Limits...
Involvement of GABA transporters in atropine-treated myopic retina as revealed by iTRAQ quantitative proteomics.

Barathi VA, Chaurasia SS, Poidinger M, Koh SK, Tian D, Ho C, Iuvone PM, Beuerman RW, Zhou L - J. Proteome Res. (2014)

Bottom Line: 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.These results were further validated with immunohistochemistry and Western blot analysis.The GABAergic transmission in the neural retina plays a pivotal role in the maintenance of axial eye growth in mammals.

View Article: PubMed Central - PubMed

Affiliation: Singapore Eye Research Institute , 11 Third Hospital Avenue, Singapore 168751, Singapore.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Clustering analysis on up-regulated or down-regulatedproteinscommon to four sets of data showing the similar trend of the expressionprofile in control, myopia, and atropine treatment using the ShortTime-series Expression Miner (STEM) software. (A) Down-regulated inmyopia and increased after atropine treatment. (B) Up-regulated inmyopia and decreased after atropine treatment. (C) No change in myopiaand increased after atropine treatment. (D) No change in myopia anddecreased after atropine treatment.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4227558&req=5

fig5: Clustering analysis on up-regulated or down-regulatedproteinscommon to four sets of data showing the similar trend of the expressionprofile in control, myopia, and atropine treatment using the ShortTime-series Expression Miner (STEM) software. (A) Down-regulated inmyopia and increased after atropine treatment. (B) Up-regulated inmyopia and decreased after atropine treatment. (C) No change in myopiaand increased after atropine treatment. (D) No change in myopia anddecreased after atropine treatment.

Mentions: Among 3882retina proteins, 3291 proteins can be quantified (2831 proteins quantifiedwith two or more peptides, Supplemental Table2 in the Supporting Information). The workflow for the entirequantitative analysis of the iTRAQ results is illustrated in Figure 4. Briefly, data with CV% >30% from two technicalruns were removed, and iTRAQ ratios from Run1 and Run2 were used tocalculate the geometric mean as the final ratio for each protein.The global mean ratio and standard deviation for the whole data setwere determined, and one standard deviation (SD) of the global meanratio was defined as the cutoff for differentially expression (globalmean ratio ±1 SD).38,39 Only those proteinsin common for Batch1 and Batch2 were listed as differentially expressedproteins between myopic group and control group. The same criteriawas used for myopic group and atropine group when we determined whetherthose protein levels were in response to the atropine treatment. Thirtyproteins were found to be up-regulated (ratio for myopia/control >global mean +1 SD, Supplemental Table 3A in theSupporting Information), and 28 proteins were down-regulated(ratio for myopia/control < global mean −1 SD, Supplemental Table 3B in the Supporting Information) in all four runs (two biological replicates and two technical replicates)in myopic eyes as compared with control eyes. The changes of retinaprotein profiles between myopia and atropine treated myopic groupswere revealed by Expression Miner software and as described in Figure 5. Using control as the baseline, 13 retina proteinswere up-regulated in myopia but were found to be down-regulated (ratiofor treatment/myopia < global mean −1 SD) after atropinetreatment (Figure 5A, SupplementTable 3A in the Supporting Information). Similarly, 10 retinaproteins were down-regulated in myopia but gradually showed up-regulation(ratio for treatment/myopia > global mean +1 SD) after atropinetreatment(Figure 5B, SupplementTable 3B in the Supporting Information). Additionally, tworetina proteins showed no significant changes between myopia and controlbut up-regulated after atropine treatment (ratio for treatment/myopia> global mean +1 SD, Figure 5C, Supplement Table 3C in the Supporting Information), whereas 17 retina proteins showed no significant changes betweenmyopia and control but down-regulated after atropine treatment (ratiofor treatment/myopia < global mean −1 SD, Figure 5D, Supplement Table 3C in theSupporting Information).


Involvement of GABA transporters in atropine-treated myopic retina as revealed by iTRAQ quantitative proteomics.

Barathi VA, Chaurasia SS, Poidinger M, Koh SK, Tian D, Ho C, Iuvone PM, Beuerman RW, Zhou L - J. Proteome Res. (2014)

Clustering analysis on up-regulated or down-regulatedproteinscommon to four sets of data showing the similar trend of the expressionprofile in control, myopia, and atropine treatment using the ShortTime-series Expression Miner (STEM) software. (A) Down-regulated inmyopia and increased after atropine treatment. (B) Up-regulated inmyopia and decreased after atropine treatment. (C) No change in myopiaand increased after atropine treatment. (D) No change in myopia anddecreased after atropine treatment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4227558&req=5

fig5: Clustering analysis on up-regulated or down-regulatedproteinscommon to four sets of data showing the similar trend of the expressionprofile in control, myopia, and atropine treatment using the ShortTime-series Expression Miner (STEM) software. (A) Down-regulated inmyopia and increased after atropine treatment. (B) Up-regulated inmyopia and decreased after atropine treatment. (C) No change in myopiaand increased after atropine treatment. (D) No change in myopia anddecreased after atropine treatment.
Mentions: Among 3882retina proteins, 3291 proteins can be quantified (2831 proteins quantifiedwith two or more peptides, Supplemental Table2 in the Supporting Information). The workflow for the entirequantitative analysis of the iTRAQ results is illustrated in Figure 4. Briefly, data with CV% >30% from two technicalruns were removed, and iTRAQ ratios from Run1 and Run2 were used tocalculate the geometric mean as the final ratio for each protein.The global mean ratio and standard deviation for the whole data setwere determined, and one standard deviation (SD) of the global meanratio was defined as the cutoff for differentially expression (globalmean ratio ±1 SD).38,39 Only those proteinsin common for Batch1 and Batch2 were listed as differentially expressedproteins between myopic group and control group. The same criteriawas used for myopic group and atropine group when we determined whetherthose protein levels were in response to the atropine treatment. Thirtyproteins were found to be up-regulated (ratio for myopia/control >global mean +1 SD, Supplemental Table 3A in theSupporting Information), and 28 proteins were down-regulated(ratio for myopia/control < global mean −1 SD, Supplemental Table 3B in the Supporting Information) in all four runs (two biological replicates and two technical replicates)in myopic eyes as compared with control eyes. The changes of retinaprotein profiles between myopia and atropine treated myopic groupswere revealed by Expression Miner software and as described in Figure 5. Using control as the baseline, 13 retina proteinswere up-regulated in myopia but were found to be down-regulated (ratiofor treatment/myopia < global mean −1 SD) after atropinetreatment (Figure 5A, SupplementTable 3A in the Supporting Information). Similarly, 10 retinaproteins were down-regulated in myopia but gradually showed up-regulation(ratio for treatment/myopia > global mean +1 SD) after atropinetreatment(Figure 5B, SupplementTable 3B in the Supporting Information). Additionally, tworetina proteins showed no significant changes between myopia and controlbut up-regulated after atropine treatment (ratio for treatment/myopia> global mean +1 SD, Figure 5C, Supplement Table 3C in the Supporting Information), whereas 17 retina proteins showed no significant changes betweenmyopia and control but down-regulated after atropine treatment (ratiofor treatment/myopia < global mean −1 SD, Figure 5D, Supplement Table 3C in theSupporting Information).

Bottom Line: 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.These results were further validated with immunohistochemistry and Western blot analysis.The GABAergic transmission in the neural retina plays a pivotal role in the maintenance of axial eye growth in mammals.

View Article: PubMed Central - PubMed

Affiliation: Singapore Eye Research Institute , 11 Third Hospital Avenue, Singapore 168751, Singapore.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus