Hypoxia-induced activation of calpain and oxidative stress in mitochondria leads to RGC death in human iPSC-derived retinal organoids.

Proteolysis by calpain enzyme contributes to retinal ganglion cell (RGC) death in hypoxic monkey and human retinal explants, although the mechanism is not fully understood yet. The present experiments are to determine if calpain activation in mitochondria and the subsequent oxidative stress were underlying mechanism driving RGC death in a hypoxia/regeneration culture model, using retinal organoids derived from human induced pluripotent stem (iPS) cells. Retinal organoids were differentiated from human iPS cells. RGCs labeled with tdTomato were purified with magnetic-activated cell sorting. Cellular localization of calpain-related proteins was observed by immunohistochemistry. For example, α-spectrin breakdown product 150 (SBDP150) was detected as a marker for cytosolic calpain activation. Retinal organoids and purified RGCs were cultured with or without calcium chelator BAPTA, calpain inhibitor SNJ-1945, or NRF2 activator NK252 under hypoxia/reoxygenation. Truncated apoptosis-inducing factor (tAIF), a marker for mitochondrial calpain activation was determined by immunoblotting. Mitochondrial membrane potential (MMP) was measured with MT-1 dye. Thiol levels were assessed with Thiol Assay Kit. Hypoxia/reoxygenation induced an increase in the cytoplasmic calpain activation marker SBDP150 and the mitochondrial calpain activation marker tAIF, leading to RGC death. Additionally, it led to a decrease in thiol levels and MMP impairment. These changes were inhibited by BAPTA and calpain inhibitor SNJ-1945. NK252 prevented RGC death but did not inhibit calpain-mediated proteolysis. Our findings with human iPS-drived RGC culture model demonstrate that calcium influx in hypoxic RGCs activates mitochondrial calpain, which induced the depletion of thiol levels and the collapse of MMP, ultimately leading to cell death.