Proliferation Potential of Müller Glia after Retinal Damage Varies between Mouse Strains

Retinal Müller glia can serve as a source for regeneration of damaged retinal neurons in fish, birds and mammals. However, the proliferation rate of Müller glia has been reported to be low in the mammalian retina. To overcome this problem, growth factors and morphogens have been studied as potent promoters of Müller glial proliferation, but the molecular mechanisms that limit the proliferation of Müller glia in the mammalian retina remain unknown. In the present study, we found that the degree of damage-induced Müller glia proliferation varies across mouse strains. In mouse line 129×1/SvJ (129), there was a significantly larger proliferative response compared with that observed in C57BL/6 (B6) after photoreceptor cell death. Treatment with a Glycogen synthase kinase 3 (GSK3) inhibitor enhanced the proliferation of Müller glia in 129 but not in B6 mouse retinas. We therefore focused on the different gene expression patterns during retinal degeneration between B6 and 129. Expression levels of Cyclin D1 and Nestin correlated with the degree of Müller glial proliferation. A comparison of genome-wide gene expression between B6 and 129 showed that distinct sets of genes were upregulated in the retinas after damage, including immune response genes and chromatin remodeling factors.     

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.     

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.     

Epoxypukalide Induces Proliferation and Protects against Cytokine-Mediated Apoptosis in Primary Cultures of Pancreatic β-Cells

There is an urgency to find new treatments for the devastating epidemic of diabetes. Pancreatic β-cells viability and function are impaired in the two most common forms of diabetes, type 1 and type 2. Regeneration of pancreatic β-cells has been proposed as a potential therapy for diabetes. In a preliminary study, we screened a collection of marine products for β-cell proliferation. One unique compound (epoxypukalide) showed capability to induce β-cell replication in the cell line INS1 832/13 and in primary rat cell cultures. Epoxypukalide was used to study β-cell proliferation by [³H]thymidine incorporation and BrdU incorporation followed by BrdU/insulin staining in primary cultures of rat islets. AKT and ERK1/2 signalling pathways were analyzed. Cell cycle activators, cyclin D2 and cyclin E, were detected by western-blot. Apoptosis was studied by TUNEL and cleaved caspase 3. β-cell function was measured by glucose-stimulated insulin secretion. Epoxypukalide induced 2.5-fold increase in β-cell proliferation; this effect was mediated by activation of ERK1/2 signalling pathway and upregulation of the cell cycle activators, cyclin D2 and cyclin E. Interestingly, epoxypukalide showed protection from basal (40% lower versus control) and cytokine-induced apoptosis (80% lower versus control). Finally, epoxypukalide did not impair β-cell function when measured by glucose-stimulated insulin secretion. In conclusion, epoxypukalide induces β-cell proliferation and protects against basal and cytokine-mediated β-cell death in primary cultures of rat islets. These findings may be translated into new treatments for diabetes.     

Induction of Retinal Progenitors and Neurons from Mammalian Müller Glia under Defined Conditions

Vision impairment caused by loss of retinal neurons affects millions of people worldwide, and currently, there is no effective treatment. Müller glia of mammalian retina may represent an under-recognized and potential source for regeneration of a wide range of retinal cell types, including retinal ganglion cells and photoreceptors. Here, we demonstrated that mouse Müller glia cells have the capacity to be reprogrammed into the retinal neuronal cell fate and are competent to give rise to photoreceptors under a defined culture condition. Inactivation of p53 released proliferation restriction of Müller glia and significantly enhanced the induction of retinal progenitor from Müller glia in culture. Moreover, following the ocular transplantation, the Müller glia-derived progenitors were differentiated toward the fates of photoreceptors and retinal ganglion cells. Together, these results demonstrate the feasibility of using Müller glia as a potential source for retinal repair and regeneration.     

Müller Glia Activation in Response to Inherited Retinal Degeneration Is Highly Varied and Disease-Specific

Despite different aetiologies, most inherited retinal disorders culminate in photoreceptor loss, which induces concomitant changes in the neural retina, one of the most striking being reactive gliosis by Müller cells. It is typically assumed that photoreceptor loss leads to an upregulation of glial fibrilliary acidic protein (Gfap) and other intermediate filament proteins, together with other gliosis-related changes, including loss of integrity of the outer limiting membrane (OLM) and deposition of proteoglycans. However, this is based on a mix of both injury-induced and genetic causes of photoreceptor loss. There are very few longitudinal studies of gliosis in the retina and none comparing these changes across models over time. Here, we present a comprehensive spatiotemporal assessment of features of gliosis in the degenerating murine retina that involves Müller glia. Specifically, we assessed Gfap, vimentin and chondroitin sulphate proteoglycan (CSPG) levels and outer limiting membrane (OLM) integrity over time in four murine models of inherited photoreceptor degeneration that encompass a range of disease severities (Crb1^rd8/rd8, Prph2^+/Δ307, Rho^-/-, Pde6b^rd1/rd1). These features underwent very different changes, depending upon the disease-causing mutation, and that these changes are not correlated with disease severity. Intermediate filament expression did indeed increase with disease progression in Crb1^rd8/rd8 and Prph2^+/Δ307, but decreased in the Prph2^+/Δ307 and Pde6b^rd1/rd1 models. CSPG deposition usually, but not always, followed the trends in intermediate filament expression. The OLM adherens junctions underwent significant remodelling in all models, but with differences in the composition of the resulting junctions; in Rho^-/- mice, the adherens junctions maintained the typical rod-Müller glia interactions, while in the Pde6b^rd1/rd1 model they formed predominantly between Müller cells in late stage of degeneration. Together, these results show that gliosis and its associated processes are variable and disease-dependent.     

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.     

Expression and Characterization of Purinergic Receptors in Rat Middle Meningeal Artery–Potential Role in Migraine

The dura mater and its vasculature have for decades been central in the hypothesis of migraine and headache pathophysiology. Although recent studies have questioned the role of the vasculature as the primary cause, dural vessel physiology is still relevant in understanding the complex pathophysiology of migraine. The aim of the present study was to isolate the middle meningeal artery (MMA) from rodents and characterize their purinergic receptors using a sensitive wire myograph method and RT-PCR. The data presented herein suggest that blood flow through the MMA is, at least in part, regulated by purinergic receptors. P2X1 and P2Y6 receptors are the strongest contractile receptors and, surprisingly, ADPβS caused contraction most likely via P2Y1 or P2Y13 receptors, which is not observed in other arteries. Adenosine addition, however, caused relaxation of the MMA. The adenosine relaxation could be inhibited by {"type":"entrez-protein","attrs":{"text":"SCH58261","term_id":"1052882304","term_text":"SCH58261"}}SCH58261 (A2A receptor antagonist) and caffeine (adenosine receptor antagonist). This gives one putative molecular mechanism for the effect of caffeine, often used as an adjuvant remedy of cranial pain. Semi-quantitative RT-PCR expression data for the receptors correlate well with the functional findings. Together these observations could be used as targets for future understanding of the in vivo role of purinergic receptors in the MMA.     

Müller Glia as an Active Compartment Modulating Nervous Activity in the Vertebrate Retina: Neurotransmitters and Trophic Factors

Müller cells represent the main type of glia present in the retina interacting with most, if not all neurons in this tissue. Müller cells have been claimed to function as optic fibers in the retina delivering light to photoreceptors with minimal distortion and low loss [Franze et al (2007) Proc Natl Acad Sci 104:8287–8292]. Most of the mediators found in the brain are also detected in the retinal tissue, and glia cells are active players in the synthesis, release, signaling and uptake of major mediators of synaptic function. Müller glia trophic factors may regulate many different aspects of neuronal circuitry during synaptogenesis, differentiation, neuroprotection and survival of photoreceptors, Retinal Ganglion Cells (RGCs) and other targets in the retina. Here we review the role of several transmitters and trophic factors that participate in the neuron-glia loop in the retina. Special issue article in honor of Dr. Ricardo Tapia.     

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).     

Role of Müller glia in neuroprotection and regeneration in the retina

Glial cells are thought to protect neurons from various neurological insults. When there is injury to retina, Müller cells, which are the predominant glial element in the retina, undergo significant morphological, cellular and molecular changes. Some of these changes reflect Müller cell involvement in protecting the retina from further damage. Müller cells express growth factors, neurotransmitter transporters and antioxidant agents that could have an important role in preventing excitotoxic damage to retinal neurons. Moreover, Müller cells contact to endothelial cells to facilitate the neovascularization process during hypoxic conditions. Finally, recent studies have pointed to a role of Müller cells in retina regeneration after damage, dedifferentiating to progenitor cells and then giving rise to different neuronal cell types. In this article we will review the role of Müller glia in neuroprotection and regeneration after damage in the retina.     

DNA Damage and p53 Restrict Proliferation of Müller Cells in the Mouse Retina in Response to the Influence of N-Methyl-N-Nitrosourea

Biophysics - Systemic administration of N-methyl-N-nitrosourea in rats resulted in the death of retinal photoreceptors followed by differentiation of retinal Müller glial cells into...     

Primary Cilia Reconsidered in the Context of Ciliopathies: Extraciliary and Ciliary Functions of Cilia Proteins Converge on a Polarity theme?

Once dismissed as vestigial organelles, primary cilia have garnered the interest of scientists, given their importance in development/signaling, and for their implication in a new disease category known as ciliopathies. However, many, if not all, “cilia” proteins also have locations/functions outside of the primary cilium. These extraciliary functions can complicate the interpretation of a particular ciliopathy phenotype: it may be a result of defects at the cilium and/or at extraciliary locations, and it could be broadly related to a unifying cellular process for these proteins, such as polarity. Assembly of a cilium has many similarities to the development of other polarized structures. This evolutionarily preserved process for the assembly of polarized cell structures offers a perspective on how the cilium may have evolved. We hypothesize that cilia proteins are critical for cell polarity, and that core polarity proteins may have been specialized to form various cellular protrusions, including primary cilia.

     

Embryonic Stem Cell-Derived Microvesicles Induce Gene Expression Changes in Müller Cells of the Retina

Cell-derived microvesicles (MVs), recognized as important components of cell-cell communication, contain mRNAs, miRNAs, proteins and lipids and transfer their bioactive contents from parent cells to cells of other origins. We have studied the effect that MVs released from embryonic stem cells (ESMVs) have on retinal progenitor Müller cells. Cultured human Müller cells were exposed to mouse ESMVs every 48 hours for a total of 9 treatments. Morphological changes were observed by light microscopy in the treated cells, which grew as individual heterogeneous cells, compared to the uniform, spindle-like adherent cellular sheets of untreated cells. ESMVs transferred to Müller cells embryonic stem cell (ESC) mRNAs involved in the maintenance of pluripotency, including Oct4 and Sox2, and the miRNAs of the 290 cluster, important regulators of the ESC-specific cell cycle. Moreover, ESMV exposure induced up-regulation of the basal levels of endogenous human Oct4 mRNA in Müller cells. mRNA and miRNA microarrays of ESMV-treated vs. untreated Müller cells revealed the up-regulation of genes and miRNAs involved in the induction of pluripotency, cellular proliferation, early ocular genes and genes important for retinal protection and remodeling, as well as the down-regulation of inhibitory and scar-related genes and miRNAs involved in differentiation and cell cycle arrest. To further characterize the heterogeneous cell population of ESMV-treated Müller cells, their expression of retinal cell markers was compared to that in untreated control cells by immunocytochemistry. Markers for amacrine, ganglion and rod photoreceptors were present in treated but not in control Müller cells. Together, our findings indicate that ESMs induce de-differentiation and pluripotency in their target Müller cells, which may turn on an early retinogenic program of differentiation.     

A Novel Adeno-Associated Viral Variant for Efficient and Selective Intravitreal Transduction of Rat Müller Cells

Background

The pathologies of numerous retinal degenerative diseases can be attributed to a multitude of genetic factors, and individualized treatment options for afflicted patients are limited and cost-inefficient. In light of the shared neurodegenerative phenotype among these disorders, a safe and broad-based neuroprotective approach would be desirable to overcome these obstacles. As a result, gene delivery of secretable-neuroprotective factors to Müller cells, a type of retinal glia that contacts all classes of retinal neurons, represents an ideal approach to mediate protection of the entire retina through a simple and innocuous intraocular, or intravitreal, injection of an efficient vehicle such as an adeno-associated viral vector (AAV). Although several naturally occurring AAV variants have been isolated with a variety of tropisms, or cellular specificities, these vectors inefficiently infect Müller cells via intravitreal injection.

Methodology/Principal Findings

We have previously applied directed evolution to create several novel AAV variants capable of efficient infection of both rat and human astrocytes through iterative selection of a panel of highly diverse AAV libraries. Here, in vivo and in vitro characterization of these isolated variants identifies a previously unreported AAV variant ShH10, closely related to AAV serotype 6 (AAV6), capable of efficient, selective Müller cell infection through intravitreal injection. Importantly, this new variant shows significantly improved transduction relative to AAV2 (>60%) and AAV6.

Conclusions/Significance

Our findings demonstrate that AAV is a highly versatile vector capable of powerful shifts in tropism from minor sequence changes. This isolated variant represents a new therapeutic vector to treat retinal degenerative diseases through secretion of neuroprotective factors from Müller cells as well as provides new opportunities to study their biological functions in the retina.     

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.