Abstract
Purpose :
Age-related macular degeneration (AMD) is a degenerative disorder of the macula, the region of central retina responsible for the greatest visual acuity. Oxidative stress and aging of retinal pigment epithelial (RPE) cells are the major reason of AMD. 4-Hydroxynonenal (4-HNE) is a major product of lipid peroxidation which takes part in ferroptosis of cells. Also, 4-HNE is accumulated in aging cells and could be related to several age-related diseases. The mechanism of RPE cell death under oxidative stress is still controversial. The goal of the current study is to determine RPE cell death mechanisms using 4-HNE and RSL3 treatment models.
Methods :
ARPE-19 or human primary RPE cells were treated with 4-HNE or RSL3, and cell viability was tested 24 hours later. The effect of apoptosis, necroptosis, pyroptosis and ferroptosis pathways on cell survival was tested using specific inhibitors. Cellular ATP and ROS levels were measured. Cell morphology was observed under a light microscopy, PYCARD and RIPK3 expression was used to visualize inflammasomes and necrosomes that are involved in pyroptosis and necroptosis, respectively. Lipid ROS, a ferroptosis marker, was tested using BODIPY reagent.
Results :
1. 4-HNE induces RPE cell death in a concentration-dependent manner which can be rescued by ferroptosis inhibitors (Lip-1 and Fer-1) and both upstream and downstream necroptosis inhibitors (RIPK1 inhibitor Nec-1 and MLKL inhibitor NSA respectively), but not apoptosis or pyroptosis inhibitors; 2. Ferroptosis inducer RSL3 induces RPE cell death in a concentration-dependent manner which can be rescued by Lip-1, Fer-1, and Nec-1, but not NSA; 3. Both 4-HNE and RSL3 induce RIPK3 activation in RPE cells which can be inhibited by Lip-1, Fer-1, and Nec-1; 4. Both 4-HNE and RSL3 induce lipid ROS accumulation in RPE cells which can be inhibited by Lip-1, Fer-1, and Nec-1 but not NSA.
Conclusions :
Both RSL3 and 4-HNE can induce RPE ferroptosis. Ferroptosis is associated with RIPK3 activation, therefore representing one type of necroptosis. However, 4-HNE but not RSL3-induced RPE death can be inhibited by MLKL inhibitor NSA. RIPK1 inhibitor Nec-1 is likely a better inhibitor for RPE ferroptosis compared to MLKL inhibitor NSA, possibly because RIPK1/3 activation can induce other types of cell death when MLKL is inhibited.
This is a 2021 ARVO Annual Meeting abstract.