Abstract
Purpose :
We previously cultured monkey and human retinal explants under hypoxia/reoxygenation. Calpain-activated proteolysis was discovered to be a mechanism for retinal ganglion cell (RGC) death. Further details on how calpain causes cell death were needed. The purpose of the present experiment was to determine the detailed molecular mechanisms for calpain-induced RGC death.
Methods :
Retinal organoids were differentiated from human iPS cells. Retinal organoids and RGCs purified with magnetic activated cell sorting were confirmed by quantitative PCR using specific markers. Localization of calpain-related proteins was observed by immunohistochemistry (IHC). Retinal organoids and purified RGCs labeled with tdTomato were cultured with or without calcium chelator BAPTA or calpain inhibitor SNJ-1945 under hypoxia/reoxygenation. TdTomato-positive RGCs were counted with confocal microscopy or flow cytometry. Spectrin breakdown product 150 (SBDP150, specific marker for calpain activation) was visualized by IHC and western blotting (WB). Apoptosis inducing factor (AIF) was determined by WB.
Results :
Specific markers for differentiated retinal cells were expressed in retinal organoids. RGC marker Brn3B co-localized with tdTomato-labeled cells in retinal organoids and purified RGCs. Calpains 1 and 2, endogenous inhibitor calpastatin, and calpain substrate spectrin were localized in RGCs. Hypoxia/reoxygenation caused calpain activation and cell death. These changes were inhibited by BAPTA and SNJ-1945. Truncated AIF (tAIF) appeared in hypoxic retinal organoids, and this was ameliorated by SNJ-1945.
Conclusions :
Calpain caused proteolysis of AIF in addition to structural proteins. This suggests that activated calpains in mitochondria as well as in cytosol could contribute to cell death in hypoxic RGCs. iPS-derived human retinal organoids and purified RGCs were useful in such studies on cell death mechanisms.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.