Evaluation of Sirtuin Role in Neuroprotection of Retinal Ganglion Cells in Hypoxia

PURPOSE. Hypoxia-induced apoptosis is responsible for reduced retinal ganglion cell (RGC) viability in a variety of chronic ocular disorders. Sirtuin 1 (SIRT1) plays an important role in preserving cell viability during hypoxia. We investigated the role of SIRT1 in sustaining RGC viability in an in vitro model of hypoxia. METHODS. Staurosphorine-differentiated RGCs (RGC-5) received varying hypoxic concentrations (100-500 mu M) of cobalt chloride (CoCl2) for 24 hours. Hypoxia-induced cell viability was assessed by WST-1 assay. The role of SIRT1 in promoting viability was determined indirectly via sirtinol (SIRT1 inhibitor). Hypoxia-induced apoptosis was evaluated by measuring stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) and caspase 3 activity. Vascular endothelial growth factor (VEGF) was measured to ascertain the influence of SIRT1. RESULTS. CoCl2 concentrations greater than 100 mu M resulted in significantly reduced RGC viability (P = 0.01). CoCl2 treatment increased SIRT1 levels significantly (P < 0.01): 100 (6.5-fold), 200 (6-fold), 300 (3.5-fold), and 400 mu M (4.5-fold). Phosphorylated SAPK/JNK increased 36-fold (200 mu M CoCl2 concentration), then plateaued at the 300- (25-fold) and 400-mu M (27.8-fold) CoCl2 concentrations (P < 0.01). CoCl2 and sirtinol treatment increased Caspase 3 activity (P < 0.05). VEGF release was significantly higher than control at the 100-mu M CoCl2 concentrations (P < 0.01). Sirtinol reduced RGC viability, SIRT1 levels, and VEGF release (P < 0.01) while having greater effect on SAPK/JNK phosphorylation. CONCLUSIONS. SIRT1 significantly influences RGC viability. Sirtinol's effect reflects the interaction SIRT1 has with apoptotic signaling proteins. This investigation demonstrated SIRT1 importance in forestalling the effects of hypoxia-induced apoptosis. (Invest Ophthalmol Vis Sci. 2012;53:4315-4322) DOI:10.1167/iovs.11-9259