P004 - ANALYSIS OF BETA-AMYLOID CYTOTOXICITY AS A FUNCTION OF STRUCTURE AND AGGREGATION CONDITIONS (ID 1700)
Abstract
Aims
Beta-amyloid aggregation is proposed to play a crucial role in the pathogenesis of Alzheimer’s disease (AD). Data from several in vitro studies suggest that the aggregates of amyloid-β peptide (Aβ) show a wide range of structural diversity. These structures can be formed simultaneously and even have the tendency to convert into each other, making their characterization even more challenging. Our aim was to investigate how the aggregation condition influences the overall structural composition and toxicity of Aβ42 aggregates. We also addressed in the comparative study the potential use of mHippoE-14, an immortalized embryonic mouse hippocampal cell line as model for Aβ cytotoxicity studies.
Methods
Recombinant Aβ42 was expressed in E. coli BL21 cells. The peptide was aggregated under wide range of conditions: pH 5.5-8.5, 0-500 mM NaCl, 4-50 ºC with or without shaking. The structural composition of the samples was analyzed by ThT fluorescence measurements, transmission electron microscopy, and circular dichroism spectroscopy. Their toxicity was assessed by using immortalized and primary rodent cell lines.
Results
The widest structural variety was found among aggregates formed under physiological-like pH. Higher amyloid content resulted in lower cytotoxicity. We are the first to report that mHippoE-14 cell line showed sensitivity to toxic Aβ42 aggregates and interestingly, pH 8.5 aggregates were more toxic to these cells, while pH 7.5 samples decreased the viability of SH-S5HY cells more.
Conclusions
Our results can help to understand the relationship between Aβ42 structure and toxicity, and also provide support for AD researchers in choosing the right aggregation and testing conditions.
