June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Chronic activation of the unfolded protein response attenuates protein synthesis in the retinas of rd16 mice
Author Affiliations & Notes
  • Christopher R Starr
    Optometry and Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Priyamvada M Pitale
    Optometry and Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Marina Gorbatyuk
    Optometry and Vision Sciences, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Footnotes
    Commercial Relationships   Christopher Starr, None; Priyamvada Pitale, None; Marina Gorbatyuk, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4778. doi:
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    • Get Citation

      Christopher R Starr, Priyamvada M Pitale, Marina Gorbatyuk; Chronic activation of the unfolded protein response attenuates protein synthesis in the retinas of rd16 mice. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4778.

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

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Abstract

Purpose : In recent years, studies have demonstrated that chronic activation of the unfolded protein response (UPR) is involved in the pathogeneses of varied retinal dystrophies and plays a role in the activation of pro-inflammatory and aberrant calcium signaling that results in apoptotic photoreceptor (PR) cell death. The goal of this study is to test the hypothesis of whether a persistently activated UPR can negatively regulate protein synthesis in degenerating PRs.

Methods : Retinas were isolated from C57BL/6 (WT) and rd16 mice at postnatal (p) day 15 and 20 to assess activation of the UPR by western blot analysis. To study the rate of protein synthesis, we used the surface sensing of translation (SUnSET) method. Mice were intraperitoneally injected with puromycin (Pu) (0.04μmol/g body weight), and after 30 min, retinas were harvested for western blot analysis using an anti-Pu antibody. Intensity of incorporated Pu was normalized to the total protein level detected by coomassie staining of the membrane. In addition, primary rod PRs were isolated from WT p20 mice. Primary PRs were treated with 3μM thapsigargin for 2h to activate the UPR followed by exposure to 20μM Pu for 30min. Levels of nascent protein synthesis were assessed by immunocytochemical analysis and quantitative fluorescence microscopy.

Results : The UPR markers PERK, BiP, ATF4, CHOP and ATF6 were significantly upregulated in rd16 at p15 and p20; the elevation was in the range from 85% to 266%. Phospho-eIF2α, a hallmark of translational arrest, was elevated at p15 and p20 by 180 and 108%, respectively. In addition, phospho-4EBP1, whose level correlates with translation, was reduced by 30% at both p15 and p20. These levels were associated with a 26% reduction in protein synthesis at p15 as detected by the SUnSET technique. Results of the in vivo study were confirmed by experiments with Pu-treated primary PRs demonstrated that UPR activation inhibits the rate of newly synthesized proteins by over 3-fold.

Conclusions : Rd16 retinas with a persistently activated UPR undergo translational inhibition. Future experiments may shed light on the role of impaired protein synthesis in the function of degenerating PRs. Strategies that reduce the duration of the PERK-phospho-eIF2α branch and restore protein synthesis could be applied as a novel therapeutic approach to retard retinal degeneration.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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