Development of new fusion proteins for visualizing amyloid-? oligomers in vivo.

ABSTRACT: The intracellular accumulation of amyloid-? (A?) 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 A? oligomers in vivo is key for drug discovery research, however, it has been challenging because A? aggregation inhibits the fluorescence from fusion proteins. Here, we developed A?1-42-GFP fusion proteins that are oligomerized and visualize their dynamics inside cells even when aggregated. We examined the aggregation states of A?-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 A? 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 A?-GFP plasmids and A?-GFP transgenic C. elegans. We found that A?-GFP fusion proteins induced cell death in COS7 cells. These results suggested that novel A?-GFP fusion proteins could be utilized for studying the physiological functions of A? oligomers in living cells and animals, and for drug screening by analyzing A? toxicity.

SUBMITTER: Ochiishi T 

PROVIDER: S-EPMC4793674 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature