Hirata Masayuki, Kishimoto Yayoi, Shearer Thomas R, Azuma Mitsuyoshi
Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co., Ltd., 6-4-3, Minatojima-Minamimachi, Chuo-Ku, Kobe, Hyogo, 650-0047, Japan.
Department of Oral Rehabilitation and Biosciences, Oregon Health & Science University, 2730 S Moody Ave, Portland, OR, 97201, United States.
Exp Eye Res. 2025 Oct;259:110511. doi: 10.1016/j.exer.2025.110511. Epub 2025 Jul 11.
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.
钙蛋白酶的蛋白水解作用导致缺氧的猴和人类视网膜外植体中的视网膜神经节细胞（RGC）死亡，尽管其机制尚未完全明确。本实验旨在确定在缺氧/再灌注培养模型中，线粒体中钙蛋白酶的激活及随后的氧化应激是否是导致RGC死亡的潜在机制，实验使用了源自人类诱导多能干细胞（iPS）的视网膜类器官。视网膜类器官由人类iPS细胞分化而来。用磁珠分选法纯化标记有tdTomato的RGC。通过免疫组织化学观察钙蛋白酶相关蛋白的细胞定位。例如，检测α-血影蛋白裂解产物150（SBDP150）作为胞质钙蛋白酶激活的标志物。视网膜类器官和纯化的RGC在缺氧/复氧条件下，分别在添加或不添加钙螯合剂BAPTA、钙蛋白酶抑制剂SNJ-1945或NRF2激活剂NK252的情况下进行培养。通过免疫印迹法测定线粒体钙蛋白酶激活的标志物——截短的凋亡诱导因子（tAIF）。用MT-1染料测量线粒体膜电位（MMP）。用硫醇检测试剂盒评估硫醇水平。缺氧/复氧诱导胞质钙蛋白酶激活标志物SBDP150和线粒体钙蛋白酶激活标志物tAIF增加，导致RGC死亡。此外，它还导致硫醇水平降低和MMP损伤。这些变化被BAPTA和钙蛋白酶抑制剂SNJ-1945抑制。NK252可防止RGC死亡，但不抑制钙蛋白酶介导的蛋白水解作用。我们在人类iPS衍生的RGC培养模型中的研究结果表明，缺氧RGC中的钙内流激活线粒体钙蛋白酶，进而导致硫醇水平耗竭和MMP崩溃，最终导致细胞死亡。
