April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
βA3/A1-crystallin: A New Protein Involved In Degradation Of Rod Outer Segments During Rpe Phagocytosis
Author Affiliations & Notes
  • Debasish Sinha
    Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, Maryland
  • J S. Zigler, Jr.
    Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, Maryland
  • Cheng Zhang
    Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, Maryland
  • Rhonda Grebe
    Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, Maryland
  • Yuri V. Sergeev
    OGVFB, National Eye Institute, Bethesda, Maryland
  • Donald J. Zack
    Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, Maryland
  • James T. Handa
    Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, Maryland
  • Gerard A. Lutty
    Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, Maryland
  • Footnotes
    Commercial Relationships  Debasish Sinha, None; J. S. Zigler, Jr., None; Cheng Zhang, None; Rhonda Grebe, None; Yuri V. Sergeev, None; Donald J. Zack, None; James T. Handa, None; Gerard A. Lutty, None
  • Footnotes
    Support  EY018636 (DS), EY019037 (DS), EY019037-S (DS), EY09357 (GAL), EY016151-5 (GAL), EY14005 (JTH), EY000476-01 (YVS), EY01765 (Wilmer Imaging Core), HRF (DS) and RPB.
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 4786. doi:
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      Debasish Sinha, J S. Zigler, Jr., Cheng Zhang, Rhonda Grebe, Yuri V. Sergeev, Donald J. Zack, James T. Handa, Gerard A. Lutty; βA3/A1-crystallin: A New Protein Involved In Degradation Of Rod Outer Segments During Rpe Phagocytosis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4786.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : To demonstrate that βA3/A1-crystallin, a major structural protein of the ocular lens, is expressed in retinal pigmented epithelium (RPE) cells. Further, by utilizing the Nuc1 rat in which the βA3/A1-crystallin gene is mutated, we show that this protein is required by RPE cells for proper degradation of outer segment discs that have been internalized in phagosomes.

Methods: : Expression of βA3/A1-crystallin in RPE was analyzed by in situ hybridization, real-time PCR of laser capture micro-dissected RPE and also by western blotting (WB) and immunohistochemistry (IHC) during post-natal eye development of wild type (WT) and Nuc1 rats. Structural changes in the RPE were evaluated using electron microscopy. Immuno-electron microscopy was used for intra-cellular localization of βA3/A1-crystallin. Expression of autophagy proteins and lysosomal enzymes in the RPE was assessed by IHC and WB. Autofluorescence and Oil red-O staining was used to demonstrate abnormal lipid accumulation in RPE. Molecular modeling was used to investigate the effect of the Nuc1 mutation on putative lysosomal sorting signals of the βA3/A1-crystallin protein.

Results: : We demonstrate, for the first time, that βA3/A1-crystallin, a major structural protein of the ocular lens, is expressed in RPE cells. In the normal RPE, βA3/A1-crystallin is localized to the lysosomes. In the RPE of Nuc1 rats, βA3/A1-crystallin fails to translocate into the lysosomes and is found in the cytosol, perhaps because misfolding of the mutant protein masks sorting signals required for proper trafficking. Electron microscopy clearly shows that undigested outer segment material and cellular waste products accumulate in the mutant RPE cells. The digestion of phagocytized outer segments requires a high level of lysosomal enzyme activity, and Cathepsin D, the major enzyme responsible for proteolysis of the outer segments, is decreased in mutant RPE cells. Interestingly, our results also indicate a defect in the autophagy process in the Nuc1 RPE, probably also linked to impaired lysosomal function, since phagocytosis and autophagy may share common mechanisms in degradation of their targets.

Conclusions: : Our findings implicate βA3/A1-crystallin as a novel lysosomal component required by the RPE for the proper degradation of shed photoreceptor outer segment discs.

Keywords: retinal pigment epithelium • phagocytosis and killing • photoreceptors 
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