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Gulgun Tezel, Xiangjun Yang, Jian Cai, Jon B Klein; Proteomics analysis of astroglia-driven neuroinflammatory responses in mouse glaucoma. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2560. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Recent studies of clinical/postmortem samples from glaucomatous and non-glaucomatous human donors and studies of experimental models have accumulated evidence that supports a connection of glial inflammatory responses to neurodegeneration in glaucoma. Since improved molecular understanding of glia-driven neuroinflammation can enable us for therapeutic protection of neurons from inflammatory neurotoxicity, this study aimed for molecular characterization of astroglia responses in experimental mouse glaucoma.
Intraocular pressure elevation was induced by microbead/viscoelastic injections into the anterior chamber. Through an experimental period of up to 12 weeks, we isolated retina and optic nerve head astroglia from mice with or without ocular hypertension by two-step immunomagnetic cell selection. Isolated astroglia proteins were pooled from samples matched for the IOP-time integral (and neuron injury within each group) and were analyzed by isotope labeling-based quantitative mass spectrometry.
Our updated methodology allowed increased protein yields (~10 µg of astroglia protein from 5 mouse retinas or optic nerve heads), and Western blot analysis of isolated astroglia proteins for specific cell markers verified more than 95% purity. The quantitative LC-MS/MS analysis identified thousands of proteins (based on >3 unique peptide matches and a false discovery rate of <1%), including those with over two-fold up-regulated or down-regulated expression with ocular hypertension (p<0.05). The Ingenuity Pathways Analysis of high-throughput datasets linked the ocular hypertension-induced molecular alterations to inflammatory activation through TLR signaling, cytokine signaling, and NF-κB activation pathways.
Our high-confident datasets support the feasibility of astroglia-specific sampling and verify the sensitivity of astroglia-specific analysis to characterize distinct molecular processes in mouse glaucoma. Since increased release of inflammatory mediators by activated glia (in the retina, optic nerve head, and optic nerve axons) causes neurotoxicity, inhibition of glia-driven neuroinflammation may consequently reduce secondary injury at multiple sites and protect both RGC somas and axons. Ongoing studies of cell type-targeted conditional transgenic models for functional testing of the specific molecular outcomes should value them as treatment target for immunomodulation in glaucoma.
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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