-
Cells Aug 2021Retinal detachment (RD) is a sight-threatening condition, leading to photoreceptor cell death; however, only a few studies provide insight into its effects on the entire...
Retinal detachment (RD) is a sight-threatening condition, leading to photoreceptor cell death; however, only a few studies provide insight into its effects on the entire retinal region. We examined the spatiotemporal changes in glial responses in a mouse RD model. In electroretinography, a- and b-waves were reduced in a time-dependent manner. Hematoxylin and eosin staining revealed a gradual decrease in the outer nuclear layer throughout the retinal region. Terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) assay showed that TUNEL-positive photoreceptors increased 5 days after RD and decreased by 14 days. Glial response was evaluated by immunohistochemistry using antibodies against glial fibrillary acidic protein (GFAP, Müller glial marker) and Iba-1 (microglial marker) and osteopontin (OPN, activated microglial marker). GFAP immunoreactivity increased after 7 days in complete RD, and was retained for 14 days. OPN expression increased in microglial cells 3-7 days after RD, and decreased by 14 days in the detached and border regions. Although OPN was not expressed in the intact region, morphologically activated microglial cells were observed. These retinal glial cell responses and photoreceptor degeneration in the border and intact regions suggest that the effects of RD in the border and intact retinal regions need to be understood further.
Topics: Animals; Calcium-Binding Proteins; Disease Models, Animal; Electroretinography; Ependymoglial Cells; Glial Fibrillary Acidic Protein; Male; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microglia; Osteopontin; Retina; Retinal Detachment; Up-Regulation
PubMed: 34440741
DOI: 10.3390/cells10081972 -
Experimental Eye Research Jan 2024The epiretinal membrane is a fibrocontractile tissue that forms on the inner surface of the retina, causing visual impairment ranging from mild to severe, and even...
Inhibition of Rho kinase (ROCK) impairs cytoskeletal contractility in human Müller glial cells without effects on cell viability, migration, and extracellular matrix production.
The epiretinal membrane is a fibrocontractile tissue that forms on the inner surface of the retina, causing visual impairment ranging from mild to severe, and even retinal detachment. Müller glial cells actively participate in the formation of this membrane. Current research is constantly seeking for new therapeutic approaches that aim to prevent or treat cellular dysfunctions involved in the progression of this common fibrosis condition. The Rho GTPases signaling pathway regulates several processes associated with the epiretinal membrane, such as cell proliferation, migration, and contraction. Rho kinase (ROCK), an effector of the RhoA GTPase, is an interesting potential therapeutic target. This study aimed to evaluate the effects of a ROCK inhibitor (Y27632) on human Müller cells viability, growth, cytoskeletal organization, expression of extracellular matrix components, myofibroblast differentiation, migration, and contractility. Müller cells of the MIO-M1 lineage were cultured and treated for different periods with the inhibitor. Viability was evaluated by MTT assay and trypan blue exclusion method, and growth was evaluated by growth curve and BrdU incorporation assay. The actin cytoskeleton was stained with fluorescent phalloidin, intermediate filaments and microtubules were analyzed with immunofluorescence for vimentin and α-tubulin. Gene and protein expression of collagens I and V, laminin and fibronectin were evaluated by rt-PCR and immunofluorescence. Chemotactic and spontaneous cell migration were studied by transwell assay and time-lapse observation of live cells, respectively. Cell contractility was assessed by collagen gel contraction assay. The results showed that ROCK inhibition by Y27632 did not affect cell viability, but decreased cell growth and proliferation after 72 h. There was a change in cell morphology and organization of F-actin, with a reduction in the cell body, disappearance of stress fibers and formation of long, branched cell extensions. Microtubules and vimentin filaments were also affected, possibly because of F-actin alterations. The inhibitor also reduced gene expression and immunoreactivity of smooth muscle α-actin, a marker of myofibroblasts. The expression of extracellular matrix components was not affected by the inhibitor. Chemotactic cell migration showed no significant changes, while cell contractility was substantially reduced. No spontaneous migration of MIO-M1 cells was observed. In conclusion, pharmacological inhibition of ROCK in Müller cells could be a potentially promising approach to treat epiretinal membranes by preventing cell proliferation, contractility and transdifferentiation, without affecting cell viability.
Topics: Humans; rho-Associated Kinases; Actins; Ependymoglial Cells; Vimentin; Cell Survival; Epiretinal Membrane; Cells, Cultured; Extracellular Matrix
PubMed: 38043763
DOI: 10.1016/j.exer.2023.109745 -
Developmental Dynamics : An Official... Jul 2016Retinal dystrophies are a major cause of blindness for which there are currently no curative treatments. Transplantation of stem cell-derived neuronal progenitors to... (Review)
Review
Retinal dystrophies are a major cause of blindness for which there are currently no curative treatments. Transplantation of stem cell-derived neuronal progenitors to replace lost cells has been widely investigated as a therapeutic option. Another promising strategy would be to trigger self-repair mechanisms in patients, through the recruitment of endogenous cells with stemness properties. Accumulating evidence in the past 15 year0s has revealed that several retinal cell types possess neurogenic potential, thus opening new avenues for regenerative medicine. Among them, Müller glial cells have been shown to be able to undergo a reprogramming process to re-acquire a stem/progenitor state, allowing them to proliferate and generate new neurons for repair following retinal damages. Although Müller cell-dependent spontaneous regeneration is remarkable in some species such as the fish, it is extremely limited and ineffective in mammals. Understanding the cellular events and molecular mechanisms underlying Müller cell activities in species endowed with regenerative capacities could provide knowledge to unlock the restricted potential of their mammalian counterparts. In this context, the present review provides an overview of Müller cell responses to injury across vertebrate model systems and summarizes recent advances in this rapidly evolving field. Developmental Dynamics 245:727-738, 2016. © 2015 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc.
Topics: Animals; Ependymoglial Cells; Humans; Regeneration; Retina; Stem Cells
PubMed: 26661417
DOI: 10.1002/dvdy.24375 -
EBioMedicine Mar 2022Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but...
BACKGROUND
Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons.
METHODS
We used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation.
FINDINGS
We first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids.
INTERPRETATION
We show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.
FUNDING
This work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926).
Topics: Adult Stem Cells; Animals; Cell Differentiation; Ependymoglial Cells; Humans; Mammals; Mice; Neuroglia; Retina
PubMed: 35278743
DOI: 10.1016/j.ebiom.2022.103914 -
Investigative Ophthalmology & Visual... Aug 2016Müller cells (MCs) are a major source of VEGF in diabetic retinopathy (DR). Vascular endothelial growth factor is the main therapeutic target for treating DR. This...
PURPOSE
Müller cells (MCs) are a major source of VEGF in diabetic retinopathy (DR). Vascular endothelial growth factor is the main therapeutic target for treating DR. This study aimed to investigate whether autophagy is involved in MC response under high glucose (HG).
METHODS
Rat retinal Müller cells (rMCs) were exposed to normal or high glucose in and out of presence of pharmacologic inhibitors and activators and small interfering RNA (siRNA) for p62/SQTSM1 for 24 hours.
RESULTS
High glucose induces increase of early and late autophagic markers, accumulation of p62/SQTSM1 and endoplasmic reticulum (ER) stress response associated with apoptosis augmentation (P < 0.01). The inhibition of autophagy in HG leads to higher rMC apoptotic rate (P < 0.001). By silencing the p62/SQTSM1, ER stress is ameliorated (p<0.0001), preventing apoptosis. Retinal MCs in HG treated with rapamycin (mTOR inhibitor) show autophagy machinery activation and reestablishment of cargo degradation, protecting cells from apoptosis (P < 0.0001). Rapamycin improves lysosomal proteolytic activity by improving cathepsin L activity restoring autophagic cargo degradation, and preventing increased VEGF release (P < 0.0001). In experimental model of diabetes, Beclin-1 and p62/SQTSM-1 were found to be marked increased in retinas from diabetic Wystar Kyoto rats compared with control group (P < 0.003) with reduction of cathepsin L activity.
CONCLUSIONS
High glucose upregulates autophagy but accumulates p62/SQTSM1 cargo due to lysosomal dysfunction, leading to massive VEGF release and cell death of rMCs. Lysosomal impairment and autophagic dysfunction are early events present in the pathogenesis of diabetic retinopathy (DR). This might be valuable for developing a novel therapeutic strategy to treat DR.
Topics: Animals; Apoptosis; Autophagy; Blotting, Western; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Ependymoglial Cells; Gene Expression Regulation; Glucose; Microscopy, Electron, Transmission; Oxidative Stress; RNA, Small Interfering; Rats; Retina; Sequestosome-1 Protein; Sweetening Agents
PubMed: 27564518
DOI: 10.1167/iovs.16-19197 -
Immunobiology May 2022The present study was intended to investigate the role of embryonic stem cell-derived exosomes (ESC-Exos) in Müller cell retrodifferentiation and their specific...
OBJECTIVE
The present study was intended to investigate the role of embryonic stem cell-derived exosomes (ESC-Exos) in Müller cell retrodifferentiation and their specific mechanism.
METHODS
Following co-incubation of the extracted ESC-Exos and Müller cells, their effects on the retrodifferentiation and proliferation of Müller cells were measured by EdU staining, immunofluorescence, and western blot. ESCs transfected with small interfering RNA of BDNF were co-incubated with Müller cells to determine Müller cell proliferation and retrodifferentiation. β-catenin expression in the nucleus and GSK-3β phosphorylation were measured to determine the role of the Wnt pathway in Müller cells. The function of the retina in RCS rats was observed using flash electroretinogram.
RESULTS
Co-incubation of ESCs with Müller cells or overexpression of BDNF contributed to Müller cell retrodifferentiation and proliferation, as evidenced by increased cell proliferation, fluorescence intensities of proliferation markers and retinal stem cell markers, and expression of BDNF and β-catenin, and suppressed GSK-3β phosphorylation. However, co-incubation with ESCs silencing BDNF or treatment with GW4869 inhibited the proliferation and retrodifferentiation of retinal Müller cells. In addition, exosome injection increased BDNF, BrdU, PH3, SOX2, and Pax6 expression, enhanced β-catenin expression in the nucleus, diminished GSK-3β, and improved retinal degeneration in RCS rats.
CONCLUSION
ESC-Exos accelerated Müller cell retrodifferentiation and proliferation through Wnt pathway activation by delivering BDNF protein to Müller cells.
Topics: Animals; Brain-Derived Neurotrophic Factor; Cell Proliferation; Embryonic Stem Cells; Ependymoglial Cells; Exosomes; Eye Diseases, Hereditary; Glycogen Synthase Kinase 3 beta; Rats; Retinal Degeneration; Vision Disorders; Wnt Signaling Pathway; beta Catenin
PubMed: 35390666
DOI: 10.1016/j.imbio.2022.152211 -
Translational Vision Science &... Jan 2022Because the importance of glia in regulating brain functions has been demonstrated, genetic technologies that manipulate glial cell-specific gene expression in the brain...
PURPOSE
Because the importance of glia in regulating brain functions has been demonstrated, genetic technologies that manipulate glial cell-specific gene expression in the brain have become essential and have made great progress. However, it is unknown whether the same strategy that is used in the brain can be applied to the retina because retinal glia differs from glia in the brain. Here, we aimed to find a method for selective gene expression in Müller cells (characteristic glial cells in the retina) and identified Mlc1 as a specific promoter of Müller cells.
METHODS
Mlc1-tTA::Yellow-Cameleon-NanotetO/tetO (YC-Nano) mice were used as a reporter line. YC-Nano, a fluorescent protein, was ectopically expressed in the cell type controlled by the Mlc1 promotor. Immunofluorescence staining was used to identify the cell type expressing YC-Nano protein.
RESULTS
YC-Nano-positive (+) signals were observed as vertical stalks in the sliced retina and spanned from the nerve fiber layer through the outer nuclear layer. The density of YC-Nano+ cells was higher around the optic nerve head and lower in the peripheral retina. The YC-Nano+ signals colocalized with vimentin, a marker of Müller cells, but not with the cell markers for blood vessels, microglia, neurons, or astrocytes.
CONCLUSIONS
The Mlc1 promoter allows us to manipulate gene expression in Müller cells without affecting astrocytes in the retina.
TRANSLATIONAL RELEVANCE
Gene manipulation under control of Mlc1 promoter offers novel technique to investigate the role of Müller cells.
Topics: Animals; Astrocytes; Ependymoglial Cells; Gene Expression; Membrane Proteins; Mice; Neuroglia; Retina
PubMed: 35040915
DOI: 10.1167/tvst.11.1.25 -
FEBS Letters Dec 2017The mammalian cerebral cortex is responsible for higher cognitive functions such as perception, consciousness, and acquiring and processing information. The neocortex is... (Review)
Review
The mammalian cerebral cortex is responsible for higher cognitive functions such as perception, consciousness, and acquiring and processing information. The neocortex is organized into six distinct laminae, each composed of a rich diversity of cell types which assemble into highly complex cortical circuits. Radial glia progenitors (RGPs) are responsible for producing all neocortical neurons and certain glia lineages. Here, we discuss recent discoveries emerging from clonal lineage analysis at the single RGP cell level that provide us with an inaugural quantitative framework of RGP lineage progression. We further discuss the importance of the relative contribution of intrinsic gene functions and non-cell-autonomous or community effects in regulating RGP proliferation behavior and lineage progression.
Topics: Animals; Cell Differentiation; Cell Lineage; Cell Tracking; Ependymoglial Cells; Humans; Neural Stem Cells; Neurogenesis; Signal Transduction; Single-Cell Analysis
PubMed: 29121403
DOI: 10.1002/1873-3468.12906 -
Redox Biology Jun 2019Hyperhomocysteinemia (Hhcy), or increased levels of the excitatory amino acid homocysteine (Hcy), is implicated in glaucoma, a disease characterized by increased...
Hyperhomocysteinemia (Hhcy), or increased levels of the excitatory amino acid homocysteine (Hcy), is implicated in glaucoma, a disease characterized by increased oxidative stress and loss of retinal ganglion cells (RGCs). Whether Hhcy is causative or merely a biomarker for RGC loss in glaucoma is unknown. Here we analyzed the role of NRF2, a master regulator of the antioxidant response, in Hhcy-induced RGC death in vivo and in vitro. By crossing Nrf2 mice and two mouse models of chronic Hhcy (Cbs and Mthfr mice), we generated CbsNrf2 and MthfrNrf2 mice and performed systematic analysis of retinal architecture and visual acuity followed by assessment of retinal morphometry and gliosis. We observed significant reduction of inner retinal layer thickness and reduced visual acuity in Hhcy mice lacking NRF2. These functional deficits were accompanied by fewer RGCs and increased gliosis. Given the key role of Müller glial cells in maintaining RGCs, we established an ex-vivo indirect co-culture system using primary RGCs and Müller cells. Hhcy-exposure decreased RGC viability, which was abrogated when cells were indirectly cultured with wildtype (WT) Müller cells, but not with Nrf2 Müller cells. Exposure of WT Müller cells to Hhcy yielded a robust mitochondrial and glycolytic response, which was not observed in Nrf2 Müller cells. Taken together, the in vivo and in vitro data suggest that deleterious effects of Hhcy on RGCs are likely dependent upon the health of retinal glial cells and the availability of an intact retinal antioxidant response mechanism.
Topics: Animals; Biomarkers; Cell Count; Coculture Techniques; Disease Models, Animal; Electroretinography; Ependymoglial Cells; Glycolysis; Hyperhomocysteinemia; Intraocular Pressure; Mice; Mice, Knockout; NF-E2-Related Factor 2; Retina; Retinal Ganglion Cells
PubMed: 31026769
DOI: 10.1016/j.redox.2019.101199 -
Journal of Molecular Endocrinology Jan 2022The pro-inflammatory cytokines secreted by Müller cells aggregate retinal cell loss and vascularization in diabetic retinopathy (DR). The deubiquitinase...
The pro-inflammatory cytokines secreted by Müller cells aggregate retinal cell loss and vascularization in diabetic retinopathy (DR). The deubiquitinase BRCA1-BRCA2-containing complex subunit 3 (BRCC3)-mediated nucleotide-binding domain and leucine-rich repeat receptor containing a pyrin domain 3 (NLRP3) inflammasome activation participate in this progress. This study aims to clarify whether the E3 ubiquitin ligase synoviolin (SYVN1) relieves DR via regulating the BRCC3/NLRP3 axis. The DR model was established using streptozotocin-induced mice. Immunofluorescence staining with anti-CD31, anti-glutamine synthetase, and anti-vimentin was performed to identify DR and Müller cells. Levels of pro-inflammatory cytokines, including interleukin-1β, tumor necrosis factor-α, IL-6, and IL-18, in murine serum and Müller cell supernatants were determined. Co-immunoprecipitation (Co-IP) and ubiquitination assays were used to clarify the interactions among SYVN1, BRCC3, and NLRP3. SYVN1 was reduced and BRCC3 was increased in DR retina and high glucose (HG)-induced Müller cells. Overexpressing 1 promoted the ubiquitination and degradation of BRCC3 and reduced the secretion of proinflammatory cytokines in HG-induced Müller cells. The simultaneous overexpression of 1 and Brcc3 restored the reduction of pro-inflammatory cytokines caused by the overexpression of 1 alone. Co-IP experiments confirmed the interaction between BRCC3 and NLRP3. SYVN1-mediated BRCC3 downregulation promoted NLRP3 ubiquitination and reduced pro-inflammatory cytokine secretion. 1 overexpression reduced retinal vascularization and inflammatory cytokine secretion in DR mice. SYVN1 has a protective effect on DR, whose molecular mechanisms are partly through SYVN1-mediated ubiquitination of BRCC3 and the subsequent downregulation of NLRP3.
Topics: Animals; Diabetic Retinopathy; Ependymoglial Cells; Inflammasomes; Interleukin-1beta; Mice; NLR Family, Pyrin Domain-Containing 3 Protein; Ubiquitination
PubMed: 34874278
DOI: 10.1530/JME-21-0123