FK962 protects retinal ganglion cell under hypoxia/reoxygenation: possible involvement of glial cell line-derived neurotrophic factor signaling pathway.

Loss of retinal ganglion cells (RGCs) is the cause of visual impairment and blindness in glaucoma. Previously, our studies showed that FK962 (N-[1-acetylpiperidin-4-yl]-4-fluorobenzamide) promoted neurite elongation in rat RGCs and trigeminal ganglion (TG) cells. In TG cells, glial cell line-derived neurotrophic factor (GDNF) is known to be involved in the mechanism. The purpose of the present study is to investigate whether, 1) FK962 shows an RGC-protective effect under hypoxia/reoxygenation (H/R) and 2) GDNF is involved in the neuroprotective mechanism of FK962. Rat primary retinal cells were cultured under 24-hour hypoxia/24-hour reoxygenation conditions, with or without FK962, recombinant GDNF, GDNF antibody and RET receptor tyrosine kinase inhibitor, GSK3179106. Cells were co-immunostained with RBPMS and Neurofilament 200 as a RGC marker, and the number of survived RGCs was counted. Results showed H/R treatment decreased the number of survived RGCs. FK962 promoted RGC survival under H/R by a bell-shaped dose response, with the highest RGC-protective effect of 10-8 M. The protective effect was the same level with 10-12M exogenous GDNF. Addition of GDNF antibody or GSK3179106 counteracted the neuroprotective effect of FK962. From these results, it is suggested that FK962 ameliorates RGC death under H/R, possibly via a GDNF signaling pathway.

Calpain-specific breakdown fragment in human drusen.

PURPOSE: With aging and age-related macular dystrophy (AMD), proteolytic fragments are deposited in extracellular drusen located between the RPE and Bruch's membrane. Localized hypoxia may be a risk factor for AMD. Our hypothesis is that following hypoxia, activation of proteolytic enzymes called calpains may cause proteolysis/degeneration of retinal cells and RPE. No direct evidence has yet demonstrated activation of calpains in AMD. The purpose of the present study was to identify calpain-cleaved proteins in drusen. METHODS: Seventy-six (76) drusen were analyzed in human eye sections from six normal and twelve AMD human donor eyes. The sections were subjected to immunofluorescence for the calpain-specific 150 kDa breakdown product from α-spectrin, SBDP150 - a marker for calpain activation, and for recoverin - a marker for photoreceptor cells. RESULTS: Among 29 nodular drusen, 80% from normal eyes and 90% from AMD eyes stained positive for SBDP150. Among 47 soft drusen, mostly from AMD eyes, 72% stained positive for SBDP150. Thus, the majority of both soft and nodular drusen from AMD donors contained SBDP150. CONCLUSIONS: SBDP150 was detected for the first time in soft and nodular drusen from human donors. Our results suggest that calpain-induced proteolysis participates in the degeneration of photoreceptors and/or RPE cells during aging and AMD. Calpain inhibitors may ameliorate AMD progression.

Activation of Cytosolic Calpain, Not Caspase, Is Underlying Mechanism for Hypoxic RGC Damage in Human Retinal Explants.

Purpose: Activation of proteolytic enzymes, calpains and caspases, have been observed in many models of retinal disease. We previously demonstrated calpain activation in monkey retinal explants cultured under hypoxia. However, cellular responses are often species-specific. The purpose of the present study was to determine whether calpains or caspase-3 was involved in retinal ganglion cell (RGC) damage caused by hypoxia/reoxygenation in human retinal explants. The explant model was improved by use of an oxygen-controlled chamber. Methods: Human and monkey retinal explants were cultured under hypoxic conditions in an oxygen-controlled chamber and then reoxygenated. Calpain inhibitor SNJ-1945 was maintained throughout the culture period. Immunohistochemistry and immunoblotting were performed for calpains 1 and 2, calpastatin, α-spectrin, calpain-specific α-spectrin breakdown product at 150 kDa (SBDP150), caspase-3, and apoptosis-inducing factor (AIF). Propidium iodide (PI) staining measured membrane disruption, and TUNEL staining detected DNA fragmentation. Results: Activation of calpains in nerve fibers and increases of PI-positive RGCs were observed in retinal explants incubated for 16-hour hypoxia/8-hour reoxygenation. Except for autolysis of calpain 2, SNJ-1945 ameliorated these changes. In longer incubations under 24-hour hypoxia/16-hour reoxygenation, TUNEL-positive cells appeared, although activated caspase-3 and truncated AIF were not observed. DNA fragmentation was inhibited by SNJ-1945. Conclusions: An improved human retinal explant model showed that calpains, not caspase-3, were involved in cell damage induced by hypoxia/reoxygenation. This finding could be relevant for patient treatment with a calpain inhibitor if calpain activation is documented in human retinal ischemic diseases.