title: Characterization of oligomer formation of amyloid-beta peptide using a split-luciferase complementation assay.
abstract: Amyloid-β peptide (Aβ) is the amyloid component of senile plaques in Alzheimer disease (AD) brains. Recently a soluble oliomeric form of Aβ in Aβ precursor protein transgenic mouse brains and AD brains was identified as a potential causative molecule for memory impairment, suggesting that soluble Aβ oligomers cause neurodegeneration in AD. Further characterization of this species has been hampered, however, because the concentrations are quite small and it is difficult to monitor Aβ oligomers specifically. Here we developed a novel method for monitoring Aβ oligomers using a split-luciferase complementation assay. In this assay, the N- and C-terminal fragments of Gaussia luciferase (Gluc) are fused separately to Aβ. We found that conditioned media from both N- and C-terminal fragments of Gluc-tagged Aβ1-42 doubly transfected HEK293 cells showed strong luminescence. We used gel filtration analyses to analyze the size of oligomers formed by the luciferase complementation assay, and found that it matched closely with oligomers formed by endogenous Aβ in Tg2576 neurons. Large oligomers (24-36-mers), 8-mers, trimers, and dimers predominate. In both systems, Aβ formed oligomers intracellularly, which then appear to be secreted as oligomers. We then evaluated several factors that might impact oligomer formation. The level of oligomerization of Aβ1-40 was similar to that of Aβ1-42. Homodimers formed more readily than heterodimers. The level of oligomerization of murine Aβ1-42 was similar to that of human Aβ1-42. As expected, the familial AD-linked Arctic mutation (E22G) significantly enhanced oligomer formation. These data suggest that Gluc-tagged Aβ enables the analysis of Aβ oligomers.
authors.affiliation: Department of Neurology, Alzheimer's Disease Research Unit, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.
authors.forename: Tadafumi
authors.initials: T
authors.lastname: Hashimoto
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Adams
authors.forename: Zhanyun
authors.initials: Z
authors.lastname: Fan
authors.forename: Pamela J
authors.initials: PJ
authors.lastname: McLean
authors.forename: Bradley T
authors.initials: BT
authors.lastname: Hyman
mesh_terms: D016229:Amyloid beta-Peptides / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001704:Biopolymers / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D002850:Chromatography, Gel; D017931:DNA Primers; D018076:DNA, Complementary; D004797:Enzyme-Linked Immunosorbent Assay; D005816:Genetic Complementation Test; D006801:Humans; D008156:Luciferases / Q000235:genetics*
chemical_list: D016229:Amyloid beta-Peptides; D001704:Biopolymers; D017931:DNA Primers; D018076:DNA, Complementary; D008156:Luciferases