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Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium
Published on: February 27, 2011
Rod outer segments mediate mitochondrial DNA damage and apoptosis in human retinal pigment epithelium
G F Jin1, J S Hurst, B F Godley
1Department of Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston 77555-0787, USA.
Purpose:
To investigate the interrelationships between DNA damage, mitochondrial activity, and apoptosis in retinal pigment epithelial cells (RPE) after exposure to rod outer segments (ROS).
Methods:
After incubation of cultured human RPE with ROS, mitochondrial redox function was evaluated from MTT reduction. Mitochondrial (mt) and nuclear (n) DNA damage were determined by quantitative polymerase chain reactions (QPCR). Apoptotic RPE cells were detected by binding of annexin V to phosphatidyl serine (PS) using fluorescence microscopy. The expression of the pro-apoptotic proteins, p53 and p21(waf-1), and DNA repair enzymes, apurinic/apyrimidinic endonuclease (APE(ref-1)) and DNA polymerase beta (beta-pol) were quantitatively determined by Western blotting analysis.
Results:
Mitochondrial function decreased by 20 +/- 5% and annexin V immunofluorscent binding was enhanced after exposure of cells to physiological levels of ROS (3.8 x 10(6)cm(-2)) for 4 h. MtDNA was preferentially damaged after exposure to ROS with increased lesion frequencies of 1.49 +/- 0.37 and 2.2 +/- 0.14 per 10 kb base pairs (bp), respectively after 5 and 7 h contact, compared to untreated controls (zero class damage). APE(ref-1)expression increased more than 340% above controls after exposure to ROS for 7 and 24 h. The expression of beta-pol in cultures increased 110% above controls after 24 h contact with the ROS. The expression of p53 and p21 in cells increased 100 and 38% above controls after 24 h exposure to the ROS.
Conclusions:
Exposure of ROS to ROS induced mtDNA damage and dysfunction and activated nDNA repair pathways, which did not prevent apoptosis.
Insights
Exposure to rod outer segments (ROS) damages mitochondrial DNA and function in retinal cells, activating DNA repair but still leading to apoptosis.
Area of Science:
- Ophthalmology
- Cell Biology
- Molecular Biology
Background:
- Retinal pigment epithelial cells (RPE) are crucial for photoreceptor health.
- Rod outer segments (ROS) are shed daily and can potentially induce cellular stress.
- Understanding RPE response to ROS is vital for retinal disease research.
Purpose of the Study:
- To investigate the relationship between DNA damage, mitochondrial function, and apoptosis in RPE cells.
- To determine the cellular response to exposure to rod outer segments (ROS).
Main Methods:
- Cultured human RPE cells were exposed to ROS.
- Mitochondrial function was assessed via MTT reduction.
- Mitochondrial and nuclear DNA damage were quantified using qPCR.
- Apoptosis was detected using annexin V staining.
- Expression of p53, p21, APE(ref-1), and beta-pol was analyzed by Western blotting.
Main Results:
- ROS exposure decreased mitochondrial function by 20% and increased apoptosis.
- Mitochondrial DNA (mtDNA) showed preferential damage after ROS exposure.
- Expression of DNA repair enzymes APE(ref-1) and beta-pol significantly increased.
- Pro-apoptotic proteins p53 and p21 expression also elevated after ROS exposure.
Conclusions:
- ROS exposure induces mtDNA damage and mitochondrial dysfunction in RPE cells.
- DNA repair pathways are activated in response to ROS-induced damage.
- Despite DNA repair activation, apoptosis is not prevented, suggesting complex cellular responses to ROS.
