July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Alterations in Optic Nerve Head (ONH) Endoplasmic Reticulum (ER) Stress Response Proteins in Non-Human Primate (NHP) Early Experimental Glaucoma (EG).
Author Affiliations & Notes
  • Cheri Stowell
    Discoveries in Sight, Devers Eye Institute, Beaverton, Oregon, United States
  • Geeng-Fu Jang
    Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
    Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States
  • Lei Zhang
    Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
    Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States
  • Jack Crabb
    Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
    Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States
  • Juan Reynaud
    Discoveries in Sight, Devers Eye Institute, Beaverton, Oregon, United States
  • Stuart Keith Gardiner
    Discoveries in Sight, Devers Eye Institute, Beaverton, Oregon, United States
  • belinda Willard
    Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States
  • Nicholas Marsh-Armstrong
    Department of Ophthalmology, University of California, Davis, California, United States
  • John W Crabb
    Cole Eye Institute, Cleveland Clinic, Cleveland, Ohio, United States
    Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio, United States
  • Claude F Burgoyne
    Discoveries in Sight, Devers Eye Institute, Beaverton, Oregon, United States
  • Footnotes
    Commercial Relationships   Cheri Stowell, None; Geeng-Fu Jang, None; Lei Zhang, None; Jack Crabb, None; Juan Reynaud, None; Stuart Gardiner, None; belinda Willard, None; Nicholas Marsh-Armstrong, None; John Crabb, None; Claude Burgoyne, None
  • Footnotes
    Support  NIH Grants R01-EY011610 (CFB), EY021840 (JWC), EY022134 (JWC), Legacy Good Samaritan Foundation, AGS Mid-Career Award (CFB), Alcon Research Institute (CFB), Sears Medical Trust (CFB), Research to Prevent Blindness Challenge Grant (JWC), RPB Senior Investigator Award (JWC)
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3514. doi:
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      Cheri Stowell, Geeng-Fu Jang, Lei Zhang, Jack Crabb, Juan Reynaud, Stuart Keith Gardiner, belinda Willard, Nicholas Marsh-Armstrong, John W Crabb, Claude F Burgoyne; Alterations in Optic Nerve Head (ONH) Endoplasmic Reticulum (ER) Stress Response Proteins in Non-Human Primate (NHP) Early Experimental Glaucoma (EG).. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3514.

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

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Abstract

Purpose : ER stress has been implicated in the pathophysiology of glaucomatous ONH damage. Here we test the hypothesis that ONH ER stress response protein expression is altered in NHP early EG using quantitative proteomics and immunohistochemistry (qIHC).

Methods : For proteomics, ONH, including a 2 mm nerve stump, were excised from the early EG and control eyes of 3 adult NHPs. Protein was extracted, digested with trypsin, labeled with iTRAQ tags, fractionated by chromatography, analyzed by LC MS/MS, and identified using the UniProt human database. Average ratios ≥ 1 standard deviation from the mean (p values < 0.05) were considered significantly altered. Bioinformatic analyses were done using DAVID 6.8 Functional Annotation Tool. For qIHC, EEG NHPs (n=4) were perfusion fixed with paraformaldehyde, early EG and Control eye ONHs were trephined (10 mm), sectioned at 5 um, stained, and imaged at 40x for fluorescence or 10x for polarization. Polarized images were delineated, a grid pattern created and average IHC density quantified using in-house software. Statistical analyses were performed using Generalized Estimating Equation in R.

Results : DAVID analysis identified a clustering of ONH ER/protein folding proteins that were significantly increased (PRA1 family protein 3, calnexin, calreticulin, clusterin, 78 kDa glucose regulated protein, heterogeneous nuclear ribonucleoprotein R, malectin, Peptidyl-prolyl cis-trans isomerase B, protein disulfide isomerase, reticulocalbin 3, and heterogeneous nuclear ribonucleoprotein Q) and decreased (epoxide hydrolase 1 and heat shock-related 70 kDa protein 2) in the early EG compared to control eyes. To date, significant increases (p <0.05) in the retinal nerve fiber layer and retrolaminar nerve for calreticulin and clusterin as well as increases in the prelamina, lamina and scleral for clusterin have been confirmed by qIHC. qIHC reticulocalbin-3 studies are underway.

Conclusions : Chaperone proteins that are part of the ER stress response system are altered within the ONH in NHP early EG. Their roles may include the clearance of misfolded proteins through regulation of macrophages1 as well as alterations in collagen synthesis and folding in fibrosis2-4.
Ni Fhlathartaigh et al Acta Neuropathol Commun. 2013
Wilson et al. Mol Cell Proteomics 2012
Martinez-Martinez et al. Sci Rep. 2017
Marie et al. J Proteomics 2015

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

 

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