The Role of eIF5A in TNF– induced Apoptosis of Lamina Cribrosa Cells

Abstract

Abstract: : Purpose: To determine the role of eIF5A in the process of TNF–α induced apoptosis of lamina cribrosa (LC) cells. Methods: LC cells were isolated from the optic nerve heads of two human male eye donors. The LC cells were treated with TNF–α and camptothecin and the incidence of apoptosis was scored by Hoescht staining. The expression of eIF5A protein in response to camptothecin or a combination of camptothecin and TNF–α was determined by Western blot analysis. The ability of siRNAs directed against eIF5A to protect LC cells from TNF–α– induced apoptosis was also determined by Hoescht and TUNEL–staining of transfected LC cells. Results: Exposure to TNF–α and camptothecin resulted in the death of 45 % of LC cells after 48 hours as determined by Hoescht staining. Expression of eIF5A protein was found to increase significantly following 8 to 24 hours exposure to TNF–α and camptothecin but not in response to camptothecin alone. siRNAs directed against eIF5A were able to reduce apoptosis of LC cells in response to TNF–α and camptothecin by between 35 % and 69 % as determined by Hoescht staining. An siRNA against GAPDH, intended as a control, was also able to reduce apoptosis of LC cells by 42 %. Similarly, TUNEL labelling of transfected LC cells treated with TNF–α and camptothecin revealed an 80 % reduction in apoptosis with an siRNA against eIF5A. Conclusions: TNF–α induces apoptosis in human LC cells. eIF5A is upregulated by LC cells in response to TNF–α, and siRNAs against eIF5A protect LC cells from apoptosis. Thus, eIF5A appears to be a novel pro–apoptotic protein in the TNF pathway and a possible target for neuroprotection in glaucoma.

Keywords: apoptosis/cell death • astroglia: optic nerve head • lamina cribrosa