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Karen Eastlake, Wendy Heywood, Dhani C Tracey-White, Erika Aquino, Emily Bliss, Gerardo Vasta, Kevin Mills, Peng Tee Khaw, Mariya Moosajee, G. Astrid Limb; Comparison of zebrafish retinal proteins during experimental degeneration and regeneration using quantitative proteomics. Invest. Ophthalmol. Vis. Sci. 2017;58(8):365.
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© ARVO (1962-2015); The Authors (2016-present)
Zebrafish have the ability to regenerate retina after injury. Although many studies have investigated the retinal gene profile in this species during regeneration, understanding mechanisms involved in this process is far from complete as few studies have investigated the biological function at protein level. To address this we compared the proteomic profile of the zebrafish retina after injury and upon regeneration.
Adult longfin wildtype zebrafish eyes were injected with 200μM Ouabain to induce degeneration. Protein isolated from retina excised at 0, 3 and 18 days after injection -corresponding to normal, degenerated and regenerating states- were analysed using two-dimensional difference gel electrophoresis (2D-DIGE) and label-free quantitative proteomics using quadrupole time of flight (QToF) LC-MS/MS. Protein identification and bioinformatics analysis were performed using Protein Lynx Global Server (PLGS), Non-Linear dynamics Progenesis software and online Panther gene ontology database. Results were compared to RT-PCR and western blots from excised retina
Across all specimens, 2042 proteins were identified. Proteins showing the highest fold upregulation in the degenerated retina included fibrinogen gamma polypeptide, apolipoproteins A-Ib and A-II, galectin-1 and vitellogenin-6. In addition, gene ontology analysis indicates reduced metabolic processing, and an increase in fibrin clot formation. In the regenerating retina, cytoskeleton and membrane transport proteins were considerably altered, with the highest fold upregulation observed for tubulin beta 2A, histone H2B and brain type fatty acid binding protein. RT-PCR and western blot analysis confirmed that galectin-1 expression was significantly upregulated in the degenerated retina as compared to control or regenerating retina.
Investigations on the role of key proteins identified in the regenerating zebrafish retina may aid not only to understand mechanisms that prevent regeneration of the adult human retina, but to the design of new approaches to stimulate endogenous repair mechanisms following retinal degenerative disease.
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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