Retinal Müller Glial Research Articles

Cyr61 activates retinal cells and prolongs photoreceptor survival in rd1 mouse model of retinitis pigmentosa

Subretinal injections with glial cell line-derived neurotrophic factor (GDNF) rescue morphology as well as function of rod cells in mouse and rat animal models of retinitis pigmentosa. At the same time, it is postulated that this effect is indirect, mediated by activation of retinal Müller glial (RMG) cells. Here, we show that Cyr61/CCN1, one of the secreted proteins up-regulated in primary RMG after glial cell line-derived neurotrophic factor stimulation, provides neuroprotective and pro-survival capacities: Recombinant Cyr61 significantly reduced photoreceptor (PR) cells death in organotypic cultures of Pde6b(rd1) retinas. To identify stimulated pathways in the retina, we treated Pde6b(rd1) retinal explants with Cyr61 and observed an overall increase in activated Erk1/2 and Stat3 signalling molecules characterized by activation-site-specific phosphorylation. To identify Cyr61 retinal target cells, we isolated primary porcine PR, RMG and retinal pigment epithelium (RPE) cells and exposed them separately to Cyr61. Here, RMG as well as RPE cells responded with induced phosphorylation of Erk1/2, Stat3 and Akt. In PR, no increase in phosphorylation in any of the studied proteins was detected, suggesting an indirect neuroprotective effect of Cyr61. Cyr61 may thus act as an endogenous pro-survival factor for PR, contributing to the complex repertoire of neuroprotective activities generated by RMG and RPE cells. We propose the following model of Cyr61 neuroprotection within the retina: Cyr61 stimulates retinal Müller glial (RMG) and retinal pigment epithelium (RPE) cells and activates PI3K/Akt, mitogen-activated protein kinase(MAPK)/Erk and Janus kinase(JAK)/Stat-signalling pathways in these cells. Phosphorylated Stat3 and Erk1/2 presumably translocate to the nucleus, induce transcriptional changes, which increase secretion of neuroprotective agents that protect photoreceptors (PR) from mutation-induced death.

The role of inflammation in the pathogenesis of macular edema secondary to retinal vascular diseases.

Macular edema (ME) is a nonspecific sign of numerous retinal vascular diseases. This paper is an updated overview about the role of inflammatory processes in the genesis of both diabetic macular edema (DME) and ME secondary to retinal vein occlusion (RVO). We focus on the inflammatory mediators implicated, the effect of the different intravitreal therapies, the recruitment of leukocytes mediated by adhesion molecules, and the role of retinal Müller glial (RMG) cells.

Distinct effects of inflammation on gliosis, osmohomeostasis, and vascular integrity during amyloid beta‐induced retinal degeneration

In normal retinas, amyloid-β (Aβ) accumulates in the subretinal space, at the interface of the retinal pigment epithelium, and the photoreceptor outer segments. However, the molecular and cellular effects of subretinal Aβ remain inadequately elucidated. We previously showed that subretinal injection of Aβ(1-42) induces retinal inflammation, followed by photoreceptor cell death. The retinal Müller glial (RMG) cells, which are the principal retinal glial cells, are metabolically coupled to photoreceptors. Their role in the maintenance of retinal water/potassium and glutamate homeostasis makes them important players in photoreceptor survival. This study investigated the effects of subretinal Aβ(1-42) on RMG cells and of Aβ(1-42)-induced inflammation on retinal homeostasis. RMG cell gliosis (upregulation of GFAP, vimentin, and nestin) on day 1 postinjection and a proinflammatory phenotype were the first signs of retinal alteration induced by Aβ(1-42). On day 3, we detected modifications in the protein expression patterns of cyclooxygenase 2 (COX-2), glutamine synthetase (GS), Kir4.1 [the inwardly rectifying potassium (Kir) channel], and aquaporin (AQP)-4 water channels in RMG cells and of the photoreceptor-associated AQP-1. The integrity of the blood-retina barrier was compromised and retinal edema developed. Aβ(1-42) induced endoplasmic reticulum stress associated with sustained upregulation of the proapoptotic factors of the unfolded protein response and persistent photoreceptor apoptosis. Indomethacin treatment decreased inflammation and reversed the Aβ(1-42)-induced gliosis and modifications in the expression patterns of COX-2, Kir4.1, and AQP-1, but not of AQP-4 or GS. Nor did it improve edema. Our study pinpoints the adaptive response to Aβ of specific RMG cell functions.

Direct comparison of MS‐based label‐free and SILAC quantitative proteome profiling strategies in primary retinal Müller cells

To better understand the involvement of retinal Müller glial (RMG) cells in retinal diseases, we phenotyped primary porcine RMGs in dependence of cultivation time using different quantitative proteomic strategies. A well-established LC-MS/MS-based quantification method was employed: stable isotope labeling by amino acids in cell culture (SILAC) and directly compared to label-free (LF) quantifications, based on total peak intensities using two different programs (MaxQuant and Progenesis LC-MS). The overall numbers of detected proteins were largely similar (overlap of 1324 proteins), only a total of 173 proteins were significantly altered between the different culture conditions. However, among these, only 21 proteins were shared between the three analytical strategies. Hence, the majority of altered proteins only reached significance thresholds in one of the applied analyses with a larger overlap between the two LF approaches. Among the shared, differentially abundant proteins were known RMG markers as well as new proteins associated with glial cell transition. However, proteins correlated to cellular transitions and dedifferentiation were also found among the proteins only significant in one or two of the applied strategies. Consequently, the application of different quantification and analytical strategies could increase the analytical depths of proteomic phenotyping.

Differential Regulations of AQP4 and Kir4.1 by Triamcinolone Acetonide and Dexamethasone in the Healthy and Inflamed Retina

Glucocorticoids are used to treat macular edema, although the mechanisms underlying this effect remain largely unknown. The authors have evaluated in the normal and endotoxin-induced uveitis (EIU) rats, the effects of dexamethasone (dex) and triamcinolone acetonide (TA) on potassium channel Kir4.1 and aquaporin-4 (AQP4), the two main retinal Müller glial (RMG) channels controlling retinal fluid movement. Clinical as well as relatively low doses of dex and TA were injected in the vitreous of normal rats to evaluate their influence on Kir4.1 and AQP4 expression 24 hours later. The dose-dependent effects of the two glucocorticoids were investigated using rat neuroretinal organotypic cultures. EIU was induced by footpad lipopolysaccharide injection, without or with 100 nM intraocular dex or TA. Glucocorticoid receptor and channel expression levels were measured by quantitative PCR, Western blot, and immunohistochemistry. The authors found that dex and TA exert distinct and specific channel regulations at 24 hours after intravitreous injection. Dex selectively upregulated Kir4.1 (not AQP4) in healthy and inflamed retinas, whereas TA induced AQP4 (not Kir4.1) downregulation in normal retina and upregulation in EIU. The lower concentration (100 nM) efficiently regulated the channels. Moreover, in EIU, an inflammatory condition, the glucocorticoid receptor was downregulated in the retina, which was prevented by intravitreous injections of the low concentration of dex or TA. The results show that dex and TA are far from being equivalent to modulate RMG channels. Furthermore, the authors suggest that low doses of glucocorticoids may have antiedematous effects on the retina with reduced toxicity.

GDNF‐induced osteopontin from Müller glial cells promotes photoreceptor survival in the Pde6brd1 mouse model of retinal degeneration

Glial cell line-derived neurotrophic factor (GDNF) enhances the survival of a variety of neurons, including photoreceptors (PR) in the retina. In contrast to most other GDNF receptive neurons, GDNF does, however, not exert its neuroprotective activity directly on PR neurons but transmits it indirectly by inducing expression of yet unknown neurotrophic factors in retinal Müller glial (RMG) cells. Genome-wide differential transcriptome analyses of GDNF-treated mouse retinas revealed 30 GDNF-induced transcripts containing a total of six genes coding for secreted molecules. Among them was (OPN), a secreted glycoprotein which was expressed in mouse RMG and secreted from primary mouse RMG in culture. Furthermore, OPN secretion was significantly upregulated on GDNF treatment of primary RMG. To validate, whether OPN could qualify as a neuroprotective factor for PR, we evaluated its potential neurotrophic activity on isolated PR in vitro as well as on retinal explants from the retinal degeneration 1 (Pde6brd1) mouse mutant. OPN exerted a significant, positive survival effect on primary porcine PR cells in a concentration-dependent manner and induced activation of PI3K/Akt pro-survival pathway. Moreover, in retinal explant cultures from Pde6brd1 mice, OPN significantly reduced the percentage of apoptotic cells to levels comparable with that observed in explants from wild-type mice and led to survival of significantly more PR in long-term retinal explant cultures. Our findings suggest that RMG-derived OPN is a novel candidate protein that transmits part of the GDNF-induced neuroprotective activity of RMG to PR cells.

In Vitro Effect of TNF-α and IFN-γ in Retinal Cell Infection withToxoplasma gondii

Toxoplasma gondii is an intracellular protozoan parasite and the most common cause of infectious uveitis. This study was conducted to evaluate the in vitro effect of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in rat retinal cells infected with T. gondii. Rat retinal cells, retinal pigment epithelial (RPE) cells, and retinal Müller glial (RMG) cells were in vitro infected with T. gondii RH strain tachyzoites. Cultured cells were stimulated with various concentrations of TNF-alpha and IFN-gamma. The effect of TNF-alpha and IFN-gamma in T. gondii invasion and replication between retinal cells was determined through two different methods: measuring [(3)H]-uracil incorporation and counting infected cells by microscopic examination. Infection by T. gondii was lesser within RPE cells than within RMG cells. IFN-gamma significantly inhibits [(3)H]-uracil incorporation in RMG and RPE cells (respectively, 35%, 83%, and 87% inhibition at 0.1, 1, and 10 ng/mL for RMG cells and 0%, 30%, and 75% for RPE cells). TNF-alpha significantly inhibits [(3)H]-uracil incorporation in RPE cells (23% and 38% inhibition at 1 and 10 ng/mL), but not in RMG cells. These results were confirmed by confocal microscopic data. The percentage of infected cells decreased from 20% to 7% after IFN-gamma stimulation. Both cytokines IFN-gamma and TNF-alpha inhibited T. gondii replication in the RPE cells, whereas only IFN-gamma had an anti-Toxoplasma activity within the RMG cells. The differences in cytokine response may be the reason that RPE cells are less efficiently infected by T. gondii than are RMG cells.

Involvement of Pleiotrophin in CNTF-mediated differentiation of the late retinal progenitor cells

Ciliary neurotrophic factor (CNTF) participates in retinal development by inhibiting rod differentiation and promoting bipolar and Müller cell differentiation. In order to identify genes which are regulated by CNTF in the developing retina, we carried out a subtractive hybridization study. By this approach, we identified the Pleiotrophin (Ptn) as an upregulated gene in postnatal day 0 (P0) retinal explants upon addition of CNTF. Correlation of overall expression patterns between different retinal cell markers and Ptn in situ hybridization suggest that Ptn transcripts are initially expressed in progenitor cells then in postmitotic precursors of the INL expressing the Chx10 gene, and later in some differentiated retinal Müller glial (RMG) cells and rod–bipolar cells. Overexpression of Ptn by in vitro electroporation of P0 rat retinal explants partially blocks rod differentiation and promotes bipolar cell production, similar to effects of exogenous CNTF and leukemia inhibitory factor (LIF). Furthermore, in P0 retinal explants from mice lacking Ptn, the inhibitory effect of CNTF and LIF on rod differentiation is partially reduced and the cytokine-induced bipolar cell differentiation is largely prevented. Together, these results demonstrate that influence of CNTF family of cytokines on the differentiation of late retinal progenitor cell population is partially mediated by the release of Ptn.

Requirement for nitric oxide in retinal neuronal cell death induced by activated Müller glial cells.

Retinal Müller glial cells express the inducible isoform (-2) of nitric oxide (NO) synthase (NOS) in vitro after stimulation by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) or in vivo in some retinal pathologies. Because NO may have beneficial or detrimental effects in the retina, we have used cocultures of retinal neurons with retinal Müller glial (RMG) cells from mice disrupted for the gene of NOS-2 [NOS-2 (-/-)] to clarify the role of NO in retinal neurotoxicity. We first demonstrated that NO produced by activated RMG cells was not toxic for RMG cells themselves. Second, the NO released from LPS/IFN-gamma-stimulated RMG cells induced neuronal cell death, because no neuronal cell death has been observed in cocultures with RMG cells from NOS-2 (-/-) mice and because inhibition of NOS-2 induction by transforming growth factor-beta or blockade of NO release by different NOS inhibitors prevented neuronal cell death. Addition of urate, a peroxynitrite scavenger, or superoxide dismutase partially prevented neuronal cell death induced by NO, whereas the presence of a poly(ADP-ribose) synthetase inhibitor, caspase inhibitors, or a guanylate cyclase inhibitor had no significant effect on cell death. These results demonstrated that a large release of NO from RMG cells is responsible for retinal neuronal cell death in vitro, suggesting a neurotoxic role for NO and peroxynitrite during retinal inflammatory or degenerative diseases, where RMG cells were activated.

Control of Nitric Oxide Production by Endogenous TNF-α in Mouse Retinal Pigmented Epithelial and Muller Glial Cells

Since the induction of nitric oxide synthase (NOS) by lipopolysaccharide (LPS) has been suggested to be partially dependent of the synthesis of tumor necrosis factor α (TNFα), we have investigated in vitro the production of NO in retinal cells from mice deficient in Lymphotoxin α (LTα)/TNFα. Treatment of retinal Müller glial (RMG) and retinal pigmented epithelial (RPE) cells from both wild-type and knockout mice with LPS and interferon γ (IFNγ) induced NO synthesis as determined by nitrite release into the media and was correlated to an increase in NOS-2 mRNA levels, evaluated by RT-PCR. However, the level of nitrite and the accumulation of mRNA was always less in cells from LTα/TNFα knockout mice than in wild-type mice. Simultaneous addition of TNFα restored the level of NO synthesis by RMG and RPE cells from LTα/TNFα knockout mice stimulated with LPS and IFNγ to wild type levels. Transforming growth factor β (TGFβ) blocked LPS/IFNγ-induced NO production in RMG and RPE cells from wild-type and LTα/TNFα knockout mice. Our results demonstrate that induction of NO synthesis in RMG and RPE cells by LPS and IFNγ is dependent in part on endogenous TNFα while inhibition of NO production by TGFβ does not require a modulation of TNFα synthesis.

Tumor necrosis factor and nitric oxide production by retinal M�ller glial cells from rats exhibiting inherited retinal dystrophy

The primary cause of the inherited retinal dystrophy observed in Royal College of Surgeons (RCS) rats is located in the retinal pigmented epithelium, which is unable to phagocytize photoreceptor outer segments. We have demonstrated here that retinal Müller glial (RMG) cells obtained from RCS dystrophic rats and stimulated in vitro with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) accumulated higher levels of tumor necrosis factor (TNF) and inducible nitric oxide synthase (NOS II) mRNA and released in culture supernatants significantly higher amounts of TNF and nitrite compared to cells derived from nondystrophic controls. The TNF and NOS II mRNA expression and TNF and nitrite synthesis induced in RMG cells from both strains by LPS + IFN-gamma was significantly prevented by including transforming growth factor-beta (TGF-beta) in the culture medium. Coincubation of the stimulants with an inhibitor of NOS II, NG-monomethyl-L-arginine (L-NMMA), while inhibiting nitrite synthesis, induced an increase of TNF production in supernatants from RMG cells without increasing TNF mRNA levels. The retinal dystrophy observed in RCS dystrophic rats could result from an abnormal susceptibility of RMG cells form RCS dystrophic rats to produce TNF and NO in response to stimulants. Administration of the immunomodulatory cytokine TGF-beta or inhibitors of NOS II would provide additional research avenues for photoreceptor rescue.

Tumor necrosis factor and nitric oxide production by resident retinal glial cells from rats presenting hereditary retinal degeneration

The inherited retinal dystrophy observed in Royal College of Surgeons (RCS) rats is a widely used model for the study of the photoreceptor degeneration that occurs in retinitis pigmentosa and macular degeneration. The visual cell degeneration is accompanied by an abnormal accumulation of microglial cells in the retina of RCS rats presenting the dystrophy. In the present study, we show that combined stimulation of RCS dystrophic retinal Müller glial (RMG) cells with interferon-gamma (IFN-γ) and lipopolysaccharide (LPS) induced the release in culture supernatants of significantly higher amounts of tumor necrosis factor (TNF) and nitric oxide (NO) compared to nondystrophic congenic controls. In contrast, the levels of TNF and NO found in the supernatants from microglial cells were not significantly different in both strains. Interestingly, as shown by thymidine incorporation, microglial cells from RCS dystrophic rats have a prominent capacity of proliferation in culture medium compared to microglia isolated from RCS non dystrophic controls. Incubation of RMG cells and microglia with the stereoselective inhibitor of NOS, NG-monomethy I-L-arginine (L-NMMA), inhibited nitrite release in LPS+IFN-γ-stimulated RMG cells and microglia. The addition of TGF-β with LPS+IFN-γ clearly inhibited TNF release in supernatants from both dystrophic and control rat RMG cells and microglia. While TGF-β significantly inhibited nitrite synthesis in RMG cells, the effect on nitrite synthesis by microglia was very low. The retinal dystrophy observed in RCS dystrophic rats could result from an abnormal reactivity of RMG and microglial cells to release TNF and NO in response to stimulants. The immunomodulatory cytokine TGF-β and inhibitors of NOS could be negative regulators in the cytokine network and nitrite synthesis thus interfering with the development of photoreceptor cell death.

Induction and regulation of nitric oxide synthase in retinal Müller glial cells.

Müller glial cells from the rat retina were examined for their capacity to produce nitric oxide (NO). Treatment of retinal Müller glial (RMG) cells with lipopolysaccharide (LPS), interferon-gamma, and tumor necrosis factor-alpha induced NO synthesis as determined by nitrite release in media. Simultaneous addition of LPS, interferon-gamma, and tumor necrosis factor-alpha caused the largest increase in NO synthesis. NO biosynthesis was detected after 12 h and was dependent on the dose of LPS, interferon-gamma, and tumor necrosis factor-alpha. Stereoselective inhibitors of NO synthase (NOS), cycloheximide and transforming growth factor-beta, blocked cytokine-induced NO production. Cytosol from LPS/cytokine-treated RMG cultures, but not from unstimulated cultures, produced a calcium/calmodulin-independent conversion of L-arginine to L-citrulline that was completely blocked by NOS inhibitor. The expression of NOS in RMG cells was confirmed by northern blot analysis, in which stimulation of these cells led to an increase in NOS mRNA levels. We conclude that RMG cells can express an inducible form of NOS similar to the macrophage isoform. High NO release from activated RMG cells might represent a protection from infection but may also contribute to the development of retinal pathologies.