Abstract
Purpose :
Age-related macular degeneration (AMD) is characterised by accumulation of drusen in the sub-RPE and sub-retinal space with age. This extracellular debris is rich in complement proteins, esterified and unesterified cholesterol and apolipoprotein E (ApoE). Human APOE exists in three isoforms (E2, E3 and E4) with APOE2 found to be associated with AMD risk while APOE4 is a strong risk factor for Alzheimer’s disease. We hypothesized that the functional difference between ApoE isoforms may be tissue context-dependent, thus we compared the phagocytic function of APOE2/2, 3/3 and 4/4 human induced pluripotent stem cell (iPSC)-derived microglia using retina and brain-related substrates.
Methods :
Isogenic human iPSC lines homozygous for APOE2/2, 3/3 and 4/4 were differentiated into microglia using transcriptome-validated and optimised differentiation protocol. Fully differentiated microglia were transferred to 96-well plates for phagocytosis assay with (i) bovine photoreceptor outer segments (POS), (ii) human amyloid-beta (Aβ), and (iii) yeast-derived Zymosan A particles (as positive control), all labelled with pH-sensitive dye. Phagocytic activity was quantified by live-cell microscopy up to 4 hours and compared across timepoints using Image J.
Results :
While APOE3/3 (wildtype) and APOE2/2 microglia displayed normal ramified morphology during differentiation, APOE4/4 microglia appeared unusually rod-shaped, reminiscent of those observed in brain of patients with Alzheimer’s disease. APOE2/2 microglia appeared to divide more slowly than the other isoforms. Preliminary results from the phagocytosis assays demonstrated slower uptake of photoreceptor outer segments by APOE2/2 microglia compared with APOE3/3 and 4/4 over the time course. In contrast, APOE4/4 microglia showed impaired uptake of Aβ.
Conclusions :
Understanding the impact of APOE variants on lipid metabolism in relation to tissue-specific substrates could provide novel insights into the mechanism of neurodegeneration in AMD and dementia. Further assays will be performed to compare the functions of microglia and RPE. Targeting genetic and age-related abnormalities in lipid processing could lead to new treatments for neurodegeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.