Abstract
Abstract: :
Purpose: RPE cells are located in a highly oxidative environment, therefore there is a high risk of mitochondrial damage. Sustained mtDNA damage leads to mitochondrial damage and cell death, but this process is not well understood. In yeast, COS7 cells, and Hela cells, stress induces mitochondrial fission prior to apoptosis, but this is not known for RPE cells. This study was undertaken to determine if RPE mitochondria initiate fission when the process of apoptosis is triggered. Methods: ARPE–19 cells, incubated in 10% DMEM–F12 medium, were treated with H2O2/TNFα and examined at one–hour intervals for up to 8 h. Mitochondrial fission and apoptosis–related molecules were localized immunohistochemically, quantitated by Western blot, and compared to untreated age–matched cells. Conventional methods were used. Finally, RNA was extracted using RNA LabChips and gene expression analyzed with human genome U95Av2 GeneChip arrays. The 6–hour post–treatment time point was compared to untreated controls. Results: Gene–chip analysis revealed up–regulation of the DNA–repair gene polymerase ß, the genes for dynamin 2 and dynamin–like protein–1, fission protein, and fis–1. Also, the gene for bax delta was up–regulated and bax inhibitor 1 and mitofusin 2 down–regulated. Colocalization of the dynamin proteins and bax with mitochondria, peaking at 5–6 hours, was demonstrated immunohistochemically, while Western–blot analysis showed bcl–2 consistently low, and up–regulation of bax, beginning around 3 hours, peaking at 6 hours. The fission–related molecules dynamin 2 and dynamin–like protein 1 were also up–regulated, beginning after 3 hours and peaking at 6 hours. Conclusions: Following initiation of oxidative stress, ARPE–19 cells up–regulate genes and express proteins directed to mitochondria to induce fission. Similarly, pro–apoptotic genes are up–regulated and their products are directed to the mitochondria. At the same time, the nuclear repair enzyme DNA polymerase ß is up–regulated, indicating the occurrence of nuclear damage. This demonstrates that oxidative stress induces both mitochondrial fission and apoptosis, and may indicate that mitochondrial fission is an early event in the apoptotic pathway of these cells. This suggests that inhibition of mitochondrial fission might be a target to intervene in degenerative retinal diseases. Supported by NIH EY05121; DARPA HR0011–04–C–0068
Keywords: apoptosis/cell death • mitochondria • oxidation/oxidative or free radical damage