Dual-tagged amyloid-b precursor protein reveals distinct transport pathways of its N- and C-terminal fragments

The amyloid-bprecursor protein (APP), a type I transmembrane protein genetically associated with Alzheimer’s disease, has a complex biology that includes proteolytic processing into potentially toxic fragments, extensive trafficking and multiple, yet poorly-defined functions. We recently proposed that a significant fraction of APP is proteolytically cleaved in the neuronal soma into Nand C-terminal fragments (NTFs and CTFs), which then target independently of each other to separate destinations in the cell. Here, we prove this concept with live imaging and immunolocalization of two dual, Nand C-termini-tagged APP constructs: CFP-APP-YFP [containing the fluorescent tags, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP)] and FLAG-APP-Myc. When expressed at low levels in neuronal cells, these constructs are processed into differently tagged NTFs and CTFs that reveal distinct distributions and characteristics of transport. Like the endogenous Nand C-terminal epitopes of APP, the FLAG-tagged NTFs are present in trains of vesicles and tubules that localize to short filaments, which often immunostain for acetylated tubulin, whereas the Myc-tagged CTFs are detected on randomly distributedvesicle-likestructures.Theexperimental treatments thatselectivelydestabilize theacetylatedmicrotubules abrogate the distribution of NTFs along filaments, without altering the random distribution of CTFs. These results indicate that the NTFs and CTFs are recruited to distinct transport pathways and reach separate destinations in neurons, where they likely accomplish functions independent of the parental, full-length APP. Theyalsopoint toacompartmentassociatedwithacetylatedmicrotubules in theneuronalsoma—not theneurite terminals—as a major site of APP cleavage, and segregation of NTFs from CTFs.