RESUMO
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.
RESUMO
The tear film is a layer of body fluid that maintains the homeostasis of the ocular surface. The superior accessibility of tears and the presence of a high concentration of functional proteins make tears a potential medium for the discovery of noninvasive biomarkers in ocular diseases. Recent advances in mass spectrometry (MS) have enabled determination of an indepth proteome profile, improved sensitivity, faster acquisition speed, proven variety of acquisition methods, and identification of disease biomarkers previously lacking in the field of ophthalmology. The use of MS allows efficient discovery of tear proteins, generation of reproducible results, and, more importantly, determines changes of protein quantity and posttranslation modifications in microliter samples. The present review compared techniques for tear collection, sample preparation, and acquisition applied for the discovery of tear protein markers in normal subjects and multifactorial conditions, including dry eye syndrome, diabetic retinopathy, thyroid eye disease and primary openangle glaucoma, which require an early diagnosis for treatment. It also summarized the contribution of MS to early discovery by means of diseaserelated protein markers in tear fluid and the potential for transformation of the tear MSbased proteome to antibodybased assay for future clinical application.
