MYELIN-RELATED PROTEINS ARE DECREASED IN THE IMMEDIATE RETROLAMINAR OPTIC NERVE (ON) IN NON-HUMAN PRIMATES (NHP) IN EARLY EXPERIMENTAL GLAUCOMA (EEG)

Abstract

Purpose: To test the hypothesis that optic nerve head (ONH) remodeling in NHP EEG¹ may involve alterations in retrolaminar myelin². Quantitative changes in myelin-related proteins in EEG were assessed by mass spectrometry (MS), western blotting (WB) and immunohistochemistry (IHC).

Methods: The ONH (6 mm trephine including ~ 2 mm ON) and ON (starting ~ 2 mm behind the globe) were excised from the EEG and control eyes of n=3 NHP (IOP Max >28 mmHg). Protein was extracted in SDS and digested with trypsin. Peptides were labeled with iTRAQ tags, fractionated by chromatography, and analyzed by LC MS/MS. Proteins were identified using the Swiss-Protein human database; iTRAQ tags were quantified by the weighted averaged method using Mascot 2.4.1. Proteins were considered significantly altered if average ratios (EEG/ control) from ≥ 2 unique peptides in the 3 animals were at least 1 standard deviation (SD) from the mean ratio for all proteins in each sample (p values ≤ 0.05). WB and IHC used antibodies to 2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) and myelin basic protein (MBP). EEG NHP (n=4, IOP Max >28 mmHg) for IHC were perfusion fixed with paraformaldehyde, paraffin embed and sectioned at 5 μm. IHC for CNP and MBP was performed in matched paraffin sections from 4 control and 4 EEG ONH trephines (10 mm).

Results: MBP, CNP, myelin proteolipid protein, myelin-associated glycoprotein, myelin-oligodendrocyte glycoprotein, opalin and aspartoacylase were significantly decreased ≥ 2SD in the ONH. In the ON, MBP, myelin-associated glycoprotein and opalin were decreased >1SD but did not achieve significance. Western analyses (in progress) have so far corroborated the iTRAQ quantitation for CNP and MBP. Quantitative IHC (in progress) indicates localized decreases in MBP and CNP within the immediate retrolaminar ON.

Conclusions: Myelin-related proteins are decreased in the NHP ONH tissues in EEG by MS, WB and IHC. These changes localize to the immediate retrolaminar ON by IHC, which may explain why there is no significant change in the ON samples. Retrolaminar myelin remodeling may contribute or be secondary to axonal injury in NHP EEG.² 1. Burgoyne. Exp Eye Res 2011 2. Nguyen JV, et al. Proc Natl Acad Sci U S A 2011

Keywords: 629 optic nerve • 554 immunohistochemistry • 663 proteomics