Altered APP trafficking drives amyloidogenic processing in primary neurons from the AppNL-F knock-in mouse model of Alzheimer's disease

Self-assembly of the 42-residue long amyloid β-peptide (Aβ42) into neurotoxic aggregates – eventually leading to formation of amyloid plaques - is a key event in Alzheimer's disease (AD) pathogenesis. Still, the intracellular mechanisms leading to Aβ42 formation and aggregation in neurons are poorly defined. Here, we used the App NL-F knock-in mouse model to analyze the effect of Aβ42-induced pathology on the subcellular location of the Aβ precursor protein (APP), its C-terminal fragments (CTFs) and Aβ42 in primary neurons. Stimulated emission depletion (STED) microscopy was used to obtain super-resolution and enable colocalization analysis. APP/CTF levels were to a high extent found in clathrin-coated vesicles in the perinuclear region in soma in both wild-type and App NL-F neurons and significantly increased in early endosomes in neurites. In distal axons, increased colocalization of APP/CTF with the synaptic vesicle protein synaptophysin was observed. Western blotting showed a three-fold decrease in mature/immature APP in App NL-F neurons, and ELISA showed a 2.7 and 7.2-fold increase in intra- and extracellular Aβ42 levels, respectively. Interestingly, LAMP1-positive vesicles were larger in App NL-F neurons than in wild-type neurons. Thus, processing of APP and axonal transport of APP/CTFs is increased in App NL-F neurons, resulting in enhanced levels of the immediate Aβ precursor (CTFβ) at the presynapse. Hence, an increase in CTFβ levels at sites with high γ-secretase activity leads to increased formation and secretion of Aβ42. This, in turn, results in enhanced re-uptake of Aβ42 and enlarged Aβ42-containing late endosomes/lysosomes in soma, causing toxic downstream effects. • Super-resolution microscopy truly resolves APP processing and trafficking in neurons. • Increased APP-C-terminal fragment levels in early endosomes in neurites in App NL-F knock-in primary neurons. • Increased APP-C-terminal fragment levels in presynapses in App NL-F knock-in primary neurons. • Increased Aβ42 production and uptake leads to lysosomal swelling.