April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Photic Injury Induces Disulfide Linkage And Dimer Formation In Visual Arrestin
Author Affiliations & Notes
  • Christopher J. Lieven
    Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin
  • Jonathan D. Ribich
    Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin
  • Leonard A. Levin
    Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin
    Ophthalmology, University of Montreal, Montreal, Quebec, Canada
  • Footnotes
    Commercial Relationships  Christopher J. Lieven, None; Jonathan D. Ribich, None; Leonard A. Levin, Wisconsin Alumni Research Foundation (P)
  • Footnotes
    Support  NIH R21EY017970, P30EY016665
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 1812. doi:
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      Christopher J. Lieven, Jonathan D. Ribich, Leonard A. Levin; Photic Injury Induces Disulfide Linkage And Dimer Formation In Visual Arrestin. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1812.

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Abstract

Purpose: : High levels of light exposure may play a role in degenerative retinal conditions. Oxidative injury occurs in light-induced retinal degeneration, and antioxidants reduce photoreceptor loss from photic injury. There are multiple pathways by which oxidative injury could signal cell death in photic injury. Our previous work demonstrated that the sulfhydryl reducing agent tris(2-carboxyethyl)phosphine (TCEP) decreased outer retinal thinning in a rat photic injury model, suggesting that formation of one or more critical disulfides could be necessary for photoreceptor death. We therefore sought to identify target proteins that formed hetero- or homodimers as a result of photic injury.

Methods: : Male Wistar rats 6 to 10 weeks of age were reared on a normal 12-light/12-dark light cycle. Rats were dark-adapted for 16 hours, then administered tris(2-carboxyethyl)phosphine (TCEP; 100 µmol/kg), saline, or nothing immediately prior to exposure to uniform white fluorescent light at 10 klux for 10 hrs, or kept in normal cyclic light conditions. Rats were sacrificed immediately after exposure, and retinas prepped for gel electrophoresis in the presence of an alkylating agent. Gels were run in non-reducing conditions, lanes excised and treated with a reducing agent, then rotated 90 degrees and run in the second dimension. Proteins forming intermolecular disulfides were identified by a decrease in weight after reduction, causing them to "fall off" the diagonal, and MALDI/TOF mass spectrometry. Quantitative studies were carried out by western blotting.

Results: : Light exposure in dark-adapted Wistar rats led to a substantial increase in the intensity of a disulfide-linked protein, identified by mass spectrometry to be visual arrestin. Western blotting confirmed a significant increase in a redox-sensitive arrestin dimer after photic injury (p = 0.005 by Wilcoxon signed-rank test). Dimer formation depended on the length of light exposure, and was prevented by treatment of the animal with TCEP immediately prior to light exposure.

Conclusions: : Visual arrestin undergoes disulfide-dependent dimerization as a result of photic injury, and TCEP prevents dimer formation and ameliorates photoreceptor death. It is unclear whether this dimerization is a necessary or sufficient step for light-induced retinal degeneration.

Keywords: retinal degenerations: cell biology • oxidation/oxidative or free radical damage • apoptosis/cell death 
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