Abstract
Purpose:
To study the molecular mechanisms underlying ONH remodeling in NHP EEG1 and discover candidate blood-borne biomarkers, we quantified ONH proteomic change in Rhesus Macaques with laser-induced, unilateral EEG (0-30% optic nerve (ON) axon loss)2.
Methods:
An ONH trephine (6mm) containing peripapillary sclera, retina and retrolaminar ON (≈2 mm behind the globe) was obtained from the EEG/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 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. Proteins were significantly altered when average ratios (EEG/ control) determined with ≥ 2 unique peptides in the 3 high (or 1 low) IOP animals were at least 1 standard deviation (SD) from the mean (p values ≤ 0.05). Bioinformatics analyses utilized Ingenuity Pathway Analysis.
Results:
Among 1122 proteins identified in the high IOP animals, 55 were significantly increased and 68 were significantly decreased in the EEG ONH. Proteins significantly increased ≥ 2SD included chondroadherin, pigment epithelium-derived factor, biglycan, heterogenous nuclear ribonucleoprotein Q and R, and Fibulin-1. Proteins significantly decreased ≥ 2SD included alpha-tubulin N-acetyltransferase, hyaluronan and proteoglycan link protein 2, NAD-dependent protein deacetylase sirtuin-2, Dihydropteridine reductase, and 7 myelin associated proteins. Altered proteins or the direction of protein alteration were often different in the low IOP EEG ONH.
Conclusions:
Top canonical pathways associated with altered ONH proteins in high IOP NHP EEG included actin cytoskeletal signaling, epithelial adherens junction signaling/remodeling and axon guidance signaling. Top networks included cellular assembly and organization. While additional animals must be studied, proteomic differences between low/high IOP EEG ONH may represent earlier/later stages of early ONH damage.3 Altered proteins provide insights into the molecular mechanisms underlying early ONH alterations in NHP EEG and suggest candidate blood-borne biomarkers for its systemic detection. 1Burgoyne. Exp Eye Res 2011 2He, et al. IOVS 2013. In Press. 3Howell, et al. J Clin Invest 2011
Keywords: 577 lamina cribrosa •
663 proteomics •
430 astrocytes: optic nerve head