The Proteome of Native Adult Müller Glial Cells From Murine Retina.
To date, the proteomic profiling of Müller cells, the dominant macroglia of the retina, has been hampered because of the absence of suitable enrichment methods. We established a novel protocol to isolate native, intact Müller 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 liquid chromatography-mass spectrometry (LC-MSMS) comparing Müller cell-enriched to depleted neuronal fractions. Pathway enrichment analyses on both data sets enabled us to identify Müller 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 Müller cell genes by quantitative real time 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 Müller cells was confirmed by measurements on vital retinal cells using the oxidative stress indicator CM-H2DCFDA. In contrast to photoreceptors or bipolar cells, Müller 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 Müller 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 Müller cells in retinal pathologies - a topic still controversially discussed.
From the ‡Insitute of Human Genetics, University of Regensburg, D-93053 Regensburg, Germany; hauck@helmholtz-muenchen.de antje.grosche@klinik.uni-regensburg.de.
Antje
http://orcid.org/0000-0003-0338-7530
A
Grosche
From the ‡Insitute of Human Genetics, University of Regensburg, D-93053 Regensburg, Germany;
Alexandra
A
Hauser
§Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), D-85764 Neuherberg, Germany.
Marlen Franziska
MF
Lepper
From the ‡Insitute of Human Genetics, University of Regensburg, D-93053 Regensburg, Germany;
Rebecca
R
Mayo
§Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), D-85764 Neuherberg, Germany.
Christine
http://orcid.org/0000-0002-4132-4322
C
von Toerne
§Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), D-85764 Neuherberg, Germany.
Juliane
http://orcid.org/0000-0002-3422-4083
J
Merl-Pham
§Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), D-85764 Neuherberg, Germany hauck@helmholtz-muenchen.de antje.grosche@klinik.uni-regensburg.de.
Stefanie M
http://orcid.org/0000-0002-1630-6827
SM
Hauck
D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D049109:Cell Proliferation / Q000235:genetics; D002469:Cell Separation; D063928:Ependymoglial Cells / Q000378:metabolism* / Q000473:pathology; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D017628:Microglia / Q000378:metabolism* / Q000473:pathology; D009457:Neuroglia / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress / Q000235:genetics; D020543:Proteome / Q000235:genetics*; D040901:Proteomics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction
D020543:Proteome; D012333:RNA, Messenger