Abstract
Purpose: :
To determine the proteomic changes that occur in the T4R RHO mutant retina at the onset of photoreceptor (PR) cell death following acute light exposure.
Methods: :
A group of 6 T4R RHO mutant dogs were exposed in the left eye for 1 min with a source of white light delivered by means of a monocular Ganzfeld (corneal irradiance 1mW/cm2). The contralateral right eye was shielded and served as a control. At time points ranging from 1 hr to 2 weeks after light exposure, the eyes were collected and processed for histology and TUNEL assay. A second group of 3 littermate T4R RHO homozygous mutant dogs were light exposed as described above, and retinas collected 6 hrs later for 2D DIGE nanoLC-MS/MS proteomic analysis. Semi-quantitative western blotting was done to confirm differential protein levels of our focus proteins.
Results: :
TUNEL positive rods were first seen 6 hrs after light exposure. Within 24-48 hrs there was massive cell death of PRs and occasional RPE cells which resulted in significant ONL thinning within 2 weeks. Proteomic analysis performed at the onset of cell death (6 hrs post light exposure) identified 40 spots that had at least a 1.5 fold difference in intensity. Of these, 17 unique proteins were identified. Bioinformatic analysis revealed 4 proteins involved in glycolysis (fructose-bisphosphate aldolase C, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose phosphate isomerase), 3 in oxidative phosphorylation (ATP synthase subunit beta mitochondrial, ATP synthase subunit O, mitochondrial, V-ATPase subunit B2), and 3 in oxidative stress response (DnaJ homolog subfamily C member 14, Glutathione S-transferase P, and peroxiredoxin 1)
Conclusions: :
One minute exposure to light causes acute PR cell death which peaks at 24 hrs in the T4R RHO mutant retina. At the onset of cell death (6 hrs), there is differential regulation of many proteins involved in energy metabolism and oxidative stress.
Keywords: proteomics • retinal degenerations: cell biology • apoptosis/cell death