Dataset Information

Identification of DES1 as a vitamin A isomerase in Muller glial cells of the retina.

ABSTRACT: Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternative visual cycle for regenerating opsins in daylight. Here we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternative pathway. DES1 is expressed in retinal Müller cells, where it coimmunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in Rpe65(-/-) mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 had very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity.

SUBMITTER: Kaylor JJ

PROVIDER: S-EPMC3522777 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina.

Kaylor Joanna J JJ Yuan Quan Q Cook Jeremy J Sarfare Shanta S Makshanoff Jacob J Miu Anh A Kim Anita A Kim Paul P Habib Samer S Roybal C Nathaniel CN Xu Tongzhou T Nusinowitz Steven S Travis Gabriel H GH

Nature chemical biology 20121111 1

Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternative visual cycle for regenerating opsins in daylight. Here we identified dihydroceramide desaturase-1 (DES1) as a retinol isom ...[more]

PMID: 23143414

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Project description: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.

Project description:The multiplex, lattice mosaic of cone photoreceptors in the adult fish retina is a compelling example of a highly ordered epithelial cell pattern, with single cell width rows and columns of cones and precisely defined neighbor relationships among different cone types. Cellular mechanisms patterning this multiplex mosaic are not understood. Physical models can provide new insights into fundamental mechanisms of biological patterning. In earlier work, we developed a mathematical model of photoreceptor cell packing in the zebrafish retina, which predicted that anisotropic mechanical tension in the retinal epithelium orients planar polarized adhesive interfaces to align the columns as cone photoreceptors are generated at the retinal margin during post-embryonic growth.With cell-specific fluorescent reporters and in vivo imaging of the growing retinal margin in transparent juvenile zebrafish we provide the first view of how cell packing, spatial arrangement, and cell identity are coordinated to build the lattice mosaic. With targeted laser ablation we probed the tissue mechanics of the retinal epithelium.Within the lattice mosaic, planar polarized Crumbs adhesion proteins pack cones into a single cell width column; between columns, N-cadherin-mediated adherens junctions stabilize Müller glial apical processes. The concentration of activated pMyosin II at these punctate adherens junctions suggests that these glial bands are under tension, forming a physical barrier between cone columns and contributing to mechanical stress anisotropies in the epithelial sheet. Unexpectedly, we discovered that the appearance of such parallel bands of Müller glial apical processes precedes the packing of cones into single cell width columns, hinting at a possible role for glia in the initial organization of the lattice mosaic. Targeted laser ablation of Müller glia directly demonstrates that these glial processes support anisotropic mechanical tension in the planar dimension of the retinal epithelium.These findings uncovered a novel structural feature of Müller glia associated with alignment of photoreceptors into a lattice mosaic in the zebrafish retina. This is the first demonstration, to our knowledge, of planar, anisotropic mechanical forces mediated by glial cells.

Project description:BackgroundThe roles of the Hedgehog (Hh) pathway in controlling vertebrate retinal development have been studied extensively; however, species- and context-dependent findings have provided differing conclusions. Hh signaling has been shown to control both population size and cell cycle kinetics of proliferating retinal progenitors, and to modulate differentiation within the retina by regulating the timing of cell cycle exit. While cell cycle exit has in turn been shown to control cell fate decisions within the retina, a direct role for the Hh pathway in retinal cell fate decisions has yet to be established in vivo.ResultsTo gain further insight into Hh pathway function in the retina, we have analyzed retinal development in leprechaun/patched2 mutant zebrafish. While lep/ptc2 mutants possessed more cells in their retinas, all cell types, except for Müller glia, were present at identical ratios as those observed in wild-type siblings. lep/ptc2 mutants possessed a localized upregulation of GFAP, a marker for 'reactive' glia, as well as morphological abnormalities at the vitreo-retinal interface, where Müller glial endfeet terminate. In addition, analysis of the over-proliferation phenotype at the ciliary marginal zone (CMZ) revealed that the number of proliferating progenitors, but not the rate of proliferation, was increased in lep/ptc2 mutants.ConclusionOur results indicate that Patched2-dependent Hh signaling does not likely play an integral role in neuronal cell fate decisions in the zebrafish retina. ptc2 deficiency in zebrafish results in defects at the vitreo-retinal interface and Müller glial reactivity. These phenotypes are similar to the ocular abnormalities observed in human patients suffering from Basal Cell Naevus Syndrome (BCNS), a disorder that has been linked to mutations in the human PTCH gene (the orthologue of the zebrafish ptc2), and point to the utility of the lep/ptc2 mutant line as a model for the study of BCNS-related ocular pathologies. Our findings regarding CMZ progenitor proliferation suggest that, in the zebrafish retina, Hh pathway activity may not affect cell cycle kinetics; rather, it likely regulates the size of the retinal progenitor pool in the CMZ.

Dataset Information

Identification of DES1 as a vitamin A isomerase in Muller glial cells of the retina.

Publications

Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina.

Similar Datasets

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