Involvement of GABA Transporters in Atropine-Treated Myopic Retina As Revealed by iTRAQ Quantitative Proteomics

Abstract

Atropine, a muscarinic antagonist, is known to inhibit myopia progression in several animal models and humans. However, the mode of action is not established yet. In this study, we compared quantitative iTRAQ proteomic analysis in the retinas collected from control and lens-induced myopic (LIM) mouse eyes treated with atropine. The myopic group received a (−15D) spectacle lens over the right eye on postnatal day 10 with or without atropine eye drops starting on postnatal day 24. Axial length was measured by optical low coherence interferometry (OLCI), AC-Master, and refraction was measured by automated infrared photorefractor at postnatal 24, 38, and 52 days. Retinal tissue samples were pooled from six eyes for each group. The experiments were repeated twice, and technical replicates were also performed for liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis. MetaCore was used to perform protein profiling for pathway analysis. We identified a total of 3882 unique proteins with <1% FDR by analyzing the samples in replicates for two independent experiments. This is the largest number of mouse retina proteome reported to date. Thirty proteins were found to be up-regulated (ratio for myopia/control > global mean ratio + 1 standard deviation), and 28 proteins were down-regulated (ratio for myopia/control < global mean ratio - 1 standard deviation) in myopic eyes as compared with control retinas. Pathway analysis using MetaCore revealed regulation of γ-aminobutyric acid (GABA) levels in the myopic eyes. Detailed analysis of the quantitative proteomics data showed that the levels of GABA transporter 1 (GAT-1) were elevated in myopic retina and significantly reduced after atropine treatment. These results were further validated with immunohistochemistry and Western blot analysis. In conclusion, this study provides a comprehensive quantitative proteomic analysis of atropine-treated mouse retina and suggests the involvement of GABAergic signaling in the antimyopic effects of atropine in mouse eyes. The GABAergic transmission in the neural retina plays a pivotal role in the maintenance of axial eye growth in mammals.

The penetration and distribution of topical atropine in animal ocular tissues

“…Atropine‐treated myopic retina has been associated with a significant reduction in the levels of GAT‐1 protein, suggesting the involvement of GABAergic mechanisms in the pathogenesis of myopia in neural retina (Barathi et al. ). Unfortunately, drug distribution to the posterior eye segment has always been a major challenge after topical administration.…”

“…A proteomic analysis of mouse retina revealed an up‐regulation of GAT‐1 in myopic retina, which decreased significantly following atropine therapy, highlighting the anti‐myopic role of the retina through atropine's interactions with the GABAergic signalling pathway (Barathi et al. ). However, topical drug delivery to the posterior segment, including the retina, has always been deemed challenging as very few drugs can maintain adequate concentrations in the posterior ocular tissues to exert its therapeutic action.…”

Integrated Comparison of GWAS, Transcriptome, and Proteomics Studies Highlights Similarities in the Biological Basis of Animal and Human Myopia

“…[14][15][16][17][18] Concurrently, the role of visual experience in adapting ocular growth to suit the environment has been extensively investigated in animals where myopia can be induced by rearing with form deprivation occluders or negatively powered defocusing lenses. 19 Transcriptome and proteomics studies of ocular expression profiles in these animal models have associated thousands of genes [20][21][22][23][24][25][26][27][28] and hundreds of proteins [29][30][31][32][33][34][35][36][37] with environmentally driven growth. Enrichment analyses have aided interpretation of the wealth of data generated by exploratory human and animal studies 20,38 ; however, the key pathways by which genes and environment control ocular growth remain largely unclear.…”

Post-translational modifications and their applications in eye research

“…Previous studies have demonstrated that the retina is the major site that receives signals and determines the extent of eyeball elongation (195,196). Thus far, proteomic research has revealed a number of candidate proteins that may be associated with myopic eye growth (197)(198)(199). However, none of these studies have specifically focused on PTMs.…”