Ectonucleotidases in Muller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling.
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ABSTRACT:
SUBMITTER: Iandiev I
PROVIDER: S-EPMC2072913 | biostudies-other | 2007 Sep
REPOSITORIES: biostudies-other
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Project description:The adult zebrafish retina possesses a robust regenerative response. In the light-damaged retina, Müller glial cell divisions precede regeneration of rod and cone photoreceptors. Neuronal progenitors, which arise from the Müller glia, continue to divide and use the Müller glial cell processes to migrate to the outer nuclear layer and replace the lost photoreceptors. We tested the necessity of Müller glial cell division for photoreceptor regeneration. As knockdown tools were unavailable for use in the adult zebrafish retina, we developed a method to conditionally inhibit the expression of specific proteins by in vivo electroporation of morpholinos. We determined that two separate morpholinos targeted against the proliferating cell nuclear antigen (PCNA) mRNA reduced PCNA protein levels. Furthermore, injection and in vivo electroporation of PCNA morpholinos immediately prior to starting intense light exposure inhibited both Müller glial cell proliferation and neuronal progenitor marker Pax6 expression. PCNA knockdown additionally resulted in decreased expression of glutamine synthetase in Müller glia and Müller glial cell death, while amacrine and ganglion cells were unaffected. Finally, histological and immunological methods showed that long-term effects of PCNA knockdown resulted in decreased numbers of Müller glia and the failure to regenerate rod photoreceptors, short single cones, and long single cones. These data suggest that Müller glial cell division is necessary for proper photoreceptor regeneration in the light-damaged zebrafish retina and are consistent with the Müller glia serving as the source of neuronal progenitor cells in regenerating teleost retinas.
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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: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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Project description: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.
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Project description:The majority of inherited retinal degenerative diseases and dry age-related macular degeneration are characterized by decay of the outer retina and photoreceptors, which leads to progressive loss of vision. The inner retina, including second- and third-order retinal neurons, also shows aberrant structural changes at all stages of degeneration. Müller glia, the major glial cells maintain retinal homeostasis, activating and rearranging immediately in response to photoreceptor stress. These phenomena are collectively known as retinal remodeling and are anatomically well described, but their impact on visual function is less well characterized. Retinal remodeling has traditionally been considered a detrimental chain of events that decreases visual function. However, emerging evidence from functional assays suggests that remodeling could also be a part of a survival mechanism wherein the inner retina responds plastically to outer retinal degeneration. The visual system´s first synapses between the photoreceptors and bipolar cells undergo rewiring and functionally compensate to maintain normal signal output to the brain. Distinct classes of retinal ganglion cells remain even after the massive loss of photoreceptors. Müller glia possess the regenerative potential for retinal recovery and possibly exert adaptive transcriptional changes in response to neuronal loss. These types of homeostatic changes could potentially explain the well-maintained visual function observed in patients with inherited retinal degenerative diseases who display prominent anatomic retinal pathology. This review will focus on our current understanding of retinal neuronal and Müller glial adaptation for the potential preservation of retinal activity during photoreceptor degeneration. Targeting retinal self-compensatory responses could help generate universal strategies to delay sensory disease progression.
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Project description:Müller glial cells (MGC) are stem cells in the retina. Although their regenerative capacity is very low in mammals, the use of MGC as stem cells to regenerate photoreceptors (PHRs) during retina degenerations, such as in retinitis pigmentosa, is being intensely studied. Changes affecting PHRs in diseased retinas have been thoroughly investigated; however, whether MGC are also affected is still unclear. We here investigated whether MGC in retinal degeneration 1 (rd1) mouse, an animal model of retinitis pigmentosa, have impaired stem cell properties or structure. rd1 MGC showed an altered morphology, both in culture and in the whole retina. Using mixed neuron-glial cultures obtained from newborn mice retinas, we determined that proliferation was significantly lower in rd1 than in wild type (wt) MGC. Levels of stem cell markers, such as Nestin and Sox2, were also markedly reduced in rd1 MGC compared to wt MGC in neuron-glial cultures and in retina cryosections, even before the onset of PHR degeneration. We then investigated whether neuron-glial crosstalk was involved in these changes. Noteworthy, Nestin expression was restored in rd1 MGC in co-culture with wt neurons. Conversely, Nestin expression decreased in wt MGC in co-culture with rd1 neurons, as occurred in rd1 MGC in rd1 neuron-glial mixed cultures. These results imply that MGC proliferation and stem cell markers are reduced in rd1 retinas and might be restored by their interaction with "healthy" PHRs, suggesting that alterations in rd1 PHRs lead to a disruption in neuron-glial crosstalk affecting the regenerative potential of MGC.
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Project description:Retinal Müller glial cells (MGs) are among the first to demonstrate metabolic changes during retinal disease and are a potential source of regenerative cells. In response to a harmful stimulus, they can dedifferentiate acquiring neural stem cells properties, proliferate and migrate to the damaged retinal layer and differentiate into lost neurons. However, it is not yet known how this reprogramming process is regulated in mammals. Since glucose and oxygen are important regulatory elements that may help directing stem cell fate, we aimed to study the effect of glucose variations and oxidative stress in Müller cells reprogramming capacity and analyze the participation the histone deacetylase SIRT6, as an epigenetic modulator of this process. We found that the combination of high glucose and oxidative stress induced a decrease in the levels of the marker glutamine synthetase, and an increase in the migration capacity of the cells suggesting that these experimental conditions could induce some degree of dedifferentiation and favor the migration ability. High glucose induced an increase in the levels of the pluripotent factor SOX9 and a decrease in SIRT6 levels accompanied by the increase in the acetylation levels of H3K9. Inhibiting SIRT6 expression by siRNA rendered an increase in SOX9 levels. We also determined SOX9 levels in retinas from mice with a conditional deletion of SIRT6 in the CNS. To further understand the mechanisms that regulate MGs response under metabolic impaired conditions, we evaluated the gene expression profile and performed Gene Ontology enrichment analysis of Müller cells from a murine model of Diabetes. We found several differentially expressed genes and observed that the transcriptomic change involved the enrichment of genes associated with glucose metabolism, cell migration, development and pluripotency. We found that many functional categories affected in cells of diabetic animals were directly related to SIRT6 function. Transcription factors enrichment analysis allowed us to predict several factors, including SOX9, that may be involved in the modulation of the differential expression program observed in diabetic MGs. Our results underline the heterogeneity of Müller cells response and the challenge that the study of metabolic impairment in vivo represents.
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Project description:Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen-induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokinesignaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis.
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Project description:BackgroundGlaucoma, a progressive neurodegenerative disease, is a leading cause of irreversible vision loss worldwide. This study aims to elucidate the critical role of Müller glia (MG) in the context of retinal ganglion cell (RGC) death, particularly focusing on the influence of peripheral MG sensitivity to high pressure (HP).MethodsCo-cultures of porcine RGCs with MG were isolated from both the central and peripheral regions of pig retinas and subjected to both normal and HP conditions. Mass spectrometry analysis of the MG-conditioned medium was conducted to identify the proteins released by MG under all conditions.ResultsPeripheral MG were found to secrete a higher quantity of neuroprotective factors, effectively promoting RGC survival under normal physiological conditions. However, under HP conditions, co-cultures with peripheral MG exhibited impaired RGC survival. Moreover, under HP conditions, peripheral MG significantly upregulated the secretion of proteins associated with apoptosis, oxidative stress, and inflammation.ConclusionsThis study provides robust evidence suggesting the involvement of MG in RGC death in glaucoma, thus paving the way for future therapeutic investigations.
| S-EPMC10770903 | biostudies-literature
Project description:PurposeMüller glial-mesenchymal transition (GMT) is reported as the fibrogenic mechanism promoted by TGF-β-SNAIL axis in Müller cells transdifferentiated into myofibroblasts. Here we show the multifaceted involvement of TGF-β in diabetic fibrovascular proliferation via Müller GMT and VEGF-A production.MethodsSurgically excised fibrovascular tissues from the eyes of patients with proliferative diabetic retinopathy were processed for immunofluorescence analyses of TGF-β downstream molecules. Human Müller glial cells were used to evaluate changes in gene and protein expression with real-time quantitative PCR and ELISA, respectively. Immunoblot analyses were performed to detect TGF-β signal activation.ResultsMüller glial cells in patient fibrovascular tissues were immunopositive for GMT-related molecular markers, including SNAIL and smooth muscle protein 22, together with colocalization of VEGF-A and TGF-β receptors. In vitro administration of TGF-β1/2 upregulated TGFB1 and TGFB2, both of which were suppressed by inhibitors for nuclear factor-κB, glycogen synthase kinase-3, and p38 mitogen-activated protein kinase. Of the various profibrotic cytokines, TGF-β1/2 application exclusively induced Müller glial VEGFA mRNA expression, which was decreased by pretreatment with small interfering RNA for SMAD2 and inhibitors for p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Supporting these findings, TGF-β1/2 stimulation to Müller cells increased the phosphorylation of these intracellular signaling molecules, all of which were also activated in Müller glial cells in patient fibrovascular tissues.ConclusionsThis study underscored the significance of Müller glial autoinduction of TGF-β as a pathogenic cue to facilitate diabetic fibrovascular proliferation via TGF-β-driven GMT and VEGF-A-driven angiogenesis.
| S-EPMC7774059 | biostudies-literature