April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Decreased Cellular Glutathione (GSH) and Increased GSH Efflux in -Crystallin Knockout Retina
S. Parameswaran; C. Spee; S. J. Ryan; R. Kannan; D. R. Hinton
Author Affiliations & Notes
  • S. Parameswaran
    Doheny Eye Institute, Arnold and Mabel Beckman Macular Research Center, Los Angeles, California
  • C. Spee
    Doheny Eye Institute, Arnold and Mabel Beckman Macular Research Center, Los Angeles, California
  • S. J. Ryan
    Doheny Eye Institute, Arnold and Mabel Beckman Macular Research Center, Los Angeles, California
    Ophthalmology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • R. Kannan
    Doheny Eye Institute, Arnold and Mabel Beckman Macular Research Center, Los Angeles, California
    Pathology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • D. R. Hinton
    Doheny Eye Institute, Arnold and Mabel Beckman Macular Research Center, Los Angeles, California
    Pathology,
    Keck School of Medicine of the University of Southern California, Los Angeles, California
  • Footnotes
    Commercial Relationships  S. Parameswaran, None; C. Spee, None; S.J. Ryan, None; R. Kannan, None; D.R. Hinton, None.
  • Footnotes
    Support  NIH Grants EY03040, EY01545, Arnold and Mabel Beckman Foundation and RPB
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 1831. doi:
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      S. Parameswaran, C. Spee, S. J. Ryan, R. Kannan, D. R. Hinton; Decreased Cellular Glutathione (GSH) and Increased GSH Efflux in -Crystallin Knockout Retina. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1831.

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Abstract

Purpose: : Knockout of -crystallins renders retinal cells more susceptible to apoptotic cell death. Decreased retinal antioxidant status could contribute to increased vulnerability to apoptosis. We sought to determine GSH regulation in -crystallin knockout retina and -crystallin-overexpressing ARPE-19 cells.

Methods: : Neural retina and RPE/choroid were dissected from 6- to 8-wk-old wild-type (WT), A crystallin (-/-) and B crystallin (-/-) mice. GSH levels were measured by colorimetric assay. Gene and protein expression of γ-glutamylcysteine synthetase (GCS) was determined by real-time PCR and Western blot. Localization of GSH expression in -crystallin (-/-) mice was evaluated by confocal microscopy. Overexpression of -crystallin in ARPE-19 cells was achieved by transfection with pcDNA3.1 -crystallin plasmid. The effect of H2O2(150µM, 24h) on these cells was examined. GSH levels in cytosolic and mitochondrial fractions in crystallin-overexpressing cells were also measured. GSH efflux was measured in RPE derived from B crystallin (-/-) mice in serum-free medium 24 h after H2O2.

Results: : Knockout of -crystallins significantly (p<0.05) decreased GSH in neural retina and RPE/choroid. Confocal microscopy revealed the intensity of GSH immunostaining in -crystallin (-/-) mice was markedly lower vs WT retina. Overexpression of -crystallins in ARPE-19 cells significantly increased GSH levels (p<0.05 vs vector control) with 2.2-fold upregulation of the catalytic subunit of GCS. The GSH increase was more pronounced in mitochondrial fraction vs cytosolic fraction with oxidant stress. Upon H2O2 challenge, cell death in -crystallin overexpressing cells was attenuated and active caspase 3 expression was decreased vs controls. GSH efflux in RPE of B crystallin (-/-) mice was twofold higher than vs WT RPE.

Conclusions: : Absence of -crystallins is accompanied by a decrease in GSH levels in mouse retina and a significant increase in GSH efflux from RPE. The protection from oxidant-induced cell death in -crystallin-overexpressed cells could be due to increased mitochondrial levels of GSH.

Keywords: oxidation/oxidative or free radical damage • crystallins • antioxidants 
© 2009, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Permission to republish any abstract or part of an abstract in any form must be obtained in writing from the ARVO Office prior to publication.
Copyright © 2025 Association for Research in Vision and Ophthalmology.
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