Stanniocalcin-1 protects retinal ganglion cells by inhibiting apoptosis and oxidative damage

Abstract

Optic neuropathy including glaucoma is one of the leading causes of irreversible vision loss, and there are currently no effective therapies. The hallmark of pathophysiology of optic neuropathy is oxidative stress and apoptotic death of retinal ganglion cells (RGCs), a population of neurons in the central nervous system with their soma in the inner retina and axons in the optic nerve. We here tested that an anti-apoptotic protein stanniocalcin-1 (STC-1) can prevent loss of RGCs in the rat retina with optic nerve transection (ONT) and in cultures of RGC-5 cells with CoCl2 injury. We found that intravitreal injection of STC-1 increased the number of RGCs in the retina at days 7 and 14 after ONT, and decreased apoptosis and oxidative damage. In cultures, treatment with STC-1 dose-dependently increased cell viability, and decreased apoptosis and levels of reactive oxygen species in RGC-5 cells that were exposed to CoCl2. The expression of HIF-1α that was up-regulated by injury was significantly suppressed in the retina and in RGC-5 cells by STC-1 treatment. The results suggested that intravitreal injection of STC-1 might be a useful therapy for optic nerve diseases in which RGCs undergo apoptosis through oxidative stress.

Figure 1. Intravitreal administration of STC-1 increased the survival of retinal ganglion cells after optic nerve transection.

(A) Immediately after optic nerve transection, 1 μg STC-1 or PBS was injected into the vitreous cavity of rats, and the retinas were evaluated for retinal ganglion cells (RGCs) at days 1, 7, 14, and 28. (B) The density of RGCs was significantly higher in the retinas treated with STC-1 compared to PBS-treated retinas at all time-points examined as counted by cells retrogradely labeled with DTMR dye (C). The values are presented as the mean ± SEM. Scale bars, 100 μm.

Figure 2. Intravitreal STC-1 administration decreased apoptosis and oxidative damage in the retina after optic nerve transection.

(A–C) ELISA analysis showed that levels of active caspase-3 and two markers for oxidative damage (nitrotyrosine and protein carbonyl) were significantly decreased in the retina by an intravitreal injection of STC-1. (D, E) Real time RT-PCR indicated that levels of transcripts for HIF-1α and caspase-3 were increased in the retinas at day 1 after injury and significantly decreased by STC-1 treatment. The expression of caspase-3 was also significantly lower in the STC-1-treated retinas at day 7. (F) Western blot analysis confirmed that the expression of HIF-1α protein was increased in the retina at day 1 after ONT, and decreased by STC-1 injection. (G) The protein levels of UCP2 in the retina were decreased by ONT and not changed by STC-1 treatment. The values are presented as the mean ± SEM.

Figure 3. STC-1 inhibited apoptosis of RGC-5 cells exposed to CoCl₂.

(A) MTT assay showed that exposure to CoCl₂ for 12 h decreased the viability of RGC-cells in a concentration-dependent manner. (B) STC-1 treatment significantly rescued RGC-5 cells that were injured by either 200 μM or 400 μM CoCl₂ in a dose-dependent manner. (C, D) Flow cytometry showed that CoCl₂ increased the numbers of PI⁺Annexin⁺ cells in RGC-5 cells in concentration- and time-dependent manners. (E, F) Both 100 and 500 ng/mL of STC-1 decreased the numbers of PI⁺Annexin⁺ cells in RGC-5 cells injured by 200 μM CoCl₂. The values are presented as the mean ± SEM.

Figure 4. STC-1 inhibited ROS levels in RGC-5 cells exposed to CoCl₂.

(A, B) Flow cytometry showed that CoCl₂ significantly increased the percentage of cells positive for both CellROX^TM and MitoTracker Green, a marker for oxidative stress, and treatment with either 100 or 500 ng/mL of STC-1 significantly decreased the percentages of CellROX⁺ MitoTracker Green⁺ cells in RGC-5 cells. (C) ELISA analysis for nitrotyrosine indicated that levels of nitrotyrosine were markedly increased in CoCl₂-injured RGC-5 cells, and significantly decreased by STC-1 or N-acetylcysteine treatment. (D) Real time RT-PCR analysis indicated that expression of HIF-1α was induced in RGC-5 cells by CoCl₂, and was significantly down-regulated by STC-1 (100 or 500 ng/mL). However, UCP2 transcripts were not increased by STC-1. (E) Western blot analysis for HIF-1α showed that HIF-1α protein was increased in RGC-5 cells after CoCl₂ injury, and was decreased by STC-1 treatment. (F) ELISA showed that the levels of UCP2 protein were not increased in CoCl₂-injured RGC-5 cells by STC-1 treatment, whereas N-acetylcysteine treatment significantly increased levels of UCP2. The values are presented as the mean ± SEM.