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Kunal Thaker, Marilyn Chwa, Shari R Atilano, Tej Patel, Cristina M Kenney; Treatment of Haplogroup H and K ARPE-19 Cybrids with β-Amyloid. Invest. Ophthalmol. Vis. Sci. 2016;57(12):256.
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Ashkenazi Jewish populations, mainly of the mitochondrial (mt) haplogroup K, have been observed to have increased rates of hypercholesterolemia, a risk factor for age-related macular degeneration (AMD), compared to other Caucasian populations. Apolipoprotein E (apoE) is a protein involved in lipid transport, and processing the pathogenic peptide amyloid-β (am-β), known to have a role in Alzheimer’s and AMD. Haplogroup H and K adult retinal pigmental epithelial (ARPE) cytoplasmic hybrids (cybrids) were treated with amyloid-β1-42 (am-β1-42) to determine if the differences in APOE expression affected cellular response.
H (n=6) and K (n=8) haplogroup cybrids were created using platelets from individuals. RNA and DNA were isolated from cells cultured to the fifth passage. GeneChip Array Analysis was performed to find the highest differential gene expression between the two haplogroups and qPCR was performed on genes of interest. Cells were plated on 96-well plates (5x104 cells per well) and treated with 20µM am-β1-42 after 24 hours. An MTT assay was done to observe cell viability 24 hours after treating. Each sample’s data was normalized to its untreated average. Statistical significance was determined through a student’s t-test.
GeneChip Array Analysis found that the K cybrids had increased gene expression in the atherosclerosis pathways compared to H cybrids. QPCR showed that the K cybrids had a 9.3-fold increase in the expression of APOE compared to the haplogroup H cybrids (p<0.0001). APOC1, COL1A1, and MMP1 were also measured but no significant difference was found between the H and K cybrids. After treatment with am-β1-42 the cell viability in H cybrids dropped 30% ± 2.3% (p<0.001), while the K cybrids decreased 16% ± 2.0% (p<0.0001) relative to their respective untreated controls. The difference in viability between the H and K cybrids post treatment was 14.7% (p<0.0001).
These findings demonstrate that cells with haplogroup K mtDNA transcribe higher levels of APOE and are more resistant to am-β1-42 toxicity compared to the cells with haplogroup H mtDNA. In other systems, apoE enhances the elimination of am-β and provides cyto-protection. Our results suggest that in human RPE cells, higher levels of APOE expression may have protective functions against the pathogenic effects of am-β. These findings indicate that mtDNA may mediate levels of APOE expression and play a role in apoE-related diseases.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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