Abstract
Purpose :
The purpose of this study was to determine the effects of Amyloid-β (Aβ)-derived oligomers i.e., 2AT and 2AT-FT-F20Cha as stressors on the ARPE-19 cell line. The rationale for this study was based on our previously published data which demonstrated Amyloid-β1-42 peptide-induced cellular and mitochondrial damage in AMD ARPE-19 cybrid cell lines. Therefore, we hypothesized that Aβ oligomers could be used as cellular stressors to screen cytoprotective agents in the ARPE-19 cell line.
Methods :
ARPE-19 cells were plated in 96-well tissue culture plates followed by treatment with 2AT Aβ oligomer and 2AT-FT-F20Cha Aβ oligomer. This was followed by treatment with: 1) MTT solution for measurement of cellular metabolic activity/ cellular viability for the MTT assay, a colorimetric test that utilizes the phenomenon of reduction of tetrazolium salts to measure cell viability. Healthy actively metabolizing cells contain NAD(P)H-dependent cellular oxidoreductase enzymes that reflect the number of viable cells present; and 2) Lactate Dehydrogenase (LDH) solution for the LDH assay. LDH released from the cell oxidizes lactate to generate NADH, which then reacts with WST dye to generate a yellow color, the intensity of which correlates directly with the number of lysed cells.
Results :
We observed that 2AT-treated ARPE-19 cells showed significant decrease in cellular metabolic activity/ cell viability compared to the untreated ARPE-19 cells that served as controls: P=0.02; Untreated: 1 ± 0.02; 2AT: 0.90 ± 0.03; 10 % decrease. In addition, 2AT-FT-F20Cha-treated ARPE-19 cells showed 27.28% decrease in cellular metabolic activity/ cell viability compared to their untreated counterparts: P=0.01; Untreated: 1 ± 0.02; 2AT-FT-F20Cha: 0.7272 ± 0.11.
Next, we observed that 2AT-treated ARPE-19 cells showed 62 % increase in LDH levels compared to the untreated ARPE-19 cells: P=0.03; Untreated: 1 ± 0.08; 2AT: 1.62 ± 0.07. In addition, 2AT-FT-F20Cha-treated ARPE-19 cells showed 41.7 % increase in LDH levels compared to their untreated counterparts: P=0.01; Untreated: 1 ± 0.08; 2AT-FT-F20Cha: 1.417 ± 0.119.
Conclusions :
In conclusion, our study demonstrated that treatment of ARPE-19 cells with 2AT and 2AT-FT-F20Cha Aβ oligomers caused cellular damage and death, thereby confirming the toxic effects of 2AT and 2AT-FT-F20Cha Aβ oligomers and demonstrating their potential as cellular stressors in the ARPE-19 cell line.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.