Alterations in Optic Nerve Head (ONH) Endoplasmic Reticulum (ER) Stress Response Proteins in Non-Human Primate (NHP) Early Experimental Glaucoma (EG).

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 fibrosis^2-4.
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This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.