Expression and processing of fluorescent fusion proteins of amyloid precursor protein (APP)

• Published in: Biochimica et biophysica acta Vol. 1833; no. 6; pp. 1562 - 1571
• Main Authors: Coughlan, Kathleen, Huang, Xiangping, He, Xiangyuan, Chung, Charlotte H.Y., Li, Guangpu, Tang, Jordan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2013
• Subjects: Amyloid beta-Protein Precursor - metabolism; Amyloid precursor protein; Amyloid-beta; Amyloidosis - metabolism; Amyloidosis - pathology; Animals; Beta-secretase; Blotting, Western; Cell Survival; Cells, Cultured; Fluorescence; Gamma-secretase; Green fluorescence protein; Green Fluorescent Proteins - metabolism; Luminescent Proteins - metabolism; Mice; Neurons - cytology; Neurons - metabolism; Peptide Fragments - metabolism; Protein Processing, Post-Translational; Protein Transport; Recombinant Fusion Proteins - metabolism; Red Fluorescent Protein; Subcellular Fractions; Gamma-secretase; APP106; Green fluorescence protein; sGappβ; sGappα; RappG; sRapp; sGapp; Beta-secretase; GappR; Amyloid precursor protein; Amyloid-beta
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/j.bbamcr.2013.03.003

Processing of β-amyloid precursor protein (APP) by β- and γ-secretases in neurons produces amyloid-β (Aβ), whose excess accumulation leads to Alzheimer's disease (AD). Knowledge on subcellular trafficking pathways of APP and its fragments is important for the understanding of AD pathogenesis. We designed fusion proteins comprising a C-terminal fragment of APP (app) and fluorescent proteins GFP (G) and DsRed (D) to permit the tracking of the fusion proteins and fragments in cells. CAD cells expressing these proteins emitted colocalized green and red fluorescence and produce ectodomains, sGapp and sRapp, and Aβ, whose level was reduced by inhibitors of β- and γ-secretases. The presence of GappR in endosomes was observed via colocalization with Rab5. These observations indicated that the fusion proteins were membrane inserted, transported in vesicles and proteolytically processed by the same mechanism for APP. By attenuating fusion protein synthesis with cycloheximide, individual fluorescent colors from the C-terminus of the fusion proteins appeared in the cytosol which was strongly suppressed by β-secretase inhibitor, suggesting that the ectodomains exit the cell rapidly (t1/2 about 20min) while the C-terminal fragments were retained longer in cells. In live cells, we observed the fluorescence of the ectodomains located between parental fusion proteins and plasma membrane, suggesting that these ectodomain positions are part of their secretion pathway. Our results indicate that the native ectodomain does not play a decisive role for the key features of APP trafficking and processing and the new fusion proteins may lead to novel insights in intracellular activities of APP. •We developed a double labeled (GFP and DsRed) truncated APP construct.•The modified APP retains similar attributes as full length APP.•A novel technique for assessing APP processing with live cell imaging was established.•Kinetics for APP processing by Memapsin 2 was established.•A unique pool of secreted APP located intracellularly was observed.