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

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.