Calcium-dependent pathway as a primary cause of hypoxic RGC damage in monkey retinal explants

Retinal ganglion cells (RGCs) loss or degeneration in the retina is a hallmark of many sight-threatening diseases, including glaucoma and retinopathy. In our previous studies, calcium-activated cysteine protease calpain induced RGC damage under hypoxia/reoxygenation in the monkey retina explants, and the calpain inhibitor SNJ-1945 partially inhibited RGC damage. Calcium-independent proteases such as cathepsins exist in the retina, although the involvement of cathepsins in hypoxia-induced RGC damage is unclear. The purpose of the present study is to determine if cathepsins are involved in RGC damage during hypoxia/reoxygenation and to elucidate the role of calcium.

The cathepsin inhibitors (Odanacatib and SID26681509) were tested for their specificity against three cathepsins in vitro. Calpain inhibitors (SNJ-1945, PD-151746, ABT-957, and C2I) were tested for their isozyme specificity in vitro. Monkey retinal explants were cultured under hypoxic conditions with 0.3% oxygen in the chamber, followed by reoxygenation. The calpain- or cathepsin-specific inhibitors were added to the hypoxic culture medium. BAPTA and BAPT-AM were also used to determine the calcium requirement in RGC damage. After the cultured period, retinal explants were prepared for flat mounts and retinal lysates. The flat mounted retinas were stained with propidium Iodide (PI) to assess RGC damage and with an antibody specific for calpain-specific α-spectrin break down product 150 kDa (SBDP150). Immunoblotting assays were performed for α-spectrin and calpains.

The cathepsin inhibitors were confirmed to be specific to cathepsin without calpain inhibitory effects in vitro. The number of PI-positive RGCs increased in the hypoxic monkey retina; however, cathepsin inhibitors did not mitigate RGC damage. In contrast, RGC damage was completely inhibited by BAPTA and partially by BAPTA-AM. In hypoxic retinas, calpain-specific SBDP150 increased in the nerve fiber layer (NFL). Immunoblotting revealed an increase in SBDP150 and the activation of calpain 1. These changes were inhibited by BAPTA or BAPTA-AM, with BAPTA demonstrating a stronger effect compared to BAPTA-AM. Calpain inhibitors demonstrated lower specificity in vitro than previously reported. In hypoxic retina, each calpain inhibitor alleviated RGC damage and reduced SBDP150-positive staining in NFL. Furthermore, calpain inhibitors attenuated the activation of calpain 1 and the breakdown of α-spectrin, as assessed by immunoblotting.

Calcium-independent cathepsins do not contribute to RGC damage in monkey retinal explants cultured under hypoxia/reperfusion. In contrast, calcium influx from extracellular sources plays a critical role in inducing RGC damage. Elevated intracellular calcium levels could activate calpains, leading to RGC damage; however, other calcium-dependent pathways might also be involved in this process.