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 μM) 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 μM 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 μM (4.5-fold). Phosphorylated SAPK/JNK increased 36-fold (200 μM CoCl2 concentration), then plateaued at the 300- (25-fold) and 400-μM (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-μM 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.
Department of Ophthalmology, University of Florida College of Medicine, Jacksonville, Florida 32209, USA. sbalaiya@ufl.edu
Sankarathi
S
Balaiya
Lee R
LR
Ferguson
Kakarla V
KV
Chalam
D017209:Apoptosis; D002454:Cell Differentiation / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects*; D002470:Cell Survival; D002478:Cells, Cultured; D006801:Humans; D000860:Hypoxia / Q000188:drug therapy* / Q000473:pathology; D008954:Models, Biological; D018696:Neuroprotective Agents / Q000494:pharmacology; D009902:Optic Neuritis / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism; D037761:Sirtuins / Q000494:pharmacology*
D018696:Neuroprotective Agents; D037761:Sirtuins