Differential Responses of Retinal Neurons and Glia Revealed via Proteomic Analysis on Primary and Secondary Retinal Ganglion Cell Degeneration.

To explore the temporal profile of retinal proteomes specific to primary and secondary retinal ganglion cell (RGC) loss. Unilateral partial optic nerve transection (pONT) was performed on the temporal side of the rat optic nerve. Temporal and nasal retinal samples were collected at 1, 4 and 8 weeks after pONT (n = 4 each) for non-biased profiling with a high-resolution hybrid quadrupole time-of-flight mass spectrometry running on label-free SWATHTM acquisition (SCIEX). An information-dependent acquisition ion library was generated using ProteinPilot 5.0 and OneOmics cloud bioinformatics. Combined proteome analysis detected 2531 proteins with a false discovery rate of <1%. Compared to the nasal retina, 10, 25 and 61 significantly regulated proteins were found in the temporal retina at 1, 4, and 8 weeks, respectively (p < 0.05, FC ≥ 1.4 or ≤0.7). Eight proteins (ALDH1A1, TRY10, GFAP, HBB-B1, ALB, CDC42, SNCG, NEFL) were differentially expressed for at least two time points. The expressions of ALDH1A1 and SNCG at nerve fibers were decreased along with axonal loss. Increased ALDH1A1 localization in the inner nuclear layer suggested stress response. Increased GFAP expression demonstrated regional reactivity of astrocytes and Muller cells. Meta-analysis of gene ontology showed a pronounced difference in endopeptidase and peptidase inhibitor activity. Temporal proteomic profiling demonstrates established and novel protein targets associated with RGC damage.

Alteration of EIF2 Signaling, Glycolysis, and Dopamine Secretion in Form-Deprived Myopia in Response to 1% Atropine Treatment: Evidence From Interactive iTRAQ-MS and SWATH-MS Proteomics Using a Guinea Pig Model.

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.

Alteration of retinal metabolism and oxidative stress may implicate myopic eye growth: Evidence from discovery and targeted proteomics in an animal model.

Myopia, the most common cause of impaired vision, may induce sight- threatening diseases or ocular complications due to axial elongation. The exact mechanisms underlying myopia development have received much attention and understanding of these is necessary for clinical prevention or therapeutics. In this study, quantitative proteomics using Isotope Coded Protein Label (ICPL) was applied to identify differentially regulated proteins in the retinas of myopic chicks and, from their presence, infer the possible pathogenesis of excessive ocular elongation. Newly hatched white leghorn chicks (n = 15) wore -10D and + 10D lenses bilaterally for 3 and 7 days, respectively, to develop progressive lens-induced myopia (LIM) and hyperopia (LIH). Retinal proteins were quantified with nano-liquid chromatography electrospray ionization coupled with tandem mass spectrometry (nanoLC-ESI-MS/MS). Bioinformatics analysis of differentially regulated proteins revealed that the majority originated from the cytoplasmic region and were related to various metabolic, glycolytic, or oxidative processes. The fold changes of four proteins of interest (vimentin, apolipoprotein A1, interphotoreceptor retinoid binding protein, and glutathione S-transferase) were further confirmed by a novel high-resolution multiple reaction monitoring mass spectrometry (MRM-HR) using a label-free approach. SIGNIFICANCE: Discovery of effective protein biomarkers of myopia has been extensively studied to inhibit myopia progression. This study first applied lens-induced hyperopia and myopia in the same chick to maximize the inter-ocular differences, aiming to discover more protein biomarker candidates. The findings provided new evidence that myopia was metabolism related, accompanied by altered energy generation and oxidative stress at retinal protein levels. The results in the retina were also compared to our previous study in vitreous using ICPL quantitative technology. We have now presented the protein changes in these two adjacent tissues, which may provide extra information of protein changes during ocular growth in myopia.

Isotope-coded protein label based quantitative proteomic analysis reveals significant up-regulation of apolipoprotein A1 and ovotransferrin in the myopic chick vitreous.

This study used isotope-coded protein label (ICPL) quantitative proteomics and bioinformatics analysis to examine changes in vitreous protein content and associated pathways during lens-induced eye growth. First, the vitreous protein profile of normal 7-day old chicks was characterized by nano-liquid chromatography electrospray ionization tandem mass spectrometry. A total of 341 unique proteins were identified. Next, myopia and hyperopia were induced in the same chick by attaching -10D lenses to the right eye and +10D lenses to the left eye, for 3 and 7 days. Protein expression in lens-induced ametropic eyes was analyzed using the ICPL approach coupled to LCMS. Four proteins (cystatin, apolipoprotein A1, ovotransferrin, and purpurin) were significantly up-regulated in the vitreous after 3 days of wearing -10D lenses relative to +10D lens contralateral eyes. The differences in protein expression were less pronounced after 7 days when the eyes approached full compensation. In a different group of chicks, western blot confirmed the up-regulation of apolipoprotein A1 and ovotransferrin in the myopic vitreous relative to both contralateral lens-free eyes and hyperopic eyes in separate animals wearing +10D lenses. Bioinformatics analysis suggested oxidative stress and lipid metabolism as pathways involved in compensated ocular elongation.

[MR DTI and DTT study on the development of optic radiation in patients with anisometropia amblyopia].

OBJECTIVE: To evaluate the development of optic radiations (ORs) in patients with anisometropia amblyopia using magnetic resonance diffusion tensor imaging (DTI) and diffusion tensor tractography (DTT), and to explore possible mechanism of pathogenesis of amblyopia. METHODS: Brain scan was performed with 3.0 Tesla scanner on 8 patients with anisometropia amblyopia and 15 control subjects with normal sights. The fractional anisotropy (FA) values, the apparent diffusion coefficient (ADC) values, the numbers of neural fiber bundle of ORs, and the voxel numbers of ORs were compared between the patients with anisometropia amblyopia and those with normal sights and between the ipsilateral ORs and the contralateral ORs in the patients with amblyopia. RESULTS: No differences in the FA values, the ADC values, the numbers of neural fiber bundle of ORs and the voxel numbers of ORs were found between the ipsilateral ORs and the contralateral ORs in the patients with amblyopia (P > 0.05). Significant decreases in the FA values and the voxel numbers of ORs were found in the patients with amblyopia compared with the controls (P < 0.05). No differences in the voxel numbers of both ORs in the anterior parts were found between the patients with amblyopia and the controls (P > 0.05). However, the patients with amblyopia had more voxel numbers of ORs in the posterior parts than the controls (P < 0.05). The differences in the ADC values and the numbers of neural fiber bundle of ORs between the patients with amblyopia and the controls were not significant (P > 0.05). CONCLUSION: The compactability, integrity and directivity of ORs decrease in patients with anisometropia amblyopia. The projection of OR fibers is abnormal. The ORs are underdeveloped, especially in the posterior parts, although no abnormal morphologic changes occur. The DTI and DTT can detect the underdevelopment of optic radiations in patients with anisometropia amblyopia indirectly.

[Analysis of ocular higher-order aberrations in refractive amblyopia].

OBJECTIVE: To compare the distribution of ocular higher-order aberrations in control group and amblyopia group and to explore the mechanism of the higher-order aberrations in refractive amblyopia. METHODS: The root-mean-square (RMS) values of the higher-order aberrations were measured 3 times across 6.5 mm dilated pupil of 74 eyes using Allegro Wave Analyzer. The results were compared in normal control group and amblyopia group which were determined by the best corrected visual acuity. RESULTS: RMS4, RMS5, RMS6 and RMSh in amblyopia were all significantly greater than those in control group. RMS values presented a degressive trend from RMS3 to RMS6, and RMS3 was the dominant higher-order aberration in the two groups. Coma and Y-axis coma had significant differences in the two groups. CONCLUSION: The high level of higher-order aberrations is related to amblyopia. Coma, particularly y-axis coma plays an important part in the corrected vision. It suggests that the diagnosis of amblyopia should include ocular higher-order aberrations.