Abstract
Purpose :
Age-related macular degeneration (AMD) is a degenerative disorder of the macula, the region of the central retina responsible for the greatest visual acuity. Oxidative stress and aging of retinal pigment epithelial (RPE) cells are the major reason for AMD. 4-Hydroxynonenal (4-HNE) is a major product of lipid peroxidation, which 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, 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. PYCARD and RIPK3 expression were used to visualize inflammasomes and necrosomes. Lipid ROS, a ferroptosis marker, was tested using BODIPY reagent. RIPK1 and MLKL knockdown RPE cell lines were established using CRISPR/Cas9 to confirm the roles of the two genes.
Results :
1. 4-HNE-induced RPE cell death 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); 2. RSL3-induced RPE ferroptosis can be rescued by 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, Nec-1 but not NSA; 5. RIPK1 and MLKL knockdown can prevent 4-HNE induced RPE cell death while RIPK1 but not MLKL knockdown can prevent RPE ferroptosis; 6. MCC950, an inflammasome (pyroptosis marker) inhibitor, couldn’t prevent RSL3 induced RPE ferroptosis alone, but the combination of NSA and MCC950 could partially rescue the cells.
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 cell death can be inhibited by MLKL inhibition. Nec-1 is likely a better inhibitor for RPE ferroptosis compared to NSA, possibly because RIPK1/3 activation can induce inflammasome activation when MLKL is inhibited.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.