Tracking the intracellular itinerary of APP and de novo Aβ generation using click chemistry

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AbstractBackgroundAmyloid‐beta (Aβ), is generated by sequential cleavage of the amyloid precursor protein (APP) by beta‐ and gamma‐secretase. The intracellular sites of cleavage are unknown due to the lack of tools that can detect de novo Aβ production.MethodUsing unnatural amino acid (UAA) mutagenesis and click chemistry, a single fluorophore (magenta) site‐specifically binds to Aβ within the APP construct. The C‐terminus of APP is fused with the green fluorescent protein mClover, allowing for a distinction between full‐length APP and Aβ, as the uncleaved APP will appear white (magenta+green) from the bifluorescence, contrasting with the magenta of Aβ alone when separated from the green mClover C‐terminus. Live cells expressing the APP‐mClover construct (green) were incubated with cell‐impermeable tetrazine‐AlexaFluor 594 (magenta) at 4°C, which click‐labeled the UAA on Aβ on cell surface APP. The cells were then incubated at 37°C, resuming endocytosis of bifluorescent APP.ResultAfter click‐labeling, the APP construct resides exclusively at the cell surface. Once warmed to 37°C, the intracellular bifluorescence increases with time, suggesting APP endocytosis (Fig 1). Using object‐based analyses, the magenta click‐label:green mClover ratio was determined from super‐resolution z‐stack images (Fig 2). The magenta:green ratio was lower in cells treated with gamma‐secretase inhibitor (GSI).ConclusionA qualitative and semi‐quantitative picture can be created of Aβ’s localization by following the separation of the bifluorescent signal into intraluminal magenta click‐label and membrane‐bound green mClover. This technology will be coupled with immunocytochemistry of subcellular markers, which will allow for further specificity on the location of Aβ travel within a cell, with regards to the endolysosomal pathway. Furthermore, this technology can be applied to examine Aβ within the unique cytoarchitecture of primary neurons which may help inform the design of new therapeutics in this cell type central to AD pathogenesis.