Abstract
Purpose: Atropine, a non-selective muscarinic antagonist, effectively slows down myopia progression in human adolescents and several animal models. However, the underlying molecular mechanism is unclear. The current study investigated retinal protein changes of form-deprived myopic (FDM) guinea pigs in response to topical administration of 1% atropine gel (10 g/L). Methods: At the first stage, the differentially expressed proteins were screened using fractionated isobaric tags for a relative and absolute quantification (iTRAQ) approach, coupled with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) (n = 24, 48 eyes) using a sample pooling technique. At the second stage, retinal tissues from another cohort with the same treatment (n = 12, 24 eyes) with significant ocular changes were subjected to label-free sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics for orthogonal protein target confirmation. The localization of Alpha-synuclein was verified using immunohistochemistry and confocal imaging. Results: A total of 1,695 proteins (8,875 peptides) were identified with 479 regulated proteins (FC ≥ 1.5 or ≤0.67) found from FDM eyes and atropine-treated eyes receiving 4-weeks drug treatment using iTRAQ-MS proteomics. Combining the iTRAQ-MS and SWATH-MS datasets, a total of 29 confident proteins at 1% FDR were consistently quantified and matched, comprising 12 up-regulated and 17 down-regulated proteins which differed between FDM eyes and atropine treated eyes (iTRAQ: FC ≥ 1.5 or ≤0.67, SWATH: FC ≥ 1.4 or ≤0.71, p-value of ≤0.05). Bioinformatics analysis using IPA and STRING databases of these commonly regulated proteins revealed the involvement of the three commonly significant pathways: EIF2 signaling; glycolysis; and dopamine secretion. Additionally, the most significantly regulated proteins were closely connected to Alpha-synuclein (SNCA). Using immunostaining (n = 3), SNCA was further confirmed in the inner margin of the inner nuclear layer (INL) and spread throughout the inner plexiform layer (IPL) of the retina of guinea pigs. Conclusion: The molecular evidence using next-generation proteomics (NGP) revealed that retinal EIF2 signaling, glycolysis, and dopamine secretion through SNCA are implicated in atropine treatment of myopia in the FDM-induced guinea pig model.
Highlights
Myopia has emerged as a worldwide public health issue (Morgan et al, 2012)
Less myopia was found in the form-deprived myopia (FDM) + A4 group compared to the FDM group (FDM + A4 vs FDM: −1.031 ± 0.773 D vs. −4.902 ± 0.997 D, mean ± SD, p = 3.03e-09, Figure 2A)
After 2-weeks atropine treatment, less myopia was found in the FDM + A2 group (PN35 to PN49), compared to the FDM group
Summary
Myopia (near-sightedness) has emerged as a worldwide public health issue (Morgan et al, 2012). The inhibitory effect of atropine on myopia progression has been found in different animal models, including the tree shrew (Wan et al, 2018), monkey (Raviola and Wiesel, 1985; Kiorpes et al, 1987; Tigges et al, 1999), and chick (Gallego et al, 2012), as well as in young children (Chia et al, 2012; Huang et al, 2016; Yam et al, 2019). The retinal dopamine (DA) level was decreased in form-deprived myopia (FDM) animal models, including in chicks (Stone et al, 1989), rhesus monkeys (Iuvone et al, 1989), guinea pigs (Mao et al, 2011), and tree shrews (McBrien et al, 2001). Antagonists of γ-aminobutyric acid (GABA) receptor, inhibited eye growth in the FDM model of chicks (Stone et al, 2003), while increased expression of GABA was found in the retinas of LIM guinea pigs (Zhao et al, 2017). The release of retinal DA was increased in FDM chicks after atropine treatment (Schwahn et al, 2000), and the retinal levels of GABA transporter 1 were significantly decreased after atropine treatment (Barathi et al, 2014)
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