High-Glucose-Induced Rab20 Upregulation Disrupts Gap Junction Intercellular Communication and Promotes Apoptosis in Retinal Endothelial and Müller Cells: Implications for Diabetic Retinopathy.

To investigate whether high glucose (HG) alters Rab20 expression and compromises gap junction intercellular communication (GJIC) and cell survival, retinal cells were studied for altered intracellular trafficking of connexin 43 (Cx43). Retinal endothelial cells (RRECs) and retinal Müller cells (rMCs) were grown in normal (N; 5 mM glucose) or HG (30 mM glucose) medium for seven days. In parallel, cells grown in HG medium were transfected with either Rab20 siRNA or scrambled siRNA as a control. Rab20 and Cx43 expression and their localization and distribution were assessed using Western Blot and immunostaining, respectively. Changes in GJIC activity were assessed using scrape load dye transfer, and apoptosis was identified using differential dye staining assay. In RRECs or rMCs grown in HG medium, Rab20 expression was significantly increased concomitant with a decreased number of Cx43 plaques. Importantly, a significant increase in the number of Cx43 plaques and GJIC activity was observed in cells transfected with Rab20 siRNA. Additionally, Rab20 downregulation inhibited HG-induced apoptosis in RRECs and rMCs. Results indicate HG-mediated Rab20 upregulation decreases Cx43 localization at the cell surface, resulting in compromised GJIC activity. Reducing Rab20 expression could be a useful strategy in preventing HG-induced vascular and Müller cell death associated with diabetic retinopathy.

High Glucose Induces Mitochondrial Dysfunction in Retinal Müller Cells: Implications for Diabetic Retinopathy.

Purpose: To investigate whether high glucose (HG) induces mitochondrial dysfunction and promotes apoptosis in retinal Müller cells. Methods: Rat retinal Müller cells (rMC-1) grown in normal (N) or HG (30 mM glucose) medium for 7 days were subjected to MitoTracker Red staining to identify the mitochondrial network. Digital images of mitochondria were captured in live cells under confocal microscopy and analyzed for mitochondrial morphology changes based on form factor (FF) and aspect ratio (AR) values. Mitochondrial metabolic function was assessed by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a bioenergetic analyzer. Cells undergoing apoptosis were identified by differential dye staining and TUNEL assay, and cytochrome c levels were assessed by Western blot analysis. Results: Cells grown in HG exhibited significantly increased mitochondrial fragmentation compared to those grown in N medium (FF = 1.7 ± 0.1 vs. 2.3 ± 0.1; AR = 2.1 ± 0.1 vs. 2.5 ± 0.2; P < 0.01). OCR and ECAR were significantly reduced in cells grown in HG medium compared to those grown in N medium (steady state: 75% ± 20% of control, P < 0.02; 64% ± 22% of control, P < 0.02, respectively). These cells also exhibited a significant increase (∼2-fold) in the number of apoptotic cells compared to those grown in N medium (P < 0.01), with a concomitant increase in cytochrome c levels (247% ± 94% of control, P < 0.05). Conclusions: Findings indicate that HG-induced mitochondrial morphology changes and subsequent mitochondrial dysfunction may contribute to retinal Müller cell loss associated with diabetic retinopathy.

Endothelin2 Induces Expression of Genes Associated with Reactive Gliosis in Retinal Müller Cells.

PURPOSE/AIM OF THE STUDY: Photoreceptor degeneration is normally accompanied by reactive gliosis and gene expression changes in Müller (glial) cells. The signaling pathway involved inducing these changes in Müller cells is not known. It has been proposed that endothelin2 (EDN2) released by degenerating photoreceptors might induce gliotic changes in Müller cells. In the present study, we directly tested the hypothesis by determining whether treatment of Müller cell cultures with EDN2 results in upregulation of genes known to be expressed in activated Müller cells in vivo. MATERIALS AND METHODS: Experiments were carried using an established rat Müller cell line (rMC-1), and gene expression was assessed by qRT-PCR. RESULTS: We observed that EDN2 treatment upregulated transcripts for glial fibrillary acidic protein (Gfap), Serpina3n and endothelin receptor B (EdnrB), three genes associated with reactive gliosis in Müller cells. Ciliary neurotrophic factor (CNTF) treatment similarly led to induction of Gfap, Serpina3n and EdnrB transcripts, whereas glutamate treatment had no significant effect. CONCLUSIONS: The finding supports a role for EDN2 as a signaling agent between photoreceptors and Müller cells.

High glucose alters Cx43 expression and gap junction intercellular communication in retinal Müller cells: promotes Müller cell and pericyte apoptosis.

PURPOSE: To investigate whether high glucose (HG) alters connexin 43 (Cx43) expression and gap junction intercellular communication (GJIC) activity in retinal Müller cells, and promotes Müller cell and pericyte loss. METHODS: Retinal Müller cells (rMC-1) and cocultures of rMC-1 and retinal pericytes were grown in normal (N) or HG (30 mM glucose) medium. Additionally, rMC-1 transfected with Cx43 small interfering RNA (siRNA) were grown as cocultures with pericytes, and rMC-1 transfected with Cx43 plasmid were grown in HG. Expression of Cx43 was determined by Western blotting and immunostaining and GJIC was assessed by scrape-loading dye transfer (SLDT) technique. Apoptosis was analyzed by TUNEL or differential staining assay, and Akt activation by assessing Akt phosphorylation. RESULTS: In monocultures of rMC-1 and cocultures of rMC-1 and pericytes, Cx43 protein level, number of Cx43 plaques, GJIC, and Akt phosphorylation were significantly reduced in HG medium. Number of TUNEL-positive cells was also significantly increased in rMC-1 monocultures and in rMC-1 and pericyte cocultures grown in HG medium. Importantly, when rMC-1 transfected with Cx43 siRNA were grown as cocultures with pericytes, a significant decrease in GJIC, and increase in TUNEL-positive cells was observed, concomitant with decreased Akt phosphorylation. Upregulation of Cx43 rescued rMC-1 from HG-induced apoptosis. CONCLUSIONS: Gap junction communication between Müller cells and pericytes is essential for their survival. Downregulation of Cx43 that is HG induced and impairment of GJIC activity in Müller cells contributes to loss of glial and vascular cells associated with the pathogenesis of diabetic retinopathy.

Gene expression changes in retinal Müller (glial) cells exposed to elevated pressure.

PURPOSE: Retinal Müller (glial) cells undergo "reactive gliosis", a stress response that is accompanied by changes in their morphology and upregulation of various cellular markers. Reactive gliosis is seen in many retinal diseases and conditions; however, it is not known whether it is a common, stereotypic response or the nature of the response varies with the type of retinal stress. To address this question, we have examined gene expression changes in Müller cells exposed to elevated pressure. MATERIALS AND METHODS: Rat Müller cells (rMC-1) were exposed to elevated pressure, and RNA was extracted and analyzed using Affymetrix GeneChip microarrays to identify pressure-responsive genes. RESULTS: Analysis of microarray data showed that at 6 h, 186 genes had > 1.5-fold change with FDR < 0.01. Of these, 62 genes were up-regulated while 124 genes were down-regulated. At 24 h, 73 genes changed > 1.5-fold. Of these, 37 genes were up-regulated while 36 genes were down-regulated. Ingenuity canonical pathway analysis showed that several signaling and metabolic pathways were significantly changed in Müller cells under high pressure. In addition, among up- and down-regulated genes, we identified eight genes-areg, bmp4, cyp1b1, gpnmb, herc2, msh2, heph, and selenbp1, that have been directly or indirectly associated with elevated intraocular pressure. Two genes, areg and gpnmb, further showed time-dependent changes in mRNA and protein expression. CONCLUSION: The results show that Müller cells in vitro respond to elevated pressure by differential regulation of expressed genes. The transcriptional profile is different from that seen with hypoxia, which indicates that Müller cells respond differentially to different microenvironmental changes in the retina.

Ciliary neurotrophic factor induces genes associated with inflammation and gliosis in the retina: a gene profiling study of flow-sorted, Müller cells.

BACKGROUND: Ciliary neurotrophic factor (CNTF), a member of the interleukin-6 cytokine family, has been implicated in the development, differentiation and survival of retinal neurons. The mechanisms of CNTF action as well as its cellular targets in the retina are poorly understood. It has been postulated that some of the biological effects of CNTF are mediated through its action via retinal glial cells; however, molecular changes in retinal glia induced by CNTF have not been elucidated. We have, therefore, examined gene expression dynamics of purified Müller (glial) cells exposed to CNTF in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Müller cells were flow-sorted from mgfap-egfp transgenic mice one or three days after intravitreal injection of CNTF. Microarray analysis using RNA from purified Müller cells showed differential expression of almost 1,000 transcripts with two- to seventeen-fold change in response to CNTF. A comparison of transcriptional profiles from Müller cells at one or three days after CNTF treatment showed an increase in the number of transcribed genes as well as a change in the expression pattern. Ingenuity Pathway Analysis showed that the differentially regulated genes belong to distinct functional types such as cytokines, growth factors, G-protein coupled receptors, transporters and ion channels. Interestingly, many genes induced by CNTF were also highly expressed in reactive Müller cells from mice with inherited or experimentally induced retinal degeneration. Further analysis of gene profiles revealed 20-30% overlap in the transcription pattern among Müller cells, astrocytes and the RPE. CONCLUSIONS/SIGNIFICANCE: Our studies provide novel molecular insights into biological functions of Müller glial cells in mediating cytokine response. We suggest that CNTF remodels the gene expression profile of Müller cells leading to induction of networks associated with transcription, cell cycle regulation and inflammatory response. CNTF also appears to function as an inducer of gliosis in the retina.

High glucose increases angiopoietin-2 transcription in microvascular endothelial cells through methylglyoxal modification of mSin3A.

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor alpha. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease.

Methylglyoxal modification of mSin3A links glycolysis to angiopoietin-2 transcription.

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here, we report that in retinal Müller cells, increased glycolytic flux causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc transferase to an mSin3A-Sp3 complex, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding of the repressor complex to a glucose-responsive GC box in the angiopoietin-2 promoter, resulting in increased Ang-2 expression. A similar mechanism involving methylglyoxal-modification of other coregulator proteins may play a role in the pathobiology of a variety of conditions associated with changes in methylglyoxal concentration, including cancer and diabetic vascular disease.

Nonsynaptic localization of the excitatory amino acid transporter 4 in photoreceptors.

Excitatory amino acid transporters (EAATs) are involved in regulating extracellular glutamate levels at synaptic regions in the CNS. EAAT1, 2, 3, and 5 have been found in the mammalian retina, but the presence of EAAT4 has remained controversial. Recently, we found a high level of EAAT4 mRNA in the human retina, and this observation lead us to examine whether EAAT4 was expressed in the mammalian retina. Immunoblotting studies showed the presence of EAAT4-immunoreactive proteins in human and mouse retinas, corresponding to EAAT4 monomers and dimers. Immunohistochemistry revealed that EAAT4 was localized in rod and cone photoreceptor outer segments in the human retina, and in the outer and inner segments of mouse and ground squirrel retinas. In no case was EAAT4 found in the outer plexiform layer or in any other layer in the retina. EAAT4 expression by photoreceptors was confirmed by immunoblotting a purified rod outer segment preparation, which showed the presence of a 50-kDa EAAT4-immunoreactive protein. In addition, the EAAT4-associated protein, GTRAP41, was found in the human, mouse, and squirrel retinas as well as in the rod outer segment preparation. Further immunocytochemical and co-immunoprecipitation experiments demonstrated that GTRAP41 was colocalized and interacted in vivo with EAAT4. Importantly, glutamate uptake and drug inhibition experiments showed that an EAAT4-like glutamate uptake system is present in the rod outer segments. Finally, we examined whether glutamate signaling mediated by EAAT4 can modulate rod outer segment phagocytosis by the retinal pigment epithelium. Results of the present study show that EAAT4 is present in the outer segments, a nonsynaptic region of photoreceptors, where it might provide a feedback mechanism for sensing extracellular glutamate or serve as an outer barrier to prevent glutamate from escaping from the retina.

Glutamate transport by retinal Muller cells in glutamate/aspartate transporter-knockout mice.

Glutamate transporters are involved in maintaining extracellular glutamate at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitotoxic damage. The mammalian retina is known to express the excitatory amino acid transporters, EAAT1-5; however, their specific role in glutamate homeostasis is poorly understood. To examine the role of the glial glutamate/aspartate transporter (GLAST) in the retina, we have studied glutamate transport by Muller cells in GLAST-/- mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the Km value for glutamate uptake was similar in wild-type and GLAST-/- mouse retinas, but the Vmax was approximately 50% lower in the mutant. In Na+-free medium, the Vmax was further reduced by 40%. In patch-clamp recordings of dissociated Muller cells from GLAST-/- mice, application of 0.1 mM glutamate evoked no current showing that the cells lacked functional electrogenic glutamate transporters. The result also indicated that there was no compensatory upregulation of EAATs in Muller cells. [3H]D-Aspartate uptake autoradiography, however, showed that Na+-dependent, high-affinity transporters account for most of the glutamate uptake by Muller cells, and that Na+-independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Muller cells in the GLAST-/- mouse retina is not due to known glutamate transporters-cystine-glutamate exchanger, ASCT-1, AGT-1, or other heteroexchangers. The present study shows that while several known glutamate transporters are expressed by mammalian Muller cells, new Na+-dependent, high-affinity glutamate transporters remain to be identified.

Retinal glucose metabolism in mice lacking the L-glutamate/aspartate transporter.

The conventional view that glucose is the substrate for neuronal energy metabolism has been recently challenged by the "lactate shuttle" hypothesis in which glutamate cycling in glial cells drives all neuronal glucose metabolism. According to this view, glutamate released by activated retinal neurons is transported into Müller (glial) cells where it triggers glycolysis. The lactate released by Müller cells serves as the energy substrate for neuronal metabolism. Because the L-Glutamate/aspartate transporter (GLAST) is the predominant, Na+-dependent, glutamate transporter expressed by Müller cells, we have used GLAST-knockout (GLAST -/-) mice to examine the relationship between lactate release and GLAST activity in the retina. We found that glucose uptake and lactate production by the GLAST -/- mouse retina was similar to that observed in the wild type mouse retina. Furthermore, addition of 1 mM glutamate and NH4Cl to the incubation medium did not further stimulate glucose uptake in either case. When lactate release was measured in the presence of the lactate uptake inhibitor, alpha-cyano-4-hydroxycinnamate, there was no significant change in the amount of lactate released by retinas from GLAST -/- mice compared to the wild type. Finally, lactate release was similar under both dark and light conditions. These results show that lactate production and release is not altered in retinas of GLAST -/- mice, which suggests that metabolic coupling between photoreceptors and Müller cells is not mediated by the glial glutamate transporter, GLAST.

Contribution of a glial glutamate transporter to GABA synthesis in the retina.

Neuronal glutamate transporters have been shown to play a role in GABA synthesis by enhancing glutamate uptake. In the present study, we have examined whether a glial glutamate transporter, GLAST, has a role in GABA synthesis in the mammalian retina. We found that the retinal GABA level was about two-fold higher in the GLAST-/- mouse retina compared to that in the wild type. Endogenous glutamate level was also increased about 2-fold in the mutant. Therefore, loss of GLAST results in a higher retinal GABA level, probably due to increased availability of its precursor, glutamate. An increase in GABAergic activity can be expected to affect trigger features such as directional selective response of neurons in the GLAST-/- mouse retina.

Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase: a role in high glucose-induced apoptosis in retinal Müller cells.

PURPOSE: A recent study demonstrated that retinal Müller cells undergo hyperglycemia-induced apoptosis in vitro. Translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the cytosol to the nucleus is a critical step in the induction of apoptosis in neuronal cells. R-(-)-deprenyl prevents nuclear translocation of GAPDH and subsequent apoptosis in neuronal cells. In this study, the role of nuclear translocation of GAPDH in hyperglycemia-induced apoptosis in retinal Müller cells and the ability of R-(-)-deprenyl to inhibit the translocation of GAPDH and apoptosis were investigated. METHODS: Transformed rat Müller cells (rMC-1) and isolated human Müller cells were cultured in normal glucose, high glucose, and high glucose plus R-(-)-deprenyl for up to 5 days. Subcellular distribution of GAPDH was determined in vitro and in vivo by immunocytochemistry. Apoptosis in tissue cultures was determined by annexin-V staining and caspase-3 activity. RESULTS: Hyperglycemia significantly increased the amount of GAPDH protein in the nucleus above normal within the first 48 hours in rMC-1 and human Müller cells. The addition of R-(-)-deprenyl to these cells incubated in high glucose reduced the amount of GAPDH protein in the nucleus and decreased hyperglycemia-induced apoptosis in both cell types. In vivo studies confirmed the accumulation of GAPDH in nuclei of Müller cells in diabetes. CONCLUSIONS: The nuclear translocation of GAPDH in rMC-1 and human Müller cells is closely associated with the induction of apoptosis. R-(-)-deprenyl inhibits nuclear accumulation of GAPDH and subsequent apoptosis in these cells. Therefore, R-(-)-deprenyl offers a strategy to explore the role of GAPDH translocation into the nucleus in the development of diabetic retinopathy.

GFAP promoter drives Müller cell-specific expression in transgenic mice.

PURPOSE: In an attempt to identify Müller cell-specific promoters and to better understand the gene regulatory mechanisms in retinal glial cells, the expression of the glial fibrillary acidic protein (GFAP) gene was studied in Müller cell cultures and in GFAP-enhanced green fluorescent protein (EGFP) transgenic mice. METHODS: A transfection assay of GFAP-luciferase constructs carrying a series of nested deletions was performed in an established Müller cell line. For in vivo analysis, transgenic mice were generated by injecting a construct carrying a 2.5-kb, 5' fragment of the mouse GFAP gene linked to the EGFP gene. Isolated retinas from transgenic mice were screened for GFP expression. Subsequently, the identity of the GFP-expressing cells was established by immunostaining cryostat sections of the retina with antibodies against Müller cell antigenic markers. Induction of the transgene and the endogenous GFAP gene was examined by injecting ciliary neurotrophic factor (CNTF) into the eye. RESULTS: The DNA transfection data suggested that proximal 5' sequences of the GFAP gene are sufficient to direct high-level reporter expression in Müller cell cultures. In transgenic mice, GFP fluorescence appeared in radially oriented processes that spanned almost the entire thickness of the retina. Immunostaining with antibodies to cellular retinaldehyde-binding protein (CRALBP) and glutamine synthetase showed that the GFP-expressing cells were Müller cells. GFP-expressing Müller cells were observed in the retinas of both albino and pigmented transgenic mice. In eyes injected with CNTF, both GFP and GFAP levels were highly elevated. These observations suggest that the 2.5-kb, 5' GFAP sequence can direct inducible reporter gene expression in Müller cells. In addition to Müller cells, a few GFP-labeled astrocytes were present in the adult retina. In the developing retina, GFP-expressing astrocytes were first present at the optic nerve head, and as development progressed, the cells gradually moved toward the periphery of the retina and acquired their adult, stellate morphology. CONCLUSIONS: The present study shows that the 2.5-kb, 5' flanking region of the mouse GFAP gene can be used to express GFP, and possibly other genes, specifically in Müller cells in the mouse retina. Furthermore, expression of the transgene can be upregulated by intravitreal injection of CNTF.

CRALBP transcriptional regulation in ciliary epithelial, retinal Müller and retinal pigment epithelial cells.

Cellular retinaldehyde binding protein (CRALBP) functions in the visual cycle and mutations in the RLBP1 gene can lead to blindness. RLBP1 promoter analyses have been pursued in vitro as an approach to deciphering the mechanism controlling cell-specific expression of CRALBP. Reporter activity of wildtype and mutant RLBP1 promoter constructs suggest that CRALBP transcriptional regulation may be similar in the ciliary epithelium (CE) and retinal pigment epithelium (RPE) but different in Müller cells. Results in RPE cells refine the location of an RLBP1 enhancer element to within -1826 to -1749 bp and a repressor element to within -702 to -635 bp.

Developmental expression of beta-catenin in mouse retina.

Recent studies have shown that catenins play a pivotal role in neuronal signalling during vertebrate development. In order to study the significance of beta-catenin in the developing mouse retina, the localization of beta-catenin was examined by immunohistochemistry from embryonic day (E) 12 to adult mice. Immunoreactivity for beta-catenin was found in ganglion cells of the retina at E12, and extended to the inner and outer plexiform layer as well as the ganglion-cell layer with the strongest immunolabelling from E16 through to postnatal day (P) 5. The immunoreactivity of ganglion cells was distributed on the cell surface. Thereafter, the immunoreactivity gradually decreased, being limited to the inner plexiform layer and ganglion-cell layer, including the nerve-fiber layer in P10. By P16, the weak immunoreactivity was detected in the inner plexiform layer and ganglion-cell layer, and almost disappeared in the adult retina. No distinct immunoreactivity was found in the retinal pigment epithelium. The reverse transcription polymerase chain reaction showed that beta-catenin messenger ribonuclic acid was detected at E12, E16, P1 and P16, and thereafter markedly decreased, being weakest in the adult. These findings show that beta-catenin is expressed during development at the sites of synaptic connections of inner and outer plexiform layers, and on the ganglion cells and their fibers in the retina, suggesting that beta-catenin might play an important role in the synapse formation and ganglion-cell development during the morphogenesis of the retina.