title: Dual-tagged amyloid-β precursor protein reveals distinct transport pathways of its N- and C-terminal fragments.
abstract: The amyloid-β precursor 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 N- and 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, N- and 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 N- and 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 distributed vesicle-like structures. The experimental treatments that selectively destabilize the acetylated microtubules 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. They also point to a compartment associated with acetylated microtubules in the neuronal soma--not the neurite terminals--as a major site of APP cleavage, and segregation of NTFs from CTFs.
authors.affiliation: Department of Pharmacology and Physiology, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07101-1709, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Villegas
authors.forename: Virgil
authors.initials: V
authors.lastname: Muresan
authors.forename: Zoia
authors.initials: Z
authors.lastname: Ladescu Muresan
mesh_terms: D016564:Amyloid beta-Protein Precursor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D005456:Fluorescent Dyes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D016501:Neurites / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D021381:Protein Transport
chemical_list: D016564:Amyloid beta-Protein Precursor; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D049452:Green Fluorescent Proteins