Muller cells of chicken retina synthesize 11-cis-retinol.
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ABSTRACT:
SUBMITTER: Das SR
PROVIDER: S-EPMC1132881 | biostudies-other | 1992 Aug
REPOSITORIES: biostudies-other
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Project description:The isomerization of all-trans retinol (vitamin A) to 11-cis retinol in the retinal pigment epithelium (RPE) is a key step in the visual process for the regeneration of the visual pigment chromophore, 11-cis retinal. LRAT and RPE65 are recognized as the minimal isomerase catalytic components. However, regulators of this rate-limiting step are not fully identified and could account for the phenotypic variability associated with inherited retinal degeneration (RD) caused by mutations in the RPE65 gene. To identify new RPE65 partners, we screened a porcine RPE mRNA library using a yeast two-hybrid assay with full-length human RPE65. One identified clone (here named FATP1c), containing the cytosolic C-terminal sequence from the fatty acid transport protein 1 (FATP1 or SLC27A1, solute carrier family 27 member 1), was demonstrated to interact dose-dependently with the native RPE65 and with LRAT. Furthermore, these interacting proteins colocalize in the RPE. Cellular reconstitution of human interacting proteins shows that FATP1 markedly inhibits 11-cis retinol production by acting on the production of all-trans retinyl esters and the isomerase activity of RPE65. The identification of this new visual cycle inhibitory component in RPE may contribute to further understanding of retinal pathogenesis.
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Project description:The mechanism of retinol isomerization in the vertebrate retina visual cycle remains controversial. Does the isomerase enzyme RPE65 operate via nucleophilic addition at C(11) of the all-trans substrate, or via a carbocation mechanism? To determine this, we modeled the RPE65 substrate cleft to identify residues interacting with substrate and/or intermediate. We find that wild-type RPE65 in vitro produces 13-cis and 11-cis isomers equally robustly. All Tyr-239 mutations abolish activity. Trp-331 mutations reduce activity (W331Y to approximately 75% of wild type, W331F to approximately 50%, and W331L and W331Q to 0%) establishing a requirement for aromaticity, consistent with cation-pi carbocation stabilization. Two cleft residues modulate isomerization specificity: Thr-147 is important, because replacement by Ser increases 11-cis relative to 13-cis by 40% compared with wild type. Phe-103 mutations are opposite in action: F103L and F103I dramatically reduce 11-cis synthesis relative to 13-cis synthesis compared with wild type. Thr-147 and Phe-103 thus may be pivotal in controlling RPE65 specificity. Also, mutations affecting RPE65 activity coordinately depress 11-cis and 13-cis isomer production but diverge as 11-cis decreases to zero, whereas 13-cis reaches a plateau consistent with thermal isomerization. Lastly, experiments using labeled retinol showed exchange at 13-cis-retinol C(15) oxygen, thus confirming enzymatic isomerization for both isomers. Thus, RPE65 is not inherently 11-cis-specific and can produce both 11- and 13-cis isomers, supporting a carbocation (or radical cation) mechanism for isomerization. Specific visual cycle selectivity for 11-cis isomers instead resides downstream, attributable to mass action by CRALBP, retinol dehydrogenase 5, and high affinity of opsin apoproteins for 11-cis-retinal.
| S-EPMC2804350 | biostudies-literature
Project description:Absorption of a photon by a rhodopsin or cone-opsin pigment isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which dissociates after a brief period of activation. Light sensitivity is restored to the resulting apo-opsin when it recombines with another 11-cis-RAL. Conversion of all-trans-RAL to 11-cis-RAL is carried out by an enzyme pathway called the visual cycle in cells of the retinal pigment epithelium. A second visual cycle is present in Müller cells of the retina. The retinol isomerase for this noncanonical pathway is dihydroceramide desaturase (DES1), which catalyzes equilibrium isomerization of retinol. Because 11-cis-retinol (11-cis-ROL) constitutes only a small fraction of total retinols in an equilibrium mixture, a subsequent step involving selective removal of 11-cis-ROL is required to drive synthesis of 11-cis-retinoids for production of visual chromophore. Selective esterification of 11-cis-ROL is one possibility. Crude homogenates of chicken retinas rapidly convert all-trans-ROL to 11-cis-retinyl esters (11-cis-REs) with minimal formation of other retinyl-ester isomers. This enzymatic activity implies the existence of an 11-cis-specific retinyl-ester synthase in Müller cells. Here, we evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this 11-cis-RE-synthase. MFAT exhibited much higher catalytic efficiency as a synthase of 11-cis-REs versus other retinyl-ester isomers. Further, we show that MFAT is expressed in Müller cells. Finally, homogenates of cells coexpressing DES1 and MFAT catalyzed the conversion of all-trans-ROL to 11-cis-RP, similar to what we observed with chicken-retina homogenates. MFAT is therefore an excellent candidate for the retinyl-ester synthase that cooperates with DES1 to drive synthesis of 11-cis-retinoids by mass action.
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Project description:Müller glia in the mature retina have the capacity to become progenitor-like cells in a many different vertebrate classes. The cell-signaling pathways that control the ability of mature Müller glia to become progenitor-like cells remain uncertain. The purpose of this study was to investigate the roles of the Mitogen-Activated Protein Kinase (MAPK) pathway in regulating the activity of Müller glia in the chicken retina. In response to acute retinal damage, we found that Müller glia accumulated phosphorylated ERK1/2 and phospho-CyclicAMP Response Element Binding-protein (pCREB), and transiently expressed immediate early genes, cFos and Egr1, that are known to be downstream of MAPK-signaling. Egr1 and pCREB were normally expressed by retinal progenitors in the circumferential marginal zone (CMZ), whereas cFos and pERK1/2 were not. In addition, small molecule inhibitors of MEK (UO126) and the FGF-receptor (SU5402) suppressed the proliferation of Müller glia-derived progenitor-like cells. These inhibitors suppressed the accumulation of Egr1 and pCREB, whereas levels of cFos were unaffected in the glial cells. These findings suggest that Egr1 and pCREB are downstream of the signaling cascade activated by FGF-receptors and ERK1/2. Further, our findings suggest that Egr1 and pCREB may promote glial proliferation. We propose that activation of both the FGF-receptor and ERK1/2-pathway is required for the proliferation and transdifferentiation of Müller glia into progenitor-like cells.
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Project description:Although biological cells are mostly transparent, they are phase objects that differ in shape and refractive index. Any image that is projected through layers of randomly oriented cells will normally be distorted by refraction, reflection, and scattering. Counterintuitively, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-detecting photoreceptor cells. Here we report on the specific optical properties of glial cells present in the retina, which might contribute to optimize this apparently unfavorable situation. We investigated intact retinal tissue and individual Müller cells, which are radial glial cells spanning the entire retinal thickness. Müller cells have an extended funnel shape, a higher refractive index than their surrounding tissue, and are oriented along the direction of light propagation. Transmission and reflection confocal microscopy of retinal tissue in vitro and in vivo showed that these cells provide a low-scattering passage for light from the retinal surface to the photoreceptor cells. Using a modified dual-beam laser trap we could also demonstrate that individual Müller cells act as optical fibers. Furthermore, their parallel array in the retina is reminiscent of fiberoptic plates used for low-distortion image transfer. Thus, Müller cells seem to mediate the image transfer through the vertebrate retina with minimal distortion and low loss. This finding elucidates a fundamental feature of the inverted retina as an optical system and ascribes a new function to glial cells.
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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.
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Project description:Degenerative retinopathies are the leading causes of irreversible visual impairment in the elderly, affecting hundreds of millions of patients. Müller glia cells (MGC), the main type of glia found in the vertebrate retina, can resume proliferation in the rodent adult injured retina but contribute weakly to tissue repair when compared to zebrafish retina. However, postnatal and adult mouse MGC can be genetically reprogrammed through the expression of the transcription factor (TF) Achaete-scute homolog 1 (ASCL1) into induced neurons (iNs), displaying key hallmarks of photoreceptors, bipolar and amacrine cells, which may contribute to regenerate the damaged retina. Here, we show that the TF neurogenin 2 (NEUROG2) is also sufficient to lineage-reprogram postnatal mouse MGC into iNs. The efficiency of MGC lineage conversion by NEUROG2 is similar to that observed after expression of ASCL1 and both TFs induce the generation of functionally active iNs. Treatment of MGC cultures with EGF and FGF2 prior to Neurog2 or Ascl1 expression enhances reprogramming efficiencies, what can be at least partially explained by an increase in the frequency of MGCs expressing sex determining region Y (SRY)-box 2 (SOX2). Transduction of either Neurog2 or Ascl1 led to the upregulation of key retina neuronal genes in MGC-derived iNs, but only NEUROG2 induced a consistent increase in the expression of putative retinal ganglion cell (RGC) genes. Moreover, in vivo electroporation of Neurog2 in late progenitors from the neonatal rat retina, which are transcriptionally similar to MGCs, also induced a shift in the generation of retinal cell subtypes, favoring neuronal differentiation at the expense of MGCs and resuming the generation of RGCs. Altogether, our data indicate that NEUROG2 induces lineage conversion of postnatal rodent MGCs into RGC-like iNs in vitro and resumes the generation of this neuronal type from late progenitors of the retina in vivo.
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Project description:To investigate whether and how the basic helix-loop-helix (bHLH) gene cNSCL2 is involved in retinal development.cNSCL2, the chick homologue of human NSCL2, was isolated and sequenced. In situ hybridization was used to examine its spatial and temporal expression pattern in the retina. Replication-competent retrovirus RCAS was used to drive cNSCL2 misexpression in the developing chick retina, and the effect of the misexpression was analyzed.Expression of cNSCL2 in the retina was restricted. Its mRNA was detected in amacrine and horizontal cells, but not in photoreceptor, bipolar, or ganglion cells. Retroviral-driven misexpression of cNSCL2 in the developing chick retina resulted in missing photoreceptor cells and gross deficits in the outer nuclear layer (ONL). These deficits were probably not because of decreased photoreceptor production, in that the ONL appeared normal in early developmental stages. TUNEL+ cells were detected in the ONL, indicating that photoreceptor cells underwent apoptosis in retinas misexpressing cNSCL2. Müller glial cells were far fewer in the experimental retina than in the control, indicating that cNSCL2 also caused Müller glia atrophy. The onset of Müller glia disappearance preceded that of photoreceptor degeneration.Expression of cNSCL2 in the chick retina was restricted to amacrine and horizontal cells. Misexpression of cNSCL2 caused severe retinal degeneration, and photoreceptor cells and Müller glia were particularly affected.
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Project description:Müller cells, the principal glial cells of the retina, play an important role in immune responses. Toll-like receptors (TLRs) are members of the pattern recognition receptor family and mediate innate and adaptive immune responses. In this study, we isolated, characterized Müller cells from mouse retina, and analyzed the expression of TLRs in these cells. We found that the mRNA of TLR2, TLR3, TLR4, and TLR5 was highly expressed by Müller cells. PAM3 and LPS, the agonists for TLR2 and TLR4, promoted Müller cells to produce the inflammatory cytokine Interleukine-6 and the chemokine MIP-2/CXCL2. These results suggest that Müller cells may be involved in innate and adaptive responses via TLR signaling in the eye. Our study should facilitate further study of the role of Müller cell in eye diseases and identification of the potential therapeutic targets.
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