Nuclear NAD+-biosynthetic enzyme NMNAT1 facilitates development and early survival of retinal neurons.

Despite mounting evidence that the mammalian retina is exceptionally reliant on proper NAD+ homeostasis for health and function, the specific roles of subcellular NAD+ pools in retinal development, maintenance, and disease remain obscure. Here, we show that deletion of the nuclear-localized NAD+ synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) in the developing murine retina causes early and severe degeneration of photoreceptors and select inner retinal neurons via multiple distinct cell death pathways. This severe phenotype is associated with disruptions to retinal central carbon metabolism, purine nucleotide synthesis, and amino acid pathways. Furthermore, transcriptomic and immunostaining approaches reveal dysregulation of a collection of photoreceptor and synapse-specific genes in NMNAT1 knockout retinas prior to detectable morphological or metabolic alterations. Collectively, our study reveals previously unrecognized complexity in NMNAT1-associated retinal degeneration and suggests a yet-undescribed role for NMNAT1 in gene regulation during photoreceptor terminal differentiation.

The Role of Caspase-12 in Retinal Bystander Cell Death and Innate Immune Responses against MCMV Retinitis.

(1) Background: caspase-12 is activated during cytomegalovirus retinitis, although its role is presently unclear. (2) Methods: caspase-12-/- (KO) or caspase-12+/+ (WT) mice were immunosup eyes were analyzed by plaque assay, TUNEL assay, immunohistochemical staining, western blotting, and real-time PCR. (3) Results: increased retinitis and a more extensive virus spread were detected in the retina of infected eyes of KO mice compared to WT mice at day 14 p.i. Compared to MCMV injected WT eyes, mRNA levels of interferons α, ß and γ were significantly reduced in the neural retina of MCMV-infected KO eyes at day 14 p.i. Although similar numbers of MCMV infected cells, similar virus titers and similar numbers of TUNEL-staining cells were detected in injected eyes of both KO and WT mice at days 7 and 10 p.i., significantly lower amounts of cleaved caspase-3 and p53 protein were detected in infected eyes of KO mice at both time points. (4) Conclusions: caspase-12 contributes to caspase-3-dependent and independent retinal bystander cell death during MCMV retinitis and may also play an important role in innate immunity against virus infection of the retina.

Hydrogen sulfide: a gaseous signaling molecule modulates tissue homeostasis: implications in ophthalmic diseases.

Hydrogen sulfide (H2S) serves as a gasotransmitter in the regulation of organ development and maintenance of homeostasis in tissues. Its abnormal levels are associated with multiple human diseases, such as neurodegenerative disease, myocardial injury, and ophthalmic diseases. Excessive exposure to H2S could lead to cellular toxicity, orchestrate pathological process, and increase the risk of various diseases. Interestingly, under physiological status, H2S plays a critical role in maintaining cellular physiology and limiting damages to tissues. In mammalian species, the generation of H2S is catalyzed by cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CSE), 3-mercapto-methylthio pyruvate aminotransferase (3MST) and cysteine aminotransferase (CAT). These enzymes are found inside the mammalian eyeballs at different locations. Their aberrant expression and the accumulation of substrates and intermediates can change the level of H2S by orders of magnitude, causing abnormal structures or functions in the eyes. Detailed investigations have demonstrated that H2S donors' administration could regulate intraocular pressure, protect retinal cells, inhibit oxidative stress and alleviate inflammation by modulating the function of intra or extracellular proteins in ocular tissues. Thus, several slow-releasing H2S donors have been shown to be promising drugs for treating multiple diseases. In this review, we discuss the biological function of H2S metabolism and its application in ophthalmic diseases.

Identification of protein kinase C α- and tyrosine hydroxylase-immunoreactive cells in the microbat retina.

A growing number of studies have revealed the functional neuroarchitecture of the microbat retina and suggested that microbats can see using their eyes. To better understand the organization of the microbat retina, quantitative analysis of protein kinase C alpha (PKCα)- and tyrosine hydroxylase (TH)-immunoreactive (IR) cells was conducted on the greater horseshoe bat (Rhinolophus ferrumequinum) retina. As a result, PKCα immunoreactivity was observed in rod bipolar cells, consistent with previous studies on other mammalian retinas. PKCα-IR cell distribution in the inner nuclear layer showed regional differences in density, with the highest density found in the nasal retina. The average density of PKCα-IR cells was 10,487±441 cells/mm² (mean ± S.D.; n=4), with a total of 43,077±1,843 cells/retina. TH-IR cells in the Rhinolophus ferrumequinum retina could be classified into four types based on soma location and ramification in the inner plexiform layer: conventional amacrine, displaced amacrine, interplexiform, and intercalated cells. The majority of TH-IR cells were conventional amacrine cells. TH-IR cells were nonrandomly distributed at low density over the retina. The average density was 29.7±3.1 cells/mm² (mean ± S.D.; n=3), with a total of 124.0±11.3 cells/retina. TH-IR processes showed varicosities and formed ring-like structures encircling AII amacrine cells. Our study provides the foundation for understanding the neurochemical architecture of the microbat retina and supports the notion that the eyes do play a role in the visual system of microbats.

Lithium promotes DNA stability and survival of ischemic retinal neurocytes by upregulating DNA ligase IV.

Neurons display genomic fragility and show fragmented DNA in pathological degeneration. A failure to repair DNA breaks may result in cell death or apoptosis. Lithium protects retinal neurocytes following nutrient deprivation or partial nerve crush, but the underlying mechanisms are not well defined. Here we demonstrate that pretreatment with lithium protects retinal neurocytes from ischemia-induced damage and enhances light response in rat retina following ischemia-reperfusion injury. Moreover, we found that DNA nonhomologous end-joining (NHEJ) repair is implicated in this process because in ischemic retinal neurocytes, lithium significantly reduces the number of γ-H2AX foci (well-characterized markers of DNA double-strand breaks in situ) and increases the DNA ligase IV expression level. Furthermore, we also demonstrate that nuclear respiratory factor 1 (Nrf-1) and phosphorylated cyclic AMP-response element binding protein-1 (P-CREB1) bind to ligase IV promoter to cause upregulation of ligase IV in neurocytes. The ischemic upregulation of Nrf-1 and lithium-induced increase of P-CREB1 cooperate to promote transcription of ligase IV. Short hairpin RNAs against Nrf-1 and CREB1 could significantly inhibit the increase in promoter activity and expression of ligase IV observed in the control oligos following lithium treatment in retinal neurocytes. More importantly, ischemic stimulation triggers the expression of ligase IV. Taken together, our results thus reveal a novel mechanism that lithium offers neuroprotection from ischemia-induced damage by enhancing DNA NHEJ repair.

Deficiency of aldose reductase attenuates inner retinal neuronal changes in a mouse model of retinopathy of prematurity.

Retinopathy of prematurity (ROP) is a leading cause of childhood blindness where vascular abnormality and retinal dysfunction are reported. We showed earlier that genetic deletion of aldose reductase (AR), the rate-limiting enzyme in the polyol pathway, reduced the neovascularization through attenuating oxidative stress induction in the mouse oxygen-induced retinopathy (OIR) modeling ROP. In this study, we further investigated the effects of AR deficiency on retinal neurons in the mouse OIR. Seven-day-old wild-type and AR-deficient mice were exposed to 75 % oxygen for 5 days and then returned to room air. Electroretinography was used to assess the neuronal function at postnatal day (P) 30. On P17 and P30, retinal cytoarchitecture was examined by morphometric analysis and immunohistochemistry for calbindin, protein kinase C alpha, calretinin, Tuj1, and glial fibrillary acidic protein. In OIR, attenuated amplitudes and delayed implicit time of a-wave, b-wave, and oscillatory potentials were observed in wild-type mice, but they were not significantly changed in AR-deficient mice. The morphological changes of horizontal, rod bipolar, and amacrine cells were shown in wild-type mice and these changes were partly preserved with AR deficiency. AR deficiency attenuated the Müller cell gliosis induced in OIR. Our observations demonstrated AR deficiency preserved retinal functions in OIR and AR deficiency could partly reduce the extent of retinal neuronal histopathology. These findings suggested a therapeutic potential of AR inhibition in ROP treatment with beneficial effects on the retinal neurons.

TNAP activity is localized at critical sites of retinal neurotransmission across various vertebrate species.

Evidence is emerging with regard to the role of tissue non-specific alkaline phosphatase (TNAP) in neural functions. As an ectophosphatase, this enzyme might influence neural activity and synaptic transmission in diverse ways. The localization of the enzyme in known neural circuits, such as the retina, might significantly advance an understanding of its role in normal and pathological functioning. However, the presence of TNAP in the retina is scarcely investigated. Our multispecies comparative study (zebrafish, cichlid, frog, chicken, mouse, rat, golden hamster, guinea pig, rabbit, sheep, cat, dog, ferret, squirrel monkey, human) using enzyme histochemistry and Western blots has shown the presence of TNAP activity in the retina of several mammalian species, including humans. Although the TNAP activity pattern varies across species, we have observed the following trends: (1) in all investigated species (except golden hamster), retinal vessels display TNAP activity; (2) TNAP activity consistently occurs in the photoreceptor layer; (3) in majority of the investigated species, marked TNAP activity is present in the outer and inner plexiform layers. In zebrafish, frog, chicken, guinea pig, and rat, TNAP histochemistry has revealed several sublayers of the inner plexiform layer. Frog, golden hamster, guinea pig, mouse, and human retinas possess a subpopulation of amacrine cells positively staining for TNAP activity. The expression of TNAP in critical sites of retinal signal transmission across a wide range of species suggests its fundamental, evolutionally conserved role in vision.

Acetylation: a lysine modification with neuroprotective effects in ischemic retinal degeneration.

Neuroretinal ischemic injury contributes to several degenerative diseases in the eye and the resulting pathogenic processes involving a series of necrotic and apoptotic events. This study investigates the time and extent of changes in acetylation, and whether this influences function and survival of neuroretinal cells following injury. Studies evaluated the time course of changes in histone deacetylase (HDAC) activity, histone-H3 acetylation and caspase-3 activation levels as well as retinal morphology and function (electroretinography) following ischemia. In addition, the effect of two HDAC inhibitors, trichostatin-A and valproic acid were also investigated. In normal eyes, retinal ischemia produced a significant increase in HDAC activity within 2 h that was followed by a corresponding significant decrease in protein acetylation by 4 h. Activated caspase-3 levels were significantly elevated by 24 h. Treatment with HDAC inhibitors blocked the early decrease in protein acetylation and activation of caspase-3. Retinal immunohistochemistry demonstrated that systemic administration of trichostatin-A or valproic acid, resulted in hyperacetylation of all retinal layers after systemic treatment. In addition, HDAC inhibitors provided a significant functional and structural neuroprotection at seven days following injury relative to vehicle-treated eyes. These results provide evidence that increases in HDAC activity is an early event following retinal ischemia, and are accompanied by corresponding decreases in acetylation in advance of caspase-3 activation. In addition to preserving acetylation status, the administration of HDAC inhibitors suppressed caspase activation and provided structural and functional neuroprotection in model of ischemic retinal injury. Taken together these data provide evidence that decrease in retinal acetylation status is a central event in ischemic retinal injury, and the hyperacetylation induced by HDAC inhibition can provide acute neuroprotection.

Arginase 2 deficiency reduces hyperoxia-mediated retinal neurodegeneration through the regulation of polyamine metabolism.

Hyperoxia treatment has been known to induce neuronal and glial death in the developing central nervous system. Retinopathy of prematurity (ROP) is a devastating disease in premature infants and a major cause of childhood vision impairment. Studies indicate that, in addition to vascular injury, retinal neurons are also affected in ROP. Using an oxygen-induced retinopathy (OIR) mouse model for ROP, we have previously shown that deletion of the arginase 2 (A2) significantly reduced neuro-glial injury and improved retinal function. In the current study, we investigated the mechanism of A2 deficiency-mediated neuroprotection in the OIR retina. Hyperoxia treatment has been known to induce neuronal death in neonates. During the hyperoxia phase of OIR, a significant increase in the number of apoptotic cells was observed in the wild-type (WT) OIR retina compared with A2-deficient OIR. Mass spectrometric analysis showed alterations in polyamine metabolism in WT OIR retina. Further, increased expression level of spermine oxidase was observed in WT OIR retina, suggesting increased oxidation of polyamines in OIR retina. These changes were minimal in A2-deficient OIR retina. Treatment using the polyamine oxidase inhibitor, N, N'-bis (2, 3-butadienyl)-1, 4-butanediamine dihydrochloride, significantly improved neuronal survival during OIR treatment. Our data suggest that retinal arginase is involved in the hyperoxia-induced neuronal degeneration in the OIR model, through the regulation of polyamine metabolism.

The morphological features and mitochondrial oxidative stress mechanism of the retinal neurons apoptosis in early diabetic rats.

This paper aims to explore the relationship of retinal neuron apoptosis and manganese superoxidase dismutase (MnSOD) at early phase of diabetic retinopathy. Sprague-Dawley rats were grouped into normal controls and diabetics. Data were collected after 4, 8, and 12 weeks (n = 12). The pathological changes and ultrastructure of the retina, the apoptosis rate of retinal neurons by TdT-mediated dUTP nick end label (TUNEL), mRNA expressions of MnSOD and copper-zinc superoxide dismutase (Cu-Zn SOD), and the activities of total SOD (T-SOD) and subtypes of SOD were tested. For the controls, there was no abnormal structure or apoptosis of retinal neurons at any time. There was no change of structure for rats with diabetes at 4 or 8 weeks, but there was a decrease of retinal ganglion cells (RGCs) number and thinner inner nuclear layer (INL) at 12 weeks. The apoptosis ratio of RGCs was higher than that of the controls at 8 and 12 weeks (P < 0.001). The activity and mRNA levels of MnSOD were lower in diabetics at 4, 8, and 12 weeks (P < 0.05). In summary, the apoptosis of the retinal neurons occurred at 8 weeks after the onset of diabetes. Retinal neuron apoptosis in early diabetic rats may be associated with the decreased activity and mRNA of MnSOD.

RIT2, a neuron-specific small guanosine triphosphatase, is expressed in retinal neuronal cells and its promoter is modulated by the POU4 transcription factors.

PURPOSE: Ras-like without CAAX 2 (RIT2), a member of the Ras superfamily of small guanosine triphosphatases, is involved in regulating neuronal function. RIT2 is a unique member of the Ras family in that RIT2 is preferentially expressed in various neurons, including retinal neurons. The mechanisms that regulate RIT2 expression in neurons were studied. METHODS: Reverse transcription-quantitative PCR (RT-qPCR), immunohistochemistry, western blotting, bioinformatic prediction, electrophoretic mobility shift assay (EMSA), and cell transfection methods were used. RESULTS: With immunohistochemistry of the mouse retina, RIT2 protein was detected in the ganglion cell layer (GCL), inner plexiform layer, inner nuclear layer, and outer plexiform layer, with the strongest staining in the GCL and the inner plexiform layer. RT-qPCR combined with laser capture microdissection detected Rit2 messenger RNA in the GCL and the inner nuclear layer. Western blot analysis showed a large increase in the RIT2 protein in the retina during maturation from newborn to adult. Transient transfection identified the 1.3 kb upstream region of human RIT2 as capable of driving expression in neuronal cell lines. Based on the known expression pattern and biological activity, we hypothesized that POU4 family factors might modulate RIT2 expression in retinal ganglion cells (RGCs). Bioinformatic analyses predicted six POU4 factor-binding sites within the 1.3 kb human RIT2 promoter region. EMSA analyses showed binding of POU4 proteins to three of the six predicted sites. Cotransfection with expression vectors demonstrated that POU4 proteins can indeed modulate the human RIT2 promoter, and that ISL1, a LIM homeodomain factor, can further modulate the activity of the POU4 factors. CONCLUSIONS: These studies confirm the expression of RIT2 in retinal neuronal cells, including RGCs, begin to reveal the mechanisms responsible for neuronal expression of RIT2, and suggest a role for the POU4 family factors in modulating RIT2 expression in RGCs.

Nuclear morphology and c-Jun N-terminal kinase 1 expression differentiate serum-starved oxidative stress signalling from hydrogen peroxide-induced apoptosis in retinal neuronal cell line.

Oxidative stress induced by serum starvation and H2O2 exposure, both triggers apoptosis in retinal neuronal cell line RGC-5 (retinal ganglion cell-5). We have examined whether, despite excess generation of ROS (reactive oxygen species) and apoptosis induction, there is any dissimilarity in nuclear morphology and apoptotic signalling pathway in RGC-5 under these conditions. Sub-confluent cells were treated either with H2O2 or maintained in SFM (serum-free medium). ROS level was detected along with nuclear morphology and ultrastructural analysis. Generation of excess intracellular ROS, nuclear localization of Bax and caspase 3 activation along with decrease of cellular viability, confirmed apoptosis induction in RGC-5 by 72 h serum starvation and 500 M H2O2 exposure for 1 h. Nuclear swelling as supported by nuclear cytoplasmic ratio and conspicuous black spots with nuclear remodelling were observed only upon SFM, but not with H2O2 treatment. Serum starvation did not alter JNK1 (c-Jun N-terminal kinase 1) expression, although nuclear translocation and higher level of pJNK (phospho-JNK) was evident. Conversely, H2O2 exposure blocked the expression and activation of JNK1 to phospho-JNK as a negligible level of pJNK was present in the cytoplasm. Despite similar ROS generation in both the conditions, difference in nuclear morphology and JNK1 expression leads to the hypothesis that RGC-5 cells may follow different signalling pathways when challenged with serum starvation and H2O2.

Selective over-expression of endothelin-1 in endothelial cells exacerbates inner retinal edema and neuronal death in ischemic retina.

The level of endothelin-1 (ET-1), a potent vasoconstrictor, was associated with retinopathy under ischemia. The effects of endothelial endothelin-1 (ET-1) over-expression in a transgenic mouse model using Tie-1 promoter (TET-1 mice) on pathophysiological changes of retinal ischemia were investigated by intraluminal insertion of a microfilament up to middle cerebral artery (MCA) to transiently block the ophthalmic artery. Two-hour occlusion and twenty-two-hour reperfusion were performed in homozygous (Hm) TET-1 mice and their non-transgenic (NTg) littermates. Presence of pyknotic nuclei in ganglion cell layer (GCL) was investigated in paraffin sections of ipsilateral (ischemic) and contralateral (non-ischemic) retinae, followed by measurement of the thickness of inner retinal layer. Moreover, immunocytochemistry of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS) and aquaporin-4 (AQP4) peptides on retinal sections were performed to study glial cell reactivity, glutamate metabolism and water accumulation, respectively after retinal ischemia. Similar morphology was observed in the contralateral retinae of NTg and Hm TET-1 mice, whereas ipsilateral retina of NTg mice showed slight structural and cellular changes compared with the corresponding contralateral retina. Ipsilateral retinae of Hm TET-1 mice showed more significant changes when compared with ipsilateral retina of NTg mice, including more prominent cell death in GCL characterized by the presence of pyknotic nuclei, elevated GS immunoreactivity in Müller cell bodies and processes, increased AQP-4 immunoreactivity in Müller cell processes, and increased inner retinal thickness. Thus, over-expression of endothelial ET-1 in TET-1 mice may contribute to increased glutamate-induced neurotoxicity on neuronal cells and water accumulation in inner retina leading to edema.

RhoA inactivation prevents photoreceptor axon retraction in an in vitro model of acute retinal detachment.

PURPOSE: An early injury response to retinal detachment is disruption of synaptic connectivity between photoreceptors and second-order neurons. Most dramatic is the retraction of rod cell axons and their terminals away from the outer synaptic layer and toward their cell bodies. This study tested whether axonal retraction in detached retina was due to the activation of the small GTPase RhoA and was preventable using RhoA antagonists. METHODS: Retinal detachments were created in in vitro preparations of porcine eyecups. RhoA activation was determined with a Rhotekin binding assay. To block axon retraction, drugs were applied to neural retinal explants either before or after detachment from the retinal pigment epithelium. Presynaptic movement was quantified by image analysis of double-labeled retinas examined with confocal microscopy. RESULTS: Active RhoA increases transiently after detachment followed by morphologic evidence of axonal retraction over the next 24 hours. Pretreating the retina with a RhoA antagonist, CT-04, or a Rho kinase inhibitor, Y27632, at multiple concentrations significantly inhibited axonal retraction. Reducing calcium influx through L-type calcium channels with nicardipine also blocked retraction. To create a more plausible therapeutic scenario, drug treatments were delayed and applied after retinal detachment. The Rho kinase inhibitor, but not nicardipine, significantly blocked rod axonal retraction when applied up to 6 hours after detachment. CONCLUSIONS: Thus, RhoA and downstream Rho kinase activity constitute part of the mechanism that produces rod axonal retraction in retinal explants. Treatments that manipulate RhoA signaling may promote synaptic stability after retinal detachment.

Insulin-like growth factor-1 and insulin inhibit caspase-3/7 activation initiated by interleukin-1ß in the rat eye retinal neurons.

Proinflammatory cytokine IL-1ß specifically stimulates caspase-3/7 in vitro in RGC-5 rat eye retinal neurons. Insulin and insulin-like growth factor-1 and their combination inhibit caspase-3/7 activation in these cells, induced by removal of the serum from culture medium and/or by IL-1ß treatment.

Cannabidiol protects retinal neurons by preserving glutamine synthetase activity in diabetes.

PURPOSE: We have previously shown that non-psychotropic cannabidiol (CBD) protects retinal neurons in diabetic rats by inhibiting reactive oxygen species and blocking tyrosine nitration. Tyrosine nitration may inhibit glutamine synthetase (GS), causing glutamate accumulation and leading to further neuronal cell death. We propose to test the hypothesis that diabetes-induced glutamate accumulation in the retina is associated with tyrosine nitration of GS and that CBD treatment inhibits this process. METHODS: Sprague Dawley rats were made diabetic by streptozotocin injection and received either vehicle or CBD (10 mg/kg/2 days). After eight weeks, retinal cell death, Müller cell activation, GS tyrosine nitration, and GS activity were determined. RESULTS: Diabetes causes significant increases in retinal oxidative and nitrative stress compared with controls. These effects were associated with Müller cell activation and dysfunction as well as with impaired GS activity and tyrosine nitration of GS. Cannabidiol treatment reversed these effects. Retinal neuronal death was indicated by numerous terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL)-labeled cells in diabetic rats compared with untreated controls or CBD-treated rats. CONCLUSIONS: These results suggest that diabetes-induced tyrosine nitration impairs GS activity and that CBD preserves GS activity and retinal neurons by blocking tyrosine nitration.

Pigment epithelium derived factor as an anti-inflammatory factor against decrease of glutamine synthetase expression in retinal Müller cells under high glucose conditions.

BACKGROUND: The pathogenesis of diabetic retinopathy (DR) is similar to that of a chronic inflammatory disease. A predominant function of Müller cells is to regulate glutamate levels, but in DR the function is compromised. The present study was performed to investigate the role of pigment epithelial derived factor (PEDF) on the expression of glutamine synthetase (GS) in rat retinal Müller cells under high glucose conditions, and to study the possible mechanism for PEDF against decrease of GS expression in retinal Müller cells under high glucose conditions. METHODS: The role of PEDF on the expressions of GS and interleukin-1beta (IL-1beta) in retinal Müller cells under normal and high glucose conditions was measured by western blotting, real-time RT-PCR, or immunocytochemistry. In order to confirm the effect of PEDF on GS against the role of IL-1beta, the PEDF siRNA method was used. RESULTS: High glucose increased the expression of IL-1beta, but decreased the expressions of GS and PEDF in retinal Müller cells. PEDF increased the expression of GS and decreased the expression of IL-1beta in retinal Müller cells under high glucose conditions. The effect of IL-1beta on expression of GS was inhibited by PEDF. Moreover, down-regulation of PEDF expression by siRNA resulted in significantly increasing the expression of IL-1beta, but decreasing the expression of GS in retinal Müller cells. CONCLUSIONS: PEDF increases expression of GS against the effect of IL-1beta in retinal Müller cells under high glucose conditions. These findings suggested that PEDF may act as an anti-inflammatory factor against decrease of GS expression in retinal Müller cells in diabetic retinopathy.

Mitogen-activated protein kinases in the porcine retinal arteries and neuroretina following retinal ischemia-reperfusion.

PURPOSE: The aim of the present study was to examine changes in the expression of intracellular signal-transduction pathways, specifically mitogen-activated protein kinases, following retinal ischemia-reperfusion. METHODS: Retinal ischemia was induced by elevating the intraocular pressure in porcine eyes, followed by 5, 12, or 20 h of reperfusion. The results were compared to those of the sham- operated fellow eye. The retinal arteries and neuroretina were isolated separately and examined. Tissue morphology and DNA fragmentation were studied using histology. Extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38, c-junNH(2)-terminal kinases (JNK), and c-jun protein and mRNA expression were examined using immunofluorescence staining, western blot, and real-time PCR techniques. RESULTS: Pyknotic cell nuclei, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells, and glial fibrillary acidic protein mRNA expression were increased in ischemia, suggesting injury. Phosphorylated ERK1/2 protein levels were increased in the neuroretina following ischemia, while mRNA levels were unaltered. p38 protein and mRNA levels were not affected by ischemia. Immunofluorescence staining for phosphorylated p38 was especially intense in the retinal blood vessels, while only weak in the neuroretina. Phosphorylated JNK protein and mRNA were slightly decreased in ischemia. Phosphorylated c-jun protein and mRNA levels were higher in the neuroretina after ischemia-reperfusion. CONCLUSIONS: Retinal ischemia-reperfusion alters expression of mitogen-activated protein kinases, particularly ERK1/2, in the neuroretina and retinal arteries. The development of pharmacological treatment targeting these intracellular transduction pathways may prevent injury to the eye following retinal circulatory failure.

Ca(2+)-modulated vision-linked ROS-GC guanylate cyclase transduction machinery.

Vertebrate phototransduction depends on the reciprocal relationship between two-second messengers, cyclic GMP and Ca(2+). The concentration of both is reciprocally regulated including the dynamic synthesis of cyclic GMP by a membrane bound guanylate cyclase. Different from hormone receptor guanylate cyclases, the cyclases operating in phototransduction are regulated by the intracellular Ca(2+)-concentration via small Ca(2+)-binding proteins. Based on the site of their expression and their Ca(2+) modulation, this sub-branch of the cyclase family was named sensory guanylate cyclases, of which the retina specific forms are named ROS-GCs (rod outer segment guanylate cyclases). This review focuses on the structure and function of the ROS-GC subfamily present in the mammalian retinal neurons: photoreceptors and inner layers of the retinal neurons. Portions and excerpts of the review are from a previous chapter (Curr Top Biochem Res 6:111-144, 2004).

Expression of Cre recombinase in retinal Müller cells.

PURPOSE: In an effort to generate inducible RPE-specific Cre mice using a 3.0-kb human vitelliform macular dystrophy-2 (VMD2) promoter, we identified a mouse line with unanticipated Cre activity in the neural retina, including Müller glial cells. Müller cells play important roles in the function and maintenance of the retina, and this mouse line would be potentially useful for conditional gene targeting in Müller glia. We therefore characterized the timing, inducibility, and cell specificity of Cre expression, as well as Müller cell-specific efficiency of Cre-mediated recombination in this mouse line. METHODS: Transgenic mice carrying cassettes of human P(VMD2)-rtTA and TRE-cre were generated. Cre expression was characterized using a Cre-activatable lacZ reporter mouse line (R26R) and a floxed interleukin six signal transducing receptor (gp130) mouse line. RESULTS: beta-Galactosidase (beta-gal) assay and immunohistochemical analysis of VMD2-cre/R26R double transgenic mice indicated that Cre activity was detected in cells located in the inner nuclear layer, with prominent expression of beta-gal in Müller cells. Cre activity was also detected in photoreceptors in the outer nuclear layer. PCR analysis demonstrated that Cre-mediated recombination initiated by embryonic day 15. Immunohistochemical analysis indicated that Cre-mediated deletion of floxed gp130 gene occurred in 52% of the retinal Müller cells. Retinal function and morphology were normal in 10-month-old VMD2-cre mice. CONCLUSION: We generated a transgenic cre mouse that is useful to study gene activation and inactivation in retinal Müller cells.