Investigating the molecular regulation and functional impact of novel regulated cell death in experimental models of age-related macular degeneration

Abstract

Age related macular degeneration (AMD) is a severe blindness-causing disease in the aging population with limited treatment options. Retinal pigment epithelial (RPE) cell death is its hallmark feature, but the detailed cell death mechanism is not yet fully elucidated. This study aimed to explore the involvement of pyroptosis and ferroptosis in different AMD models. First, I investigated whether amyloid β1-40 (Aβ1-40) could cause pyroptosis and evaluated the effects of Lycium barbarum polysaccharides (LBPs) on Aβ1-40 oligomers-induced human adult RPE 19 (ARPE-19) damage, which is an in vitro AMD model. Aβ1-40 significantly decreased ARPE-19 cell viability with obvious morphological changes under light microscopy. Scanning electron microscopy revealed swollen cells with a bubbling appearance and ruptured cell membrane, which are morphological characteristics of pyroptosis. ELISA results showed increased expression of IL-1β and IL-18, which are the final products of pyroptosis. LBP administration for 24 h had no toxic effects on ARPE-19 cells and improved cell viability and morphology. Furthermore, Aβ1-40 up-regulated the cellular immunoreactivity of pyroptosis markers including NOD-like receptors protein 3 (NLRP3), caspase-1, and membrane N-terminal cleavage product of GSDMD (GSDMD-N), which could be reversed by LBP treatment. These results showed that Aβ1-40 oligomers induced pyroptosis in ARPE-19 cells, and LBPs effectively protected this model by suppressing pyroptosis. Next, I evaluated the involvement and role of ferroptosis, another novel regulated cell death in AMD models. Although it participates in cancers and some brain degenerative diseases, its role in AMD remains elusive. Here, I found that regulation of ferroptosis has a significant impact on RPE fate in sodium iodate (SIO)-induced retinal pathology, resembling AMD in human primary RPE cells (HRPEpiC), the human ARPE-19 cell line, and mice. Pre-treatment with deferoxamine (DFO) and ferrostatin-1 (Fer-1) significantly protected against SIO-induced cell death, accompanied by alleviated lipid peroxidation in both HRPEpiC and ARPE-19 cells. DFO pre-treatment also largely ameliorated the increased intracellular labile iron and glutathione disulfide, and the decreased glutathione and glutathione peroxidase-4 (GPx-4) induced by SIO in HRPEpiC and ARPE-19 cells. However, silencing GPX-4 significantly aggravated SIO-induced cell death and lipid peroxidation with more apparent mitochondria changes in ARPE-19 cells. Most importantly, in vivo results showed that Fer-1 not only remarkably alleviated SIO-induced RPE cell loss, photoreceptor death, and retinal dysfunction but also significantly ameliorated the increased labile iron and lipid peroxidation, and decreased GSH level in RPE cells isolated from the SIO-induced AMD mouse model. These data define a distinct role for ferroptosis in controlling RPE cell death in vitro and in vivo. Taken together, my studies discovered the involvement of novel cell death mechanisms in AMD models. As therapeutic options for AMD are extremely limited, this research could provide a new avenue for identifying treatment targets for AMD.