title: Development of new fusion proteins for visualizing amyloid-β oligomers in vivo.
abstract: The intracellular accumulation of amyloid-β (Aβ) oligomers critically contributes to disease progression in Alzheimer's disease (AD) and can be the potential target of AD therapy. Direct observation of molecular dynamics of Aβ oligomers in vivo is key for drug discovery research, however, it has been challenging because Aβ aggregation inhibits the fluorescence from fusion proteins. Here, we developed Aβ1-42-GFP fusion proteins that are oligomerized and visualize their dynamics inside cells even when aggregated. We examined the aggregation states of Aβ-GFP fusion proteins using several methods and confirmed that they did not assemble into fibrils, but instead formed oligomers in vitro and in live cells. By arranging the length of the liker between Aβ and GFP, we generated two fusion proteins with "a long-linker" and "a short-linker", and revealed that the aggregation property of fusion proteins can be evaluated by measuring fluorescence intensities using rat primary culture neurons transfected with Aβ-GFP plasmids and Aβ-GFP transgenic C. elegans. We found that Aβ-GFP fusion proteins induced cell death in COS7 cells. These results suggested that novel Aβ-GFP fusion proteins could be utilized for studying the physiological functions of Aβ oligomers in living cells and animals, and for drug screening by analyzing Aβ toxicity.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Tomoyo
authors.initials: T
authors.lastname: Ochiishi
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Motomichi
authors.initials: M
authors.lastname: Doi
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Yamasaki
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Hirose
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, N21W11, Kita-ku, Sapporo 001-0021, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Kitamura
authors.affiliation: Department of Neurology, Juntendo University Urayasu Hospital, 2-1-1, Tomioka, Urayasu, Chiba 279-0021, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Urabe
authors.affiliation: Department of Neurology, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Hattori
authors.affiliation: Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, N21W11, Kita-ku, Sapporo 001-0021, Japan.
authors.forename: Masataka
authors.initials: M
authors.lastname: Kinjo
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1, Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Ebihara
authors.affiliation: Department of Neurology, Juntendo University Urayasu Hospital, 2-1-1, Tomioka, Urayasu, Chiba 279-0021, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Shimura
mesh_terms: D016229:Amyloid beta-Peptides / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D019556:COS Cells; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C075222:amyloid beta-protein (1-42); D049452:Green Fluorescent Proteins