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Jeremy Whitson, Zongbo WEI, Xiang Zhang, Mario Medvedovic, Vincent M Monnier, Xingjun Fan; Transcriptome analysis of the cataract-prone GSH-depleted LEGSKO mouse lens reveals adaptations and EMT-like response. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2039.
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© ARVO (1962-2015); The Authors (2016-present)
Glutathione(GSH) is an essential antioxidant for protecting lens proteins from post-translational modifications that result in their aggregation. With age, GSH levels decrease in the lens, promoting cataract formation. In order to study how the lens adapts to GSH-deficiency and identify new targets for cataract prevention, we analyzed gene expression using an unbiased high-throughput transcriptomic approach.
Lens epithelia and cortical fiber cells from WT, LEGSKO(GSH synthesis knockout), and LEGSKO mice treated with buthionine sulfoximine (BSO;inhibitor of GCLC) were harvested from male C57Bl/6 mice at 6 months of age (n=4). Lens epithelia and fiber cells were analyzed separately and each sample pooled tissue from both eyes of a single mouse. Transcriptomic data was obtained by Illumina RNA-Seq with polyA selection. Gene expression changes with p<0.05, false discovery rate <0.1, and at least a 2-fold up- or down-regulation were considered significant. Transcriptomic results were confirmed by performing RT-qPCR on a subset of 36 transcripts in an independent set of mice.
Transcriptomic reads were mapped to 24,415 transcripts. 441 total genes showed significantly modulated expression compared to wild-type lenses. GSH-deficient lenses showed upregulation of genes relating to detoxification, including Aldh1a1, Aldh3a1 (aldehyde dehydrogenases), Mt1, Mt2 (metallothioneins), Ces1g (carboxylesterase), and Slc14a1 (urea transporter). These proteins share substrate specificity with GSH or glutathione-S-transferase and may protect GSH-deficient lenses. Genes associated with canonical EMT pathways, including Wnt10a, Egf, and Syk, showed upregulation in GSH-deficient lens epithelia samples. Comparison of this transcriptomic data with the transcriptional profile of lens epithelia undergoing EMT revealed significant correlation (r=0.63,p<0.005) between EMT lenses and BSO-treated LEGSKO lens epithelia. RT-qPCR and RNA-Seq results were in excellent agreement (correlation coefficients between 0.87-0.94 and p<5E-6 in all cases) confirming the outcome of the transcriptome.
Lens GSH depletion is associated with upregulation of Aldh, Mt, Ces1g, and Slc14a1, indicating a protective role for these genes. Transcriptome analysis also uncovered an EMT-like signaling response in chronic mild GSH-deficiency that was strengthened in acute, but severe, GSH-deficiency.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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