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Gulgun Tezel, Xiangjun Yang, Cheng Luo, Jian Cai, David W. Powell; A Cell-Specific Proteomic Approach to Astrocyte Responses in Experimental Glaucoma. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3098.
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While RGCs and their axons are specific victims of glaucoma, assessing astrocyte responses is equally important to better understand glaucomatous neurodegeneration. This study aimed to introduce and validate a new technique to study astrocyte-specific responses.
IOP elevation was induced in rats by hypertonic saline injections into episcleral veins. Enriched samples of astrocytes were isolated through the two-step immunomagnetic cell selection process originally established to enrich RGC samples. To select astrocytes, the macrophage-depleted retinal cell suspension obtained from the first step was incubated with magnetic beads bound to a monoclonal ASTRO1 antibody in the second step. Ocular hypertensive and control samples were collected by pooling from eyes matched for the cumulative IOP exposure. Protein expression was analyzed by a label-free quantitative technique using 2D-LC-MS/MS. Bioinformatic data analysis utilized the Ingenuity Pathways Analysis.
When first tested by Western blot analysis using specific antibodies, GFAP expression was detectable, but neuronal markers (including NeuN and Thy-1.1) were undetectable in enriched samples of astrocytes. 2D-LC-MS/MS analysis of these samples identified hundreds of proteins with high confidence (based on at least two identified peptides and a greater than 99.0% probability assigned by the Protein Prophet algorithm). The MS/MS data verified GFAP expression and also presented numerous low abundant signaling molecules. Astrocyte proteins exhibiting an over two-fold increased expression in ocular hypertensive samples included those involved in energy metabolism, molecular transport, protein synthesis/folding, cellular growth/proliferation, cell structure/cytoskeleton, free radical scavenging, cell signaling, cell-cell interaction, and antigen presentation/immune response. Bioinformatic comparison analysis with the parallel RGC-specific data supported differential responses of different cell types during the experimental paradigm (stress response and cell death signaling in RGCs versus cellular activation response in astrocytes).
These findings validate the sensitivity of an astrocyte-specific proteomic approach. By dissecting the complexity of prior data obtained from whole tissue, this pioneering approach will enable specific responses of astrocytes to be defined and new treatment strategies targeting glial cells to be developed.
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