleavages of AβPP were localized to functionally and distally distinct compartments. The β-secretase cleavage was observed in early endosomes, where we were able to show that the liberated N- and C-terminal fragments were sorted into distinct vesicles budding from the early endosomes in soma. Lack of colocalization of Aβ 42 and APP-CTF in soma suggested that γ-secretase cleavage occurs in neurites. Indeed, APP-CTF was, in line with Aβ 42 in our previous study, enriched in the presynapse but absent from the postsynapse. In contrast, full-length AβPP was not detected in either the pre- or the postsynaptic side of the synapse. Furthermore, we observed that endogenously produced and endocytosed Aβ 42 were localized in different compartments. Conclusion These findings provide critical super-resolved insight into amyloidogenic AβPP processing in primary neurons.