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Claude Burgoyne, Cheri Stowell, Geeng-Fu Jang, Lei Zhang, Belinda Willard, Jack S Crabb, John W Crabb; NON-HUMAN PRIMATE (NHP) OPTIC NERVE HEAD (ONH) PROTEOMIC CHANGE IN EARLY EXPERIMENTAL GLAUCOMA (EEG). Invest. Ophthalmol. Vis. Sci. 2014;55(13):4555.
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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.
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.
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.
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
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