Zhu Ying, Bian Jing Fang, Lu Da Qian, To Chi Ho, Lam Carly Siu-Yin, Li King Kit, Yu Feng Juan, Gong Bo Teng, Wang Qiong, Ji Xiao Wen, Zhang Hong Mei, Nian Hong, Lam Thomas Chuen, Wei Rui Hua
Tianjin Key Laboratory of Retinal Functions and Diseases, Tianjin Branch of National Clinical Research Center for Ocular Disease, Eye Institute and School of Optometry, Tianjin Medical University Eye Hospital, Tianjin, China.
Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Hong Kong SAR, China.
Front Pharmacol. 2022 Jan 28;13:814814. doi: 10.3389/fphar.2022.814814. eCollection 2022.
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). 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) ( = 24, 48 eyes) using a sample pooling technique. At the second stage, retinal tissues from another cohort with the same treatment ( = 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. 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, -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 ( = 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. 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.
阿托品是一种非选择性毒蕈碱拮抗剂，可有效减缓人类青少年和多种动物模型的近视进展。然而，其潜在的分子机制尚不清楚。本研究调查了形觉剥夺性近视（FDM）豚鼠局部应用1%阿托品凝胶（10 g/L）后视网膜蛋白的变化。在第一阶段，使用相对和绝对定量的等量异位标签（iTRAQ）方法结合纳升液相色谱-串联质谱（nano-LC-MS/MS）（n = 24，48只眼），采用样本合并技术筛选差异表达蛋白。在第二阶段，对另一组接受相同治疗（n = 12，24只眼）且眼部有明显变化的视网膜组织进行无标记的全理论质谱连续窗口采集（SWATH-MS）蛋白质组学分析，以进行正交蛋白靶点确认。使用免疫组织化学和共聚焦成像验证α-突触核蛋白的定位。使用iTRAQ-MS蛋白质组学方法，在接受4周药物治疗的FDM眼和阿托品治疗眼中共鉴定出1695种蛋白质（8875个肽段），发现479种调节蛋白（FC≥1.5或≤0.67）。结合iTRAQ-MS和SWATH-MS数据集，在1%错误发现率（FDR）下共一致定量和匹配了29种可信蛋白，包括12种上调蛋白和17种下调蛋白，这些蛋白在FDM眼和阿托品治疗眼中存在差异（iTRAQ：FC≥1.5或≤0.67，SWATH：FC≥1.4或≤0.71，P值≤0.05）。使用IPA和STRING数据库对这些共同调节的蛋白进行生物信息学分析，揭示了三个共同的重要途径的参与：真核起始因子2（EIF2）信号传导；糖酵解；以及多巴胺分泌。此外，调节最显著的蛋白与α-突触核蛋白（SNCA）密切相关。通过免疫染色（n = 3），在豚鼠视网膜内核层（INL）的内边缘进一步证实了SNCA，并扩散至整个内网层（IPL）。使用下一代蛋白质组学（NGP）的分子证据表明，视网膜EIF2信号传导、糖酵解以及通过SNCA的多巴胺分泌与FDM诱导的豚鼠模型中阿托品治疗近视有关。
