Abstract
Purpose :
Caspase 3 is an effector in apoptosis, identified specifically in models of retinal pathologies. Caspase 7 has similar substrate selectivity to Caspase 3 but is known to have distinct non-overlapping roles during apoptosis. To investigate whether caspase 3 and 7 have similar roles in Age-related macular degeneration (AMD) pathology, we tested the hypothesis that blocking their activities will protect retinal cells in in vitro and in vivo models of AMD pathology.
Methods :
Control and AMD human donor eyes were sectioned and labeled with cleaved caspase 3 and 7 antibodies. Caspase 3 and 7 knockout mice aged 15-39 weeks were used. Study mice were injected with NaIO3 IV (20 mg/kg) and controls with saline. Retinal thickness was measured using manual segmentation of SD-OCT images taken pretreatment (Day 0), and post treatment (Day 7). Assessors were masked to animal genotype. Eyes were also collected for histological analysis. Human primary RPE cells were treated with 20 μm A2E to induce cell death under conditions of Caspase 3 and 7 shRNA knockdown. Cell survival was measured using Incucyte live-cell imaging from 0 to 160 hours post treatment.
Results :
Cleaved caspases 3, and 7 are present in Human retina surrounding drusen deposits. At baseline, Caspase 3-/- and Caspase 7-/- mice have significantly thinner retinas than wildtype mice. After NaIO3 treatment retinal thicknesses in Caspase 3-/- and Caspase 7-/- mice are not significantly different to wildtype mice (Day 7). However, knockout mice lose significantly less retina compared to wildtypes after treatment.This is further seen when day 7 thickness is normalized to Day 0 thickness to account for the reduced retinal thicknesses in knockout mice. Furthermore, treating human RPE cells with A2E induces cleaved Caspase 3,7 expression and cell death, which is reduced by knockdown of expression, increasing survival rates.
Conclusions :
In AMD, cleaved caspases 3 and 7 are found around drusen deposits, and are likely to contribute to cell death mechanisms leading to loss of retinal cells and blindness. Evidence from mice lacking Caspase 3 or 7 exposed to NaIO3 treatment, and human RPE cell cultures with knockdown of caspase 3 and 7 expression further supports this, providing interesting insights into their roles in AMD pathology.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.