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Hollis Cline, Lucio Schiapparelli, Sahil Shah, Danial McClatchy, YuanHui Ma, John Yates, Jeffrey L Goldberg; Synthesis and axonal transport of retinal ganglion cell proteins following optic nerve injury.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1581. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We are interested in understanding how protein synthesis and axon transport contribute to visual circuit function and response to optic nerve injury. We investigated the synthesis and transport of proteins from the retina to the superior colliculus (SC) and lateral geniculate nucleus (LGN) in adult rats by combining in vivo protein labeling strategies with quantitative mass spectrometry and proteomic bioinformatic analysis.
In one labeling strategy, we used Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) to label newly-synthesized retinal proteins by injecting the eye with a non-canonical amino acid, azidohomoalanine (AHA), which is incorporated into newly synthesized proteins (NSPs) in place of methionine. We recovered NPSs from the retina, optic nerve (ON ), and LGN. In a second protein labeling strategy, we injected eyes with NHS-biotin, which binds covalently to exposed amine groups in pre-exisitng proteins in the retina and recovered NHS-biotin labeled proteins from ON, LGN and SC. In both cases, we identified biotin-labeled proteins using a biotinylated peptide enrichment method called Direct Detection of Biotinylated Proteins (DiDBiT) that detects biotinylated proteins with high sensitivity, combined with multidimensional protein identification technology (MudPIT) to identify biotin-tagged proteins.
We identified proteins transported from retina ganglion cells (RGCs) to its major targets, the SC and LGN, called the “Transportome”. The population of proteins transported to LGN or SC is highly overlapping, but some of these proteins appear to be selectively transported to either LGN or SC. These differentially targeted proteins may contribute to the distinct properties of retinogeniculate and retinocollicular synapses and information transfer. To examine the response of axonal transport to optic nerve injury, we labeled proteins in the retina with either AHA or NHS-biotin to identify differences in NSPs or pre-existing proteins in response to ON crush in adult rats. We identified ~30 proteins whose synthesis and subsequent transport into the ON were significantly altered after ON crush compared to control ONs.
Analysis of protein dynamics and transport in healthy retinal projections and after ON injury may lead to new understanding of pathophysiology of injury and new approaches to promoting ON regeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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