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Jacky Man Kwong Kwong, Thomas Lam, Ka K. Li, Chi-ho To, Joseph Caprioli; Global proteomic analysis of retina in progressive retinal ganglion cell (RGC) degeneration. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2559. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the retinal proteome during RGC damage.
Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the optic nerve in adult Wistar rats; the contralateral eye was used as a control. The density of RGCs in the retina from 1 to 8 weeks after pONT was determined by topographical quantification with Rbpms antibody. Temporal and nasal retinal samples were collected separately from both eyes and homogenized to extract soluble proteins. Equal amount of protein lysates was trypsin-digested and subjected to proteomic profiling with a nanoLC TripleTOF mass spectrometry (MS) running on label-free SWATHTM acquisition. MS spectra were searched for protein identification (at 1% FDR) with ProteinPilot 5.0 (Sciex). Gene Ontology annotation of all identified retinal proteins was performed with PANTHER gene classification. Differential protein expressions (≥1.5 fold, P<0.05) in response to pONT at different time points were compared with a novel cloud-based Oneomics platform on BaseSpace (Illumina).
The average size of injured retinal area was 46.77±16.52% at 8 weeks after pONT. There were 0.92, 0.15, 0.1 and 0.14 TUNEL positive cells in the RGC layer per temporal retinal section at 1, 2, 4 and 8 weeks after pONT, respectively; there were 0.17, 0.17, 0.13 and 0.13 TUNEL positive cells per nasal retinal section (n=4 per time point). No significant change in RGC density was detected in the nasal quadrant at 1 week after pONT (n=8) but the percentage loss increased to 43.63±7.74% at 8 weeks (n=15; P=0.0001) while there was 78.89±5.76% and 27.7±5.99% of RGC survival in the temporal quadrant at 1 and 8 weeks respectively. This indicates secondary RGC degeneration in the nasal retina, and acute and delayed phases of RGC degeneration in the temporal retina. About 2900 distinct proteins (>22,000 peptides) were identified from a pooled retinal ion library for SWATHTM quantification. At 1 week, an aldehyde dehydrogenase 1 family protein (Aldh1a1) was significantly up-regulated in the temporal retina after pONT but no significant protein change was detected in controls. At 8 weeks, 23 proteins were differentially expressed and the majority of them belongs to crystallin family.
Differential protein expression was observed in different phases of progressive RGC degeneration. This approach may help elucidate the acute proteomic response to RGC injury.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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