Abstract
Purpose: :
Oxidative stress on RPE is thought to play a role in the pathogenesis of age–related macular degeneration (AMD). Previous studies have shown that AIF, a mitochondrial protein, appears to play a role in apoptosis of RPE following exposure to high levels of oxidative stress. Once released from mitochondria, it translocates to the nucleus to initiate 50kbp DNA fragmentation, nuclei condensation and cell death via a caspase independent mechanism. The present study examined the role of AIF as a direct mediator of oxidative stress–induced RPE cell death.
Methods: :
Green fluorescent protein (GFP) was cloned in frame to the C–terminus of mouse AIF and an E1–deleted adenoviral vector was produced to express AIF/GFP. Adenovirus expressing Lac Z was used as a control. Expression of AIF/GFP was determined by Western blot. The DNA content of control (cRPE), adenoviral LacZ transduced (LacZ–RPE) or adenoviral AIF–GFP transduced (AIF/GFP–RPE) RPE cells treated with the oxidant agent hydroquinone (HQ) was determined by FACS analysis using propidium iodium staining. DNA fragmentation was examined by pulse field gel electrophoresis. Mitochondrial potential was monitored by mitotracker fluorescence.
Results: :
Mitochondrial expression of AIF/GFP was observed at four days following transduction with adenovirus (MOI <25). Similar to Lac–Z RPE and cRPE cells, no change in DNA content was noted. HQ induced an equivalent dose dependent loss of DNA content in both cRPE and LacZ–RPE cells. At sublethal concentrations of HQ no DNA loss, DNA fragmentation or loss of mitochondrial potential in cRPE or LacZ–RPE cells were noted. However, at equivalent sublethal doses, a loss of DNA content was observed in AIF/GFP–RPE cells. This DNA damage was the result of large scale DNA fragmentation (50kbp), was accompanied by nuclear shrinkage and loss of mitochondrial membrane potential, events which were not observed in AIF/GFP–RPE cells alone.
Conclusions: :
AIF overexpression, which by itself, does not appear to induce apoptosis, sensitizes RPE cells to undergo apoptosis at low doses of oxidative damage. These results suggest that AIF may be an important therapeutic target for the treatment of RPE cell injury during AMD.
Keywords: apoptosis/cell death • age-related macular degeneration • retinal pigment epithelium