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Grosche, A.* ; Hauser, A.* ; Lepper, M.F. ; Mayo, R.* ; von Toerne, C. ; Merl-Pham, J. ; Hauck, S.M.

The proteome of native adult Muller glial cells from murine retina.

Mol. Cell. Proteomics 15, 462-480 (2016)
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Open Access Green
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To date, the proteomic profiling of Muller cells, the dominant macroglia of the retina, has been hampered due to the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Muller cells from adult murine retinae at excellent purity which retain in situ morphology and are well suited for proteomic analyses. Two different strategies of sample preparation, an in StageTips (iST) and a subcellular fractionation approach including cell surface protein profiling were used for quantitative LC MSMS comparing Muller cell enriched to depleted neuronal fractions. Pathway enrichment analyses on both datasets enabled us to identify Muller cell specific functions which included focal adhesion kinase signaling, signal transduction mediated by calcium as second messenger, transmembrane neurotransmitter transport and antioxidant activity. Pathways associated with RNA processing, cellular respiration and phototransduction were enriched in the neuronal subpopulation. Proteomic results were validated for selected Muller cell genes by quantitative RT-PCR, confirming the high expression levels of numerous members of the angiogenic and anti-inflammatory annexins and antioxidant enzymes (e.g. paraoxonase 2, peroxiredoxin 1, 4 and 6). Finally, the significant enrichment of antioxidant proteins in Muller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Muller cells were most efficiently protected against H2O2-induced reactive oxygen species formation, which is in line with the protein repertoire identified in the proteomic profiling. Our novel approach to isolate intact glial cells from adult retina in combination with proteomic profiling enabled the identification of novel Muller glia specific proteins, which were validated as markers and for their functional impact in glial physiology. This provides the basis to allow the discovery of novel glial specializations and will enable us to elucidate the role of Muller cells in retinal pathologies, a topic still controversially discussed.
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Publication type Article: Journal article
Document type Scientific Article
Keywords Assay Development ; Cell Fractionation* ; Central Nervous System ; Mass Spectrometry ; Oxidative Stress ; Quantification ; Surfaceome ; Annexins ; Radial Glia ; Retina; Oxygen-induced Retinopathy; Combined Transmembrane Topology; Signal Peptide Prediction; Label-free; Autoimmune Uveitis; Mass-spectrometry; Oxidative Stress; Mouse Retina; Rat Retina; Microglial Activation
ISSN (print) / ISBN 1535-9476
e-ISSN 1535-9484
Quellenangaben Volume: 15, Issue: 2, Pages: 462-480 Article Number: , Supplement: ,
Publisher American Society for Biochemistry and Molecular Biology
Publishing Place Bethesda
Reviewing status Peer reviewed