Dysregulation of inter‐photoreceptor retinoid‐binding protein (IRBP) after induced Müller cell disruption

Reduced expression of a ~150 kDa protein was unexpectedly observed while investigating Norrin protein in a transgenic murine model in which Müller cells can be selectively and inducibly disrupted. Isolation of this unknown protein via ion exchange and hydrophobic interaction chromatography followed by Tandem mass spectrometry identified it as Inter‐photoreceptor retinoid‐binding protein (IRBP). Significantly reduced IRBP mRNA expression was observed at the early and late stages after Müller cell disruption. IRBP protein expression was also consistently reduced to 5.7% of the control level as early as 1 week after Müller cell disruption. This down‐regulation of IRBP was accompanied by focal hyperfluorescent dots and cytotoxic N‐retinylidene‐N‐retinylethanolamine (A2E) accumulation. In vitro treatment of cone photoreceptor cell lines with conditioned medium collected from stressed Müller cells suggested that Müller cells regulated photoreceptors expression of IRBP via secreted factor(s). In vivo studies suggested that one of these secreted factors was tumour necrosis factor alpha (TNFα). These findings suggest that dysregulation of IRBP expression caused by Müller cell dysfunction may be an important early event in photoreceptor degeneration in some retinal diseases.

     

Conversion of mammalian Müller glial cells into a neuronal lineage by in vitro aggregate-culture

Mammalian Müller glial cells are major glial cells in the retina. Here we report that these glial cells can be redirected towards a neuronal lineage by an aggregate-culture in vitro. Rat and macaque Müller glial cells did not express neuronal markers except after transfer to adhesive conditions. Furthermore, this expression could only take place in the presence of platelet-derived growth factor and valproic acid. We compared a normal monolayer-culture and an aggregate-culture, and rat Müller glial cells could only differentiate into neurons under non-adhesive conditions. However, Müller glial cells did not express the photoreceptor markers in vitro. After transplantation into the subretinal space, a retina-specific niche, rat Müller glial cells expressed the photoreceptor-specific marker, opsin (RET-P1). We demonstrate the potential of mammalian Müller glial cells as a source of photoreceptors, which may possibly contribute to the treatment of degenerative retinal diseases such as retinitis pigmentosa.     

Müller cell zinc transporter-3 labeling suggests a role in outer retina zinc homeostasis

Labeling for zinc transporter protein-3 (ZnT-3), which can be found localized to glutamatergic vesicles elsewhere in the nervous system, has revealed an unexpectedly high concentration of this transporter protein in the outer limiting membrane region of the murine retina, a region that contains the mitochondria-rich portion of photoreceptor inner segments and is not involved with vesicle release. Having suggested the possibility that Müller cell apical villi forming the outer limiting membrane may be associated with the labeling observed, we used immunohistochemical techniques to look for ZnT-3 labeling of Müller cells isolated from rat and mouse retinas. With DAB labeling, rat Müller cell apical villi, soma, and endfeet exhibited ZnT-3 reactivity. FITC label and confocal analysis revealed that ZnT-3 protein appeared throughout the length of mouse Müller cells. We conclude from these observations that the dense labeling for ZnT-3 in the photoreceptor inner segment region of murine retinal slices is due to labeling of ZnT-3 protein associated with Müller cell apical villi. Based on these findings we suggest that Müller cells utilize ZnT-3 to regulate retinal zinc homeostasis and that this role is important to mitochondrial function in the photoreceptor inner segments.     

Hypoosmotic and glutamate‐induced swelling of bipolar cells in the rat retina: comparison with swelling of Müller glial cells

Regulation of cellular volume is of great importance to avoid changes in neuronal excitability resulting from a decrease in the extracellular space volume. We compared the volume regulation of retinal glial (Müller) and neuronal (bipolar) cells under hypoosmotic and glutamate‐stimulated conditions. Freshly isolated slices of the rat retina were superfused with a hypoosmotic solution (60% osmolarity; 4 min) or with a glutamate (1 mM)‐containing isoosmotic solution (15 min), and the size changes of Müller and bipolar cell somata were recorded. Bipolar cell somata, but not Müller cell somata, swelled under hypoosmotic conditions and in the presence of glutamate. The hypoosmotic swelling of bipolar cell somata might be mediated by sodium flux into the cells, because it was not observed under extracellular sodium‐free conditions, and was induced by activation of metabotropic glutamate receptors and sodium‐dependent glutamate transporters. The glutamate‐induced swelling of bipolar cell somata was mediated by sodium chloride flux into the cells induced by activation of NMDA‐ and non‐NMDA glutamate receptors, glutamate transporters, and voltage‐gated sodium channels. The glutamate‐induced swelling of bipolar cell somata was abrogated by adenosine and γ‐aminobutyric acid, but not by vascular endothelial growth factor and ATP. The data may suggest that Müller cells, in contrast to bipolar cells, possess endogenous mechanisms which tightly regulate the cellular volume in response to hypoosmolarity and prolonged glutamate exposure. Inhibitory retinal transmission may regulate the volume of bipolar cells, likely by inhibition of the excitatory action of glutamate.     

Neural stem cell properties of Müller glia in the mammalian retina: regulation by Notch and Wnt signaling

The retina in adult mammals, unlike those in lower vertebrates such as fish and amphibians, is not known to support neurogenesis. However, when injured, the adult mammalian retina displays neurogenic changes, raising the possibility that neurogenic potential may be evolutionarily conserved and could be exploited for regenerative therapy. Here, we show that Müller cells, when retrospectively enriched from the normal retina, like their radial glial counterparts in the central nervous system (CNS), display cardinal features of neural stem cells (NSCs), i.e., they self-renew and generate all three basic cell types of the CNS. In addition, they possess the potential to generate retinal neurons, both in vitro and in vivo. We also provide direct evidence, by transplanting prospectively enriched injury-activated Müller cells into normal eye, that Müller cells have neurogenic potential and can generate retinal neurons, confirming a hypothesis, first proposed in lower vertebrates. This potential is likely due to the NSC nature of Müller cells that remains dormant under the constraint of non-neurogenic environment of the adult normal retina. Additionally, we demonstrate that the mechanism of activating the dormant stem cell properties in Müller cells involves Wnt and Notch pathways. Together, these results identify Müller cells as latent NSCs in the mammalian retina and hence, may serve as a potential target for cellular manipulation for treating retinal degeneration.     

Atoh7 promotes retinal Müller cell differentiation into retinal ganglion cells

Glaucoma is one of the leading eye diseases due to the death of retinal ganglion cells. Increasing evidence suggests that retinal Müller cells exhibit the characteristics of retinal progenitor cells and can differentiate to neurons in injured retinas under certain conditions. However, the number of ganglion cells differentiated from retinal Müller cells falls far short of therapeutic needs. This study aimed to promote the differentiation of retinal Müller cells into ganglion cells by introducing Atoh7 into the stem cells dedifferentiated from retinal Müller cells. Rat retinal Müller cells were isolated and dedifferentiated into stem cells, which were transfected with PEGFP-N1 or PEGFP-N1-Atoh7 vector, and then further induced to differentiate into ganglion cells. The proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of control transfected or untransfected cells. In summary, Atoh7 promotes the differentiation of retinal Müller cells into retinal ganglion cells. This may open a new avenue for gene therapy of glaucoma by promoting optic nerve regeneration.     

SHP-1 suppresses endotoxin-induced uveitis by inhibiting the TAK1/JNK pathway

We investigated how Src-homology 2-domain phosphatase-1 (SHP-1) regulates the inflammatory response in endotoxin-induced uveitis (EIU), and the signalling pathways involved. One week after intravitreal injection of short hairpin RNA targeting SHP-1 or SHP-1 overexpression lentivirus in rats, we induced ocular inflammation with an intravitreal injection of lipopolysaccharide (LPS). We then assessed the extent of inflammation and performed full-field electroretinography. The concentrations and retinal expression of various inflammatory mediators were examined with enzyme-linked immunosorbent assays and Western blotting, respectively. SHP-1 overexpression and knockdown were induced in Müller cells to study the role of SHP-1 in the LPS-induced inflammatory response in vitro. Retinal SHP-1 expression was up-regulated by LPS. SHP-1 knockdown exacerbated LPS-induced retinal dysfunction and increased the levels of proinflammatory mediators in the retina, which was abrogated by a c-Jun N-terminal kinase (JNK) inhibitor (SP600125). SHP-1 overexpression had the opposite effects. In Müller cells, the LPS-induced inflammatory response was enhanced by SHP-1 knockdown and suppressed by SHP-1 overexpression. SHP-1 negatively regulated the activation of the transforming growth factor-β-activated kinase-1 (TAK1)/JNK pathway, but not the nuclear factor-κB pathway. These results indicate that SHP-1 represses EIU, at least in part, by inhibiting the TAK1/JNK pathway and suggest that SHP-1 is a potential therapeutic target for uveitis.     

Murine dopaminergic Müller cells restore motor function in a model of Parkinson's disease

Müller cells constitute the main glial cell type in the retina where it interacts with virtually all cells displaying relevant functions to retinal physiology. Under appropriate stimuli, Müller cells may undergo dedifferentiation, being able to generate other neural cell types. Here, we show that purified mouse Müller cells in culture express a group of proteins related to the dopaminergic phenotype, including the nuclear receptor‐related 1 protein, required for dopaminergic differentiation, as well the enzyme tyrosine hydroxylase. These dopaminergic components are active, since Müller cells are able to synthesize and release dopamine to the extracellular medium. Moreover, Müller‐derived tyrosine hydroxylase can be regulated, increasing its activity because of phosphorylation of serine residues in response to agents that increase intracellular cAMP levels. These observations were extended to glial cells obtained from adult monkey retinas with essentially the same results. To address the potential use of dopaminergic Müller cells as a source of dopamine in cell therapy procedures, we used a mouse model of Parkinson's disease, in which mouse Müller cells with the dopaminergic phenotype were transplanted into the striatum of hemi‐parkinsonian mice generated by unilateral injection of 6‐hydroxydopamine. These cells fully decreased the apomorphine‐induced rotational behavior and restored motor functions in these animals, as measured by the rotarod and the forelimb‐use asymmetry (cylinder) tests. The data indicate local restoration of dopaminergic signaling in hemi‐parkinsonian mice confirmed by measurement of striatal dopamine after Müller cell grafting.

     

Wnt/β‐catenin‐signaling and the formation of Müller glia‐derived progenitors in the chick retina

Müller glia can be stimulated to de‐differentiate, proliferate, and form Müller glia‐derived progenitor cells (MGPCs) that are capable of producing retinal neurons. The signaling pathways that influence the de‐differentiation of mature Müller glia and proliferation of MGPCs may include the Wnt‐pathway. The purpose of this study was to investigate how Wnt‐signaling influences the formation of MGPCs in the chick retina in vivo. In NMDA‐damaged retinas where MGPCs are known to form, we find dynamic changes in retinal levels of potential readouts of Wnt‐signaling, including dkk1, dkk3, axin2, c‐myc, tcf‐1, and cd44. We find accumulations of nuclear β‐catenin in MGPCs that peaks at 3 days and rapidly declines by 5 days after NMDA‐treatment. Inhibition of Wnt‐signaling with XAV939 in damaged retinas suppressed the formation of MGPCs, increased expression of ascl1a and decreased hes5, but had no effect upon the differentiation of progeny produced by MGPCs. Activation of Wnt‐signaling, with GSK3β‐inhibitors, in the absence of retinal damage, failed to stimulate the formation of MGPCs, whereas activation of Wnt‐signaling in damaged retinas stimulated the formation of MGPCs. In the absence of retinal damage, FGF2/MAPK‐signaling stimulated the formation of MGPCs by activating a signaling network that includes Wnt/β‐catenin. In FGF2‐treated retinas, inhibition of Wnt‐signaling reduced numbers of proliferating MGPCs, whereas activation of Wnt‐signaling failed to influence the formation of proliferating MGPCs. Our findings indicate that Wnt‐signaling is part of a network initiated by FGF2/MAPK or retinal damage, and activation of canonical Wnt‐signaling is required for the formation of proliferating MGPCs. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 983–1002, 2016     

Nerve growth factor inhibits osmotic swelling of rat retinal glial (Müller) and bipolar cells by inducing glial cytokine release

Osmotic swelling of neurons and glial cells contributes to the development of retinal edema and neurodegeneration. We show that nerve growth factor (NGF) inhibits the swelling of glial (Müller) and bipolar cells in rat retinal slices induced by barium‐containing hypoosmotic solution. NGF also reduced Müller and bipolar cell swelling in the post‐ischemic retina. On the other hand, NGF prevented the swelling of freshly isolated Müller cells, but not of isolated bipolar cells, suggesting that NGF induces a release of factors from Müller cells that inhibit bipolar cell swelling in retinal slices. The inhibitory effect of NGF on Müller cell swelling was mediated by activation of TrkA (the receptor tyrosine kinase A), but not p75^NTR, and was prevented by blockers of metabotropic glutamate, P2Y₁, adenosine A₁, and fibroblast growth factor receptors. Basic fibroblast growth factor fully inhibited the swelling of freshly isolated Müller cells, but only partially the swelling of isolated bipolar cells. In addition, glial cell line‐derived neurotrophic factor and transforming growth factor‐β1, but not epidermal growth factor and platelet‐derived growth factor, reduced the swelling of bipolar cells. Both Müller and bipolar cells displayed TrkA immunoreactivity, while Müller cells were also immunostained for p75^NTR and NGF. The data suggest that the neuroprotective effect of NGF in the retina is in part mediated by prevention of the cytotoxic glial and bipolar cell swelling.

     

Sonic hedgehog promotes stem-cell potential of Müller glia in the mammalian retina

Müller glia have been demonstrated to display stem-cell properties after retinal damage. Here, we report this potential can be regulated by Sonic hedgehog (Shh) signaling. Shh can stimulate proliferation of Müller glia through its receptor and target gene expressed on them, furthermore, Shh-treated Müller glia are induced to dedifferentiate by expressing progenitor-specific markers, and then adopt cell fate of rod photoreceptor. Inhibition of signaling by cyclopamine inhibits proliferation and dedifferentiation. Intraocular injection of Shh promotes Müller glia activation in the photoreceptor-damaged retina, Shh also enhances neurogenic potential by producing more rhodopsin-positive photoreceptors from Müller glia-derived cells. Together, these results provide evidences that Müller glia act as potential stem cells in mammalian retina, Shh may have therapeutic effects on these cells for promoting the regeneration of retinal neurons.     

A Proteomics Approach to Identify Candidate Proteins Secreted by Müller Glia that Protect Ganglion Cells in the Retina

The retinal Müller glial cells, can enhance the survival and activity of neurons, especially of retinal ganglion cells (RGCs), which are the neurons affected in diseases such as glaucoma, diabetes, and retinal ischemia. It has been demonstrated that Müller glia release neurotrophic factors that support RGC survival, yet many of these factors remain to be elucidated. To define these neurotrophic factors, a quantitative proteomic approach was adopted aiming at identifying neuroprotective proteins. First, the conditioned medium from porcine Müller cells cultured in vitro under three different conditions were isolated and these conditioned media were tested for their capacity to promote survival of primary adult RGCs in culture. Mass spectrometry was used to identify and quantify proteins in the conditioned medium, and osteopontin (SPP1), clusterin (CLU), and basigin (BSG) were selected as candidate neuroprotective factors. SPP1 and BSG significantly enhance RGC survival in vitro, indicating that the survival‐promoting activity of the Müller cell secretome is multifactorial, and that SPP1 and BSG contribute to this activity. Thus, the quantitative proteomics strategy identify proteins secreted by Müller glia that are potentially novel neuroprotectants, and it may also serve to identify other bioactive proteins or molecular markers.     

microRNA-320a prevent Müller cells from hypoxia injury by targeting aquaporin-4

Müller cells are closely related to diabetic retinopathy (DR). Aquaporin-4 (AQP4) can effectively promote the diffusion of water across cellular membranes. However, the dynamic balance of water plays key role in many diseases, such as cerebral edema. Meanwhile, the unusual expression and distribution of AQP4 in the retina are the significant causes of ocular hypertension and reperfusion injury. To explore the functional significance between microRNA-320a (miR-320a) and AQP4 in pathological hypoxia-induced DR related retinal edema, we hypothesized that miR-320a regulates AQP4 expression and internalization to relieve the edema of Müller cells under the pathological retinal hypoxia stress by targeting AQP4, thereby attenuate the damage of Müller cells. Results demonstrated that miR-320a mimics inhibited the expressions of AQP4 in Müller cells. Furthermore, overexpression miR-320a protected Müller cells by suppressing superoxide anion. In addition, overexpression miR-320a markedly attenuated hypoxia-induced injury, significantly increased the cell viability, and promoted the internalization of AQP4. Furthermore, miR-320a can also regulate the stable anchoring of AQP4 on the cell membrane. Our study indicated that miR-320a may be a potential modulator which can mediate AQP4 expression and attenuate the hypoxia damage of Müller cells. In conclusion, miR-320a may be a potential target for DR therapy by targeting AQP4.     

In Vitro and In Vivo Proteomic Comparison of Human Neural Progenitor Cell‐Induced Photoreceptor Survival

Retinal degenerative diseases lead to blindness with few treatments. Various cell‐based therapies are aimed to slow the progression of vision loss by preserving light‐sensing photoreceptor cells. A subretinal injection of human neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) rat model of retinal degeneration has aided in photoreceptor survival, though the mechanisms are mainly unknown. Identifying the retinal proteomic changes that occur following hNPC treatment leads to better understanding of neuroprotection. To mimic the retinal environment following hNPC injection, a co‐culture system of retinas and hNPCs is developed. Less cell death occurs in RCS retinal tissue co‐cultured with hNPCs than in retinas cultured alone, suggesting that hNPCs provide retinal protection in vitro. Comparison of ex vivo and in vivo retinas identifies nuclear factor (erythroid‐derived 2)‐like 2 (NRF2) mediated oxidative response signaling as an hNPC‐induced pathway. This is the first study to compare proteomic changes following treatment with hNPCs in both an ex vivo and in vivo environment, further allowing the use of ex vivo modeling for mechanisms of retinal preservation. Elucidation of the protein changes in the retina following hNPC treatment may lead to the discovery of mechanisms of photoreceptor survival and its therapeutic for clinical applications.     

Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling

Extracellular nucleotides mediate glia-to-neuron signalling in the retina and are implicated in the volume regulation of retinal glial (Müller) cells under osmotic stress conditions. We investigated the expression and functional role of ectonucleotidases in Müller cells of the rodent retina by cell-swelling experiments, calcium imaging, and immuno- and enzyme histochemistry. The swelling of Müller cells under hypoosmotic stress was inhibited by activation of an autocrine purinergic signalling cascade. This cascade is initiated by exogenous glutamate and involves the consecutive activation of P2Y₁ and adenosine A1 receptors, the action of ectoadenosine 5′-triphosphate (ATP)ases, and a nucleoside-transporter-mediated release of adenosine. Inhibition of ectoapyrases increased the ATP-evoked calcium responses in Müller cell endfeet. Müller cells were immunoreactive for nucleoside triphosphate diphosphohydrolases (NTPDase)2 (but not NTPDase1), ecto-5′-nucleotidase, P2Y₁, and A1 receptors. Enzyme histochemistry revealed that ATP but not adenosine 5′-diphosphate (ADP) is extracellularly metabolised in retinal slices of NTPDase1 knockout mice. NTPDase1 activity and protein is restricted to blood vessels, whereas activity of alkaline phosphatase is essentially absent at physiological pH. The data suggest that NTPDase2 is the major ATP-degrading ectonucleotidase of the retinal parenchyma. NTPDase2 expressed by Müller cells can be implicated in the regulation of purinergic calcium responses and cellular volume.     

Regulatory aspects of the in vitro development of retinal Müller glial cells

Utilizing immunochemical and biochemical methods we have examined the maturation of retinal Müller cells in vitro both in monolayer cultures of dissociated tissue as well as rotation-mediated suspension culture of reaggregated embryonic retina cells. We have manipulated heterotypic cell-cell interactions through the use of such cell surface probes as plant lectins and monoclonal antibodies. In this report we show that the succinylated derivative of Con-A is capable of blocking neuronal-glial interactions in reaggregation cultures resulting in neuronal-glial segregation and failure of glial maturation. Furthermore, we describe a new monoclonal antibody which also inhibits glial maturation in vitro. This antibody recognizes an antigen which is present on retinoblast cells in general early in development, but becomes gradually restricted to Müller cells and to a much lesser extent photoreceptor cells during tissue maturation. The results further substantiate the regulatory influence of heterotypic cell-cell interactions in the development of retinal Müller cells and establishes probes for the analysis of the molecular basis of this phenomenon.     

Sigma receptor 1 activation attenuates release of inflammatory cytokines MIP1γ, MIP2, MIP3α, and IL12 (p40/p70) by retinal Müller glial cells

The high‐affinity sigma receptor 1 (σR1) ligand (+)‐pentazocine ((+)‐PTZ) affords profound retinal neuroprotection in vitro and in vivo by a yet‐unknown mechanism. A common feature of retinal disease is Müller cell reactive gliosis, which includes cytokine release. Here, we investigated whether lipopolysaccharide (LPS) stimulates cytokine release by primary mouse Müller cells and whether (+)‐PTZ alters release. Using a highly sensitive inflammatory antibody array we observed significant release of macrophage inflammatory proteins (MIP1γ, MIP2, MIP3α) and interleukin‐12 (IL12 (p40/p70)) in LPS‐treated cells compared to controls, and a significant decrease in secretion upon (+)‐PTZ treatment. Müller cells from σR1 knockout mice demonstrated increased MIP1γ, MIP2, MIP3α and IL12 (p40/p70) secretion when exposed to LPS compared to LPS‐stimulated WT cells. We investigated whether cytokine secretion was accompanied by cytosolic‐to‐nuclear NFκB translocation and whether endothelial cell adhesion/migration was altered by released cytokines. Cells exposed to LPS demonstrated increased NFκB nuclear location, which was reduced significantly in (+)‐PTZ‐treated cells. Media conditioned by LPS‐stimulated‐Müller cells induced leukocyte‐endothelial cell adhesion and endothelial cell migration, which was attenuated by (+)‐PTZ treatment. The findings suggest that release of certain inflammatory cytokines by Müller cells can be attenuated by σR1 ligands providing insights into the retinal neuroprotective role of this receptor.