Glutamate Receptor Stimulation Up-Regulates Glutamate Uptake in Human Müller Glia Cells

Neurochemical Research - Glutamate, the main excitatory amino acid in the vertebrate retina, is a well know activator of numerous signal transduction pathways, and has been critically involved in...     

Genetic Pathways Regulating Glutamate Levels in Retinal Müller Cells

Müller cells serve many functions including the regulation of extracellular glutamate levels. The product of two genes, Slc1a3 [aka solute carrier family 1 (glial high affinity glutamate transporter), member 3] and Glul (aka glutamine synthetase) are the primary role players that transport glutamate into the Müller cell and convert it into glutamine. In this study, we sought to identify the genetic regulation of both genes. Given their tightly coupled biological functions, we predicted that they would be similarly regulated. Using an array of 75 recombinant inbred strains of mice, we determined that Slc1a3 and Glul are differentially regulated by distinct chromosomal regions. Interestingly, despite their independent regulation, gene ontology analysis of tightly correlated genes reveals that the enriched and statistically significant molecular function categories of both directed acyclic graphs have substantial overlap, indicating that the shared functions of correlates of Slc1a3 and Glul include production and usage of ATP.     

Glutamate-Induced Inhibition of D-Aspartate Uptake in Müller Glia from the Retina

Müller glial cells from the retina "in situ" and in primary culture, mainly express the high-affinity sodium-coupled glutamate/aspartate transporter GLAST-1, which dominates total retinal glutamate (Glu) uptake, suggesting a major role for these cells in the modulation of excitatory transmission. The possible involvement of ionotropic and metabotropic Glu receptors in the regulation of Glu uptake was studied in primary cultures of Müller glia. We demonstrate that exposure to 1 mM L-Glu induces a time-dependent inhibition of D-aspartate (D-Asp) uptake in a Na+-dependent manner, as a result of a reduction in the number of transporters at the plasma membrane. The inhibition of D-Asp uptake by Glu was not mimicked by agonists or modified by antagonists of ionotropic and metabotropic Glu receptors. In contrast, transport was inhibited by GLAST-1 transportable substrates threo-hydroxyaspartate and aspartate-beta-hydroxamate, but not by the nontransportable inhibitors trans-pyrrolidine dicarboxylate or DL-threo-beta-benzyloxyaspartic acid. Under the same experimental conditions, L-Glu did not affect the sodium-dependent transport systems for glycine or GABA. The present results demonstrate that the specific downregulation of glutamate/aspartate transport by L-Glu is unrelated to Glu receptor activation, and results from the internalization of transporter proteins triggered by the transport process itself. Such negative feedback of Glu on Glu transport, could contribute to retinal toxicity under pathological conditions in which high extracellular concentrations of Glu are reached.     

Beyond Polarity: Functional Membrane Domains in Astrocytes and Müller Cells

Various ependymoglial cells display varying degrees of process specialization, in particular processes contacting mesenchymal borders (pia, blood vessels, vitreous body), or those lining the ventricular surface. Within the neuropil, glial morphology, cellular contacts, and interaction partners are complex. It appears that glial processes contacting neurons, specific parts of neurons, or mesenchymal or ventricular borders are characterized by specialized membranes. We propose a concept of membrane domains in addition to the existing concept of ependymoglial polarity. Such membrane domains are equipped with certain membrane-bound proteins, enabling them to function in their specific environment. This review focuses on Müller cells and astrocytes and discusses exemplary the localization of established glial markers in membrane domains. We distinguish three functional glial membrane domains based on their typical molecular arrangement. The domain of the endfoot specifically displays the complex of dystrophin-associated proteins, aquaporin 4 and the potassium channel Kir4.1. We show that the domain of microvilli and the peripheral glial process in the Müller cell share the presence of ezrin, as do peripheral astrocyte processes. As a third domain, the Müller cell has peripheral glial processes related to a specific subtype of synapse. Although many details remain to be studied, the idea of glial membrane domains may permit new insights into glial function and pathology.     

Interruption of Wnt Signaling in Müller Cells Ameliorates Ischemia-Induced Retinal Neovascularization

Retinal Müller cells are major producers of inflammatory and angiogenic cytokines which contribute to diabetic retinopathy (DR). Over-activation of the Wnt/β-catenin pathway has been shown to play an important pathogenic role in DR. However, the roles of Müller cell-derived Wnt/β-catenin signaling in retinal neovascularization (NV) and DR remain undefined. In the present study, mice with conditional β-catenin knockout (KO) in Müller cells were generated and subjected to oxygen-induced retinopathy (OIR) and streptozotocin (STZ)-induced diabetes. Wnt signaling was evaluated by measuring levels of β-catenin and expression of its target genes using immunoblotting. Retinal vascular permeability was measured using Evans blue as a tracer. Retinal NV was visualized by angiography and quantified by counting pre-retinal nuclei. Retinal pericyte loss was evaluated using retinal trypsin digestion. Electroretinography was performed to examine visual function. No abnormalities were detected in the β-catenin KO mice under normal conditions. In OIR, retinal levels of β-catenin and VEGF were significantly lower in the β-catenin KO mice than in littermate controls. The KO mice also had decreased retinal NV and vascular leakage in the OIR model. In the STZ-induced diabetic model, disruption of β-catenin in Müller cells attenuated over-expression of inflammatory cytokines and ameliorated pericyte dropout in the retina. These findings suggest that Wnt signaling activation in Müller cells contributes to retinal NV, vascular leakage and inflammation and represents a potential therapeutic target for DR.     

Nano-mechanical Compliance of Müller Cells Investigated by Atomic Force Microscopy

It has been known that a single Müller cell displays a large variation in the cytoskeletal compositions along its cell body, suggesting different mechanical properties in different segments. Müller cells are thought to be involved in many retinal diseases such as retinoschisis, which can be facilitated by a mechanical stress. Thus, mapping of mechanical properties on localized nano-domains of Müller cells could provide essential information for understanding their structural functions in the retina and roles in their pathological progresses. Using Atomic Force Microscopy (AFM) - based bio-nano-mechanics, we have investigated the local variations of the mechanical properties of Müller cells in vitro. We have a particular interest in identifying elastic moduli in regions closer to three distinctive segments of the cells - process, endfoot, and soma. Using the modified spherical AFM probes, we were able to accurately determine mechanical properties, i.e., elastic moduli from the obtained force-distance curves. We found that the regions closer to soma were mechanically more compliant than regions closer to endfoot and process of Müller cells. We found that this lateral heterogeneity of the mechanical compliance within a single Müller cell is consistent with reports from other cell types. The local variation in mechanical compliances along a single Müller cell may support their diverse mechanical functions in the retina such as a soft mechanical embedding, mechanosensing, and neurotrophic functions for neurons.     

Potential Role of Cyr61 Induced Degeneration of Human Müller Cells in Diabetic Retinopathy

The degeneration of Müller cells has been recognized to involve in the pathogenesis of diabetic retinopathy. However, the mechanism is not yet clear. This study is to explore the potential role of Cyr61, a secreted signaling protein in extracellular matrix, in inducing human Müller cell degeneration in diabetic retinopathy (DR). Twenty patients with proliferative diabetic retinopathy (PDR) and twelve non-diabetic patients were recruited for this study. Vitreous fluid was collected during vitrectomy surgery for Cyr61 ELISA. Human Müller cell line MIO-M1 were cultured to be subconfluent, and then treated with glucose (0–20 mM) or Cyr61 (0–300 ng/ml). Cyr61 expression induced by increasing concentrations of glucose was evaluated by RT-qPCR and Western blot. Effects of Cyr61 on Müller cells viability, migration and apoptosis were observed by MTT assay, Transwell assay, and TUNEL assay. Vitreous Cyr61 levels were observed to be 8-fold higher in patients with PDR (3576.92±1574.58 pg/mL), compared with non-diabetic controls (436.14±130.69 pg/mL). Interestingly, the active PDR group was significantly higher than the quiescent PDR group (P<0.01). In retinal Müller cells culture, high glucose significantly and dose-dependently elevated Cyr61 expression at both mRNA and protein levels. Cyr61 at high concentrations dose-dependently inhibited the viability and migration of Müller cells. TUNEL assay further revealed that high concentration of Cyr61 significantly promoted the cell apoptosis. In conclusion, these findings demonstrated for the first time that the expression of Cyr61 was elevated by high glucose in Müller cells, and Cyr61 inhibited cell viability and migration while induced apoptosis, suggesting the potential role of Cyr61 in Müller cell degeneration. The elevated Cyr61 levels in vitreous fluid of PDR patients further support its role in diabetic retinopathy (DR).     

Cell Volume Regulation in Cultured Human Retinal Müller Cells Is Associated with Changes in Transmembrane Potential

Müller cells are mainly involved in controlling extracellular homeostasis in the retina, where intense neural activity alters ion concentrations and osmotic gradients, thus favoring cell swelling. This increase in cell volume is followed by a regulatory volume decrease response (RVD), which is known to be partially mediated by the activation of K⁺ and anion channels. However, the precise mechanisms underlying osmotic swelling and subsequent cell volume regulation in Müller cells have been evaluated by only a few studies. Although the activation of ion channels during the RVD response may alter transmembrane potential (V_m), no studies have actually addressed this issue in Müller cells. The aim of the present work is to evaluate RVD using a retinal Müller cell line (MIO-M1) under different extracellular ionic conditions, and to study a possible association between RVD and changes in V_m. Cell volume and V_m changes were evaluated using fluorescent probe techniques and a mathematical model. Results show that cell swelling and subsequent RVD were accompanied by V_m depolarization followed by repolarization. This response depended on the composition of extracellular media. Cells exposed to a hypoosmotic solution with reduced ionic strength underwent maximum RVD and had a larger repolarization. Both of these responses were reduced by K⁺ or Cl⁻ channel blockers. In contrast, cells facing a hypoosmotic solution with the same ionic strength as the isoosmotic solution showed a lower RVD and a smaller repolarization and were not affected by blockers. Together, experimental and simulated data led us to propose that the efficiency of the RVD process in Müller glia depends not only on the activation of ion channels, but is also strongly modulated by concurrent changes in the membrane potential. The relationship between ionic fluxes, changes in ion permeabilities and ion concentrations –all leading to changes in V_m– define the success of RVD.     

Notch and Wnt Signaling Mediated Rod Photoreceptor Regeneration by Müller Cells in Adult Mammalian Retina

Background

Evidence emerging from a variety of approaches used in different species suggests that Müller cell function may extend beyond its role of maintaining retinal homeostasis to that of progenitors in the adult retina. Enriched Müller cells in vitro or those that re-enter cell cycle in response to neurotoxin-damage to retina in vivo display multipotential and self-renewing capacities, the cardinal features of stem cells.

Methodology/Principal Findings

We demonstrate that Notch and Wnt signaling activate Müller cells through their canonical pathways and that a rare subset of activated Müller cells differentiates along rod photoreceptor lineage in the outer nuclear layer. The differentiation of activated Müller cells along photoreceptor lineage is confirmed by multiple approaches that included Hoechst dye efflux analysis, genetic analysis using retina from Nrl-GFP mice, and lineage tracing using GS-GFP lentivirus in wild type and rd mice in vitro and S334ter rats in vivo. Examination of S334ter rats for head-neck tracking of visual stimuli, a behavioral measure of light perception, demonstrates a significant improvement in light perception in animals treated to activate Müller cells. The number of activated Müller cells with rod photoreceptor phenotype in treated animals correlates with the improvement in their light perception.

Conclusion/Significance

In summary, our results provide a proof of principle for non-neurotoxin-mediated activation of Müller cells through Notch and Wnt signaling toward the regeneration of rod photoreceptors.     

Comparison of Neurosphere-like Cell Clusters Derived from Dental Follicle Precursor Cells and Retinal Müller Cells

Unrelated cells such as dental follicle precursor cells (DFPCs) and retinal Müller cells (MCs) make spheres after cultivation in serum-replacement medium (SRM). Until today, the relation and molecular processes of sphere formation from different cell types remain undescribed. Thus in this study we compared proteomes of spheres derived from MCs and DFPCs. 73% of 676 identified proteins were similar expressed in both cell types and many of them are expressed in the brain (55%). Moreover proteins are overrepresented that are associated with pathways for neural diseases such as Huntington disease or Alzheimer disease. Interestingly up-regulated proteins in DFPCs are involved in the biosynthesis of glycosphingolipids. These lipids are components of gangliosides such as GD3, which is a novel neural stem cell marker. In conclusion spheres from different types of cells have highly similar proteomes. These proteomes probably show essential cellular processes in neurosphere-like cell clusters.     

Pigment Epithelium-Derived Factor Released by Müller Glial Cells Exerts Neuroprotective Effects on Retinal Ganglion Cells

Survival of retinal ganglion cells (RGC) is compromised in several vision-threatening disorders such as ischemic and hypertensive retinopathies and glaucoma. Pigment epithelium-derived factor (PEDF) is a naturally occurring pleiotropic secreted factor in the retina. PEDF produced by retinal glial (Müller) cells is suspected to be an essential component of neuron-glial interactions especially for RGC, as it can protect this neuronal type from ischemia-induced cell death. Here we show that PEDF treatment can directly affect RGC survival in vitro. Using Müller cell-RGC-co-cultures we observed that activity of Müller-cell derived soluble mediators can attenuate hypoxia-induced damage and RGC loss. Finally, neutralizing the activity of PEDF in glia-conditioned media partially abolished the neuroprotective effect of glia, leading to an increased neuronal death in hypoxic condition. Altogether our results suggest that PEDF is crucially involved in the neuroprotective process of reactive Müller cells towards RGC.     

Anaerobic energy metabolism in the oligochaeteLumbriculus variegatus Müller

Summary Anoxia tolerance, glycogen degradation, free amino acid pool, adenylate energy charge and the accumulation and excretion of end products were monitored inLumbriculus variegatus Müller throughout 48 h of anoxia. A transition period lasting about 4 h could be distinguished from subsequent events during which malate, present in high amounts in the resting animals, is utilized, probably by conversion to succinate. Up to the 12th hour of anoxia there is an increase in concentration of free amino acids, except aspartate. Glutamate increases rapidly during the first half hour but decreases thereafter. Beginning with the second hour of anoxia the alanine concentration increases at the same rate glutamate concentration decreases, but the source of nitrogen during the first hour is unknown. It is argued that the nitrogen required for the synthesis of some of the amino acids is ultimately derived from proteolysis. After about 3 h of anoxia propionate and acetate are synthesized. At first these acids accumulate in the tissues, but after 4–6 h they are excreted into the surrounding medium. Acetate is excreted over the whole experimental period at a constant rate, whereas the excretion rate of propionate decreases slowly with time. The propionate/acetate ratio is in excess of 2. Classic malate dismutation is by far the most important mechanism in the maintenance of redox balance. Depletion of glycogen stores appears to play an important role in determining anoxic survival time. Due to extremely low activity of PEPCK the ratio of the specific activities of PK and PEPCK is very high. Further, the kinetic properties of pyruvate kinase do not support the assumption of a shift of the glycolytic carbon flow at the PEP level.Abbreviations PK Pyruvate kinase - PEPCK phosphoenolpyruvate carboxykinase - PEP phospho(enol)pyruvate - FBP fructose-1,6-bisphosphate - AEC adenylate energy charge - EMP-scheme Embden-Meyerhof-Parnas scheme of glycolysis - f _w fresh body weight - dw dry body weight     

Glutamate Uptake is Reduced in Prefrontal Cortex in Huntington’s Disease

Huntington’s disease (HD) is caused by a CAG repeat expansion in the HD gene, but how this mutation causes neuronal dysfunction and degeneration is unclear. Inhibition of glutamate uptake, which could cause excessive stimulation of glutamate receptors, has been found in animals carrying very long CAG repeats in the HD gene. In seven HD patients with moderate CAG expansions (40–52), repeat expansion and HD grade at autopsy were strongly correlated (r = 0.88, p = 0.0002). Uptake of [³H]glutamate was reduced by 43% in prefrontal cortex, but the level of synaptic (synaptophysin, AMPA receptors) and astrocytic markers (GFAP, glutamate transporter EAAT1) were unchanged. Glutamate uptake correlated inversely with CAG repeat expansion (r = −0.82, p = 0.015). The reducing agent dithiothreitol improved glutamate uptake in controls, but not in HD brains, suggesting irreversible oxidation of glutamate transporters in HD. We conclude that impairment of glutamate uptake may contribute to neuronal dysfunction and degeneration in HD. Special issue article in honor of Dr. Frode Fonnum.     

The role of Müller glia and microglia in glaucoma

Cells of Müller glia and microglia react to neuronal injury in glaucoma. The change to a reactive phenotype initiates signaling cascades that may serve a neuroprotective role, but may also proceed to promote damaging effects on retinal neurons. Both effects appear to occur most likely in parallel in glaucoma, but the underlying mechanisms and signaling pathways that specifically promote protective versus destructive roles of reactive glial cells are mostly unclear. More research is needed to understand the homeostatic signaling network in which retinal glia cells are embedded to maintain or restore neuronal function after injury.