Poster V1 - Effect of Storage Time and Temperature on Aggregation of Alzheimer's Disease Amyloid-β Protein

Introduction: Amyloid-β protein (Aβ) is widely studied due to its key role in Alzheimer’s disease (AD) pathology. One characteristic of patients suffering from AD is the deposition of amyloid plaques, which consist of Aβ aggregates, in the cerebral cortex [1] , and this deposition is associated with synapse damage [2]. Thus, studying Aβ aggregation and aggregation inhibition may provide pathological and therapeutic insights. Aggregation of Aβ is a nucleation dependent process, starting with a lag phase, followed by a growth phase, and ending in equilibrium. This process may be influenced by the presence of pre-formed Aβ aggregate seeds that can facilitate further formation of protein aggregates [3]. These seeds may appear during preparation or storage of Aβ monomer, posing impact on experiments that study the aggregation process and the ability of prospective therapeutics to disrupt that process. Despite the wide range of studies exploring the preparation of monomeric Aβ, the effects of monomer storage conditions on Aβ aggregation have not been explored. To understand the limitations of solution storage for monomeric Aβ, we have conducted a systematic study examining the impact of Aβ monomer storage time and temperature on Aβ aggregation.

Materials and

Methods: Lyophilized Aβ40 was reconstituted in 50mM NaOH and purified via size exclusion chromatography (SEC) to remove pre-existing aggregates. The resulting monomer was used immediately in aggregation assays or stored at 4°C, -20°C, or -80°C for durations of 24h, 48h, 72h, 5d, 2w, 4w, and 8w.
Aβ40 aggregation assays were performed in the presence of thioflavin T (ThT), which facilitated detection of Aβ aggregates via a shifted, enhanced fluorescence upon binding amyloid. Samples containing 10µM Aβ40, 20µM ThT, and 150mM NaCl in 40 mM Tris-HCl, pH 8.0, were prepared in a 96 well plate and agitated to induce aggregation. ThT fluorescence was monitored every 15 min. The lag time to aggregate formation and extent of aggregation at equilibrium were determined from fluorescence vs. time curves. To facilitate comparison among experiments, these values were normalized to those observed for fresh monomer.

Results, Conclusions, and Discussions: Aggregation curves, presented as ThT fluorescence vs time (Figure 1) and also normalized to the lag time and equilibrium fluorescence observed for fresh monomer, demonstrate a shift toward increased lag times and reduced equilibrium plateaus as storage time increases. These changes are most pronounced for Aβ40 monomer stored at -20°C and least pronounced for Aβ40 stored at -80°C. When Aβ40 monomer was stored at 4°C, equilibrium fluorescence values begin to exhibit observable differences at 4w. In contrast, Aβ40 monomer stored at -20°C exhibits observable lag extension and plateau reduction as early as 2w. Aβ40 monomer stored at -80°C, however, exhibits no noteworthy changes in lag and observable changes in plateau at 2w.

Despite the prominence of Aβ aggregation studies in the AD literature, there is a lack of literature information about proper storage of the protein. This work demonstrates that aggregation experiments with SEC purified Aβ40 monomer are influenced by both the time and the temperature at which the monomer is stored. Overall, SEC purified Aβ40 monomer was found to be most stable with storage at -80°C, while Aβ monomer stored at -20°C was least stable. These results both demonstrate that Aβ storage temperature and time impact aggregation experiments and establish guidelines for storage of Aβ monomer in solution.

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