Quantitative Proteomic Analysis Of Non-Human Primate (NHP) Retina In Early Experimental Glaucoma (EEG)

Abstract

Purpose: To study the molecular mechanisms underlying retinal alterations in NHP EEG and discover candidate biomarkers, we quantified proteomic change in the retina of Rhesus Macaques with laser-induced, unilateral EEG.

Methods: Retinas were isolated from the EEG and control eyes of 4 adult NHP (3 high IOP (IOP Max >28 mm Hg, and 1 low IOP (IOP Max < 20 mm Hg). Protein was extracted in SDS and digested with trypsin. Peptides were labeled with iTRAQ tags, fractionated by cation exchange chromatography, and analyzed by LC MS/MS. Proteins were identified using the Swiss-Protein human database; iTRAQ tags were quantified by the weighted average method. Proteins were significantly altered if average ratios (EEG/ control) determined with ≥ 2 unique peptides in the 3 high IOP animals (or in the low IOP animal) were at least 1 standard deviation from the mean with p values ≤ 0.05. Proteomic analysis of EEG and POAG plasma was performed in separate experiments following immunodepletion. Bioinformatics analyses utilized Ingenuity Pathway Analysis.

Results: Within the high IOP retina, 177 significantly altered proteins were identified within 1714 proteins quantified. Altered proteins were often different in the low IOP retina. Significantly increased proteins in high IOP included phosphatidylinositol transfer protein alpha, periredoxin-1, glutathione S-transferase P, and Rab GDP-dissociation inhibitor beta. Significantly decreased proteins in high IOP included NADH dehydrogenase 1 alpha subunits 6 and 13, epsin, and PDZ domain-containing protein 8,. Many of the proteins altered in high IOP were detected in NHP EEG plasma, in human POAG plasma, and in the human plasma atlas, supporting the possibility of monitoring these proteins for biomarker validation.

Conclusions: Molecular and cellular functions associated with altered retinal proteins in high IOP NHP EEG included transport, cell-to-cell signaling and interaction, and small molecule biochemistry. Top networks included cellular assembly and organization and neurological disease. While additional animals must be studied, proteomic differences appear to exist in retina from low and high IOP EEG NHPs. Altered proteins provide insights into the molecular mechanisms underlying early retinal damage in NHP EEG and candidate blood-borne biomarkers.

Keywords: 688 retina • 568 intraocular pressure • 663 proteomics