Epicatechin blocks pro-nerve growth factor (proNGF)-mediated retinal neurodegeneration via inhibition of p75 neurotrophin receptor expression in a rat model of diabetes [corrected].

AIMS/HYPOTHESIS: Accumulation of pro-nerve growth factor (NGF), the pro form of NGF, has been detected in neurodegenerative diseases. However, the role of proNGF in the diabetic retina and the molecular mechanisms by which proNGF causes retinal neurodegeneration remain unknown. The aim of this study was to elucidate the role of proNGF in neuroglial activation and to examine the neuroprotective effects of epicatechin, a selective inhibitor of tyrosine nitration, in an experimental rat model of diabetes. METHODS: Expression of proNGF and its receptors was examined in retinas from streptozotocin-induced diabetic rats, and in retinal Müller and retinal ganglion cells (RGCs). RGC death was assessed by TUNEL and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays in diabetic retinas and cell culture. Nitrotyrosine was determined using Slot-blot. Activation of the tyrosine kinase A (TrkA) receptor and p38 mitogen-activated protein kinase (p38MAPK) was assessed by western blot. RESULTS: Diabetes-induced peroxynitrite impaired phosphorylation of TrkA-Y490 via tyrosine nitration, activated glial cells and increased expression of proNGF and its receptor, p75 neurotrophin receptor (p75(NTR)), in vivo and in Müller cells. These effects were associated with activation of p38MAPK, cleaved poly-(ADP-ribose) polymerase and RGC death. Treatment of diabetic animals with epicatechin (100 mg kg(-1) day(-1), orally) blocked these effects and restored neuronal survival. Co-cultures of RGCs with conditioned medium of activated Müller cells significantly reduced RGC viability (44%). Silencing expression of p75(NTR) by use of small interfering RNA protected against high glucose- and proNGF-induced apoptosis in RGC cultures. CONCLUSIONS/INTERPRETATION: Diabetes-induced peroxynitrite stimulates p75(NTR) and proNGF expression in Müller cells. It also impairs TrkA receptor phosphorylation and activates the p75(NTR) apoptotic pathway in RGCs, leading to neuronal cell death. These effects were blocked by epicatechin, a safe dietary supplement, suggesting its potential therapeutic use in diabetic patients.

Diabetes-induced peroxynitrite impairs the balance of pro-nerve growth factor and nerve growth factor, and causes neurovascular injury.

AIMS/HYPOTHESIS: Diabetic retinopathy, the leading cause of blindness in working-age Americans, is characterised by reduced neurotrophic support and increased proinflammatory cytokines, resulting in neurotoxicity and vascular permeability. We sought to elucidate how oxidative stress impairs homeostasis of nerve growth factor (NGF) and its precursor, proform of NGF (proNGF), to cause neurovascular dysfunction in the eye of diabetic patients. METHODS: Levels of NGF and proNGF were examined in samples from human patients, from retinal Müller glial cell line culture cells and from streptozotocin-induced diabetic animals treated with and without atorvastatin (10 mg/kg daily, per os) or 5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinato iron (III) chloride (FeTPPs) (15 mg/kg daily, i.p.) for 4 weeks. Neuronal death and vascular permeability were assessed by TUNEL and extravasation of BSA-fluorescein. RESULTS: Diabetes-induced peroxynitrite formation impaired production and activity of matrix metalloproteinase-7 (MMP-7), which cleaves proNGF extracellularly, leading to accumulation of proNGF and reducing NGF in samples from diabetic retinopathy patients and experimental models. Treatment of diabetic animals with atorvastatin exerted similar protective effects that blocked peroxynitrite using FeTPPs, restoring activity of MMP-7 and hence the balance between proNGF and NGF. These effects were associated with preservation of blood-retinal barrier integrity, preventing neuronal cell death and blocking activation of RhoA and p38 mitogen-activated protein kinase (p38MAPK) in experimental and human samples. CONCLUSIONS/INTERPRETATION: Oxidative stress plays an unrecognised role in causing accumulation of proNGF, which can activate a common pathway, RhoA/p38MAPK, to mediate neurovascular injury. Oral statin therapy shows promise for treatment of diabetic retinopathy.

Neuroprotective effects of cannabidiol in endotoxin-induced uveitis: critical role of p38 MAPK activation.

PURPOSE: Degenerative retinal diseases are characterized by inflammation and microglial activation. The nonpsychoactive cannabinoid, cannabidiol (CBD), is an anti-inflammatory in models of diabetes and glaucoma. However, the cellular and molecular mechanisms are largely unknown. We tested the hypothesis that retinal inflammation and microglia activation are initiated and sustained by oxidative stress and p38 mitogen-activated protein kinase (MAPK) activation, and that CBD reduces inflammation by blocking these processes. METHODS: Microglial cells were isolated from retinas of newborn rats. Tumor necrosis factor (TNF)-alpha levels were estimated with ELISA. Nitric oxide (NO) was determined with a NO analyzer. Superoxide anion levels were determined by the chemiluminescence of luminol derivative. Reactive oxygen species (ROS) was estimated by measuring the cellular oxidation products of 2', 7'-dichlorofluorescin diacetate. RESULTS: In retinal microglial cells, treatment with lipopolysaccharide (LPS) induced immediate NADPH oxidase-generated ROS. This was followed by p38 MAPK activation and resulted in a time-dependent increase in TNF-alpha production. At a later phase, LPS induced NO, ROS, and p38 MAPK activation that peaked at 2-6 h and was accompanied by morphological change of microglia. Treatment with 1 microM CBD inhibited ROS formation and p38 MAPK activation, NO and TNF-alpha formation, and maintained cell morphology. In addition, LPS-treated rat retinas showed an accumulation of macrophages and activated microglia, significant levels of ROS and nitrotyrosine, activation of p38 MAPK, and neuronal apoptosis. These effects were blocked by treatment with 5 mg/kg CBD. CONCLUSIONS: Retinal inflammation and degeneration in uveitis are caused by oxidative stress. CBD exerts anti-inflammatory and neuroprotective effects by a mechanism that involves blocking oxidative stress and activation of p38 MAPK and microglia.

Imbalance of the nerve growth factor and its precursor as a potential biomarker for diabetic retinopathy.

Our previous studies have demonstrated that diabetes-induced oxidative stress alters homeostasis of retinal nerve growth factor (NGF) resulting in accumulation of its precursor, proNGF, at the expense of NGF which plays a critical role in preserving neuronal and retinal function. This imbalance coincided with retinal damage in experimental diabetes. Here we test the hypothesis that alteration of proNGF and NGF levels observed in retina and vitreous will be mirrored in serum of diabetic patients. Blood and vitreous samples were collected from patients (diabetic and nondiabetic) undergoing vitrectomy at Georgia Regents University under approved IRB. Levels of proNGF, NGF, and p75(NTR) shedding were detected using Western blot analysis. MMP-7 activity was also assayed. Diabetes-induced proNGF expression and impaired NGF expression were observed in vitreous and serum. Vitreous and sera from diabetic patients (n = 11) showed significant 40.8-fold and 3.6-fold increases, respectively, compared to nondiabetics (n = 9). In contrast, vitreous and sera from diabetic patients showed significant 44% and 64% reductions in NGF levels, respectively, compared to nondiabetics. ProNGF to NGF ratios showed significant correlation between vitreous and serum. Further characterization of diabetes-induced imbalance in the proNGF to NGF ratio will facilitate its utility as an early biomarker for diabetic complications.

Precocious IRBP gene expression during mouse development.

PURPOSE: To determine if the time course for the onset of gene and protein expression for interphotoreceptor binding protein (IRBP) precedes that of opsin in the developing mouse retina. METHODS: Relative mRNA levels of the IRBP and opsin genes were determined in prenatal and postnatal retinal RNA with RNase protection analysis (RPA). To determine if IRBP and opsin protein expressions are differentially regulated, dissociated retinal cells from postnatal (P) days 2 and 3 mice, that were injected with BrdU, were then double-labeled with antibodies against BrdU and either opsin or IRBP. RESULTS: With RPA, IRBP mRNA was detected on embryonic (E) day 11 at the time of cone formation, whereas opsin mRNA was not detected until P0. It took until P3 for opsin expression to reach significant levels, whereas rods already appear during embryonic development. IBRP transcription preceded that of opsin because it rapidly increased from E13 to an early postnatal day. By P20, the expression levels of IRBP and opsin achieved constancy. Double antibody labeling revealed positive staining for both IRBP and BrdU as soon as 2 hours after injection, but it took until 40 hours for double positive staining for opsin and BrdU. CONCLUSION: Because only IRBP protein expression was observed before the last mitosis of the photoreceptor precursor cells, IRBP could be essential for retinal development.

Functional dissection of the promoter of the interphotoreceptor retinoid-binding protein gene: the cone-rod-homeobox element is essential for photoreceptor-specific expression in vivo.

The essential control elements in the interphotoreceptor retinoid-binding protein gene (IRBP) promoter are located between -156 and +19. The -156/-109 sequence contains a retina-specific DNAse I footprint and shows a positive regulatory activity in transiently transfected retinoblastoma cells. The -105/-85 sequence is G/C rich, shows a non-tissue specific DNAse I hypersensitivity, and a negative regulatory activity in retinoblastoma cells. The -76/-42 sequence shows a retinal-specific footprint and contains a "cone-rod-homeobox element" (CRXE) and a "photoreceptor conserved element" (PCE). IRBP promoter fragments with mutations in either CRXE, PCE or in both were linked to reporter genes and analyzed both by transient transfection and in transgenic mice. In retinoblastoma cells, the mutated CRXE-containing promoter shows a 60% repression of the CAT activity whereas the mutated PCE-containing promoter shows a 30% repression. In HeLa cells transfected with these promoters, co-transfection of a Crx expression vector with wild-type, but not with CRXE mutant promoter, activates CAT activity 20-fold over the background activity. Mutation of PCE alone or conversion of CRXE to PCE reduces this Crx-activated CAT activity to only 4-fold over the background activity. In the transgenic mouse experiments, none of the 12 lines with CRXE mutant promoter show significant expression of lacZ in the retina. In contrast, 9 of the 17 transgenic lines with PCE mutant promoter show photoreceptor-specific lacZ expression. Thus the Crx interaction with CRXE is essential for the photoreceptor-specific activity of the IRBP promoter in vivo. This interaction does not appear to require PCE, but is enhanced when PCE is present.

Deletion of thioredoxin-interacting protein preserves retinal neuronal function by preventing inflammation and vascular injury.

BACKGROUND AND PURPOSE: Retinal neurodegeneration is an early and critical event in several diseases associated with blindness. Clinically, therapies that target neurodegeneration fail. We aimed to elucidate the multiple roles by which thioredoxin-interacting protein (TXNIP) contributes to initial and sustained retinal neurodegeneration. EXPERIMENTAL APPROACH: Neurotoxicity was induced by intravitreal injection of NMDA into wild-type (WT) and TXNIP-knockout (TKO) mice. The expression of apoptotic and inflammatory markers was assessed by immunohistochemistry, elisa and Western blot. Microvascular degeneration was assessed by periodic acid-Schiff and haematoxylin staining and retinal function by electroretinogram. KEY RESULTS: NMDA induced early (1 day) and significant retinal PARP activation, a threefold increase in TUNEL-positive nuclei and 40% neuronal loss in ganglion cell layer (GCL); and vascular permeability in WT but not TKO mice. NMDA induced glial activation, expression of TNF-α and IL-1ß that co-localized with Müller cells in WT but not TKO mice. In parallel, NMDA triggered the expression of NOD-like receptor protein (NLRP3), activation of caspase-1, and release of IL-1ß and TNF-α in primary WT but not TKO Müller cultures. After 14 days, NMDA induced 1.9-fold microvascular degeneration, 60% neuronal loss in GCL and increased TUNEL-labelled cells in the GCL and inner nuclear layer in WT but not TKO mice. Electroretinogram analysis showed more significant reductions in b-wave amplitudes in WT than in TKO mice. CONCLUSION AND IMPLICATIONS: Targeting TXNIP expression prevented early retinal ganglion cell death, glial activation, retinal inflammation and secondary neuro/microvascular degeneration and preserved retinal function. TXNIP is a promising new therapeutic target for retinal neurodegenerative diseases.

Hemoglobin Suan-Dok (alpha 2 109 (G16) Leu replaced by Arg beta 2): an unstable variant associated with alpha-thalassemia.

An abnormal hemoglobin variant was detected in a seven-year old Thai girl. The clinical and hematological pictures were indistinguishable from hemoglobin H disease. Upon cellulose acetate electrophoresis at pH 8.5 an abnormal hemoglobin variant was found together with hemoglobins A and H. The variant moved slightly behind HbF and amounted to approximately 9%. Globin chain synthesis studies of peripheral blood reticulocytes incubated with tritiated leucine revealed that the total radioactivity ratio alpha-chain + variant alpha-chain/beta-chain was 0.59, while the ratio variant alpha-chain/beta-chain was 0.12. The variant hemoglobin was somewhat unstable under heat denaturation and in the isopropanol test. Structural studies by component isolation, globin chain separation, peptide mapping and aminoacid analysis of abnormal peptides showed that the leucine residue 109 of the alpha-chain was replaced by arginine. This hemoglobin mutant has not been described before and is named Hemoglobin Suan-Dok.

Timing of interphotoreceptor retinoid-binding protein (IRBP) gene expression and hypomethylation in developing mouse retina.

We determined during photoreceptor development if there is a retina-specific hypomethylation of the mouse gene encoding interphotoreceptor retinoid-binding protein (IRBP) that is associated with its activation. Second, the role of IRBP gene and protein expression in development was assessed by determining if their expression occurs before that of opsin. Retina-specific hypomethylation of the IRBP promoter region started on Embryonic (E) Day 11, at the time of cone formation, increased from E12 to E14, at the time of rod formation, and reached a peak on Postnatal (P) Day 4, which was followed thereafter by a slow decrease. Starting on E11, IRBP and opsin mRNA levels were quantitated relative to that of the beta-actin gene with RNase protection analysis. beta-Actin and IRBP transcripts were readily detected on E11. beta-actin levels remained constant during embryonic and early postnatal stages and decreased slightly afterward. On the other hand, beginning on E13, when the rods are formed, the IRBP level markedly increased. In contrast, the opsin transcript first appeared later on P0 and then increased from P3 onward. After P6, the opsin and IRBP transcript levels became comparable and by P20 their levels reached constancy. The timing of the onset of protein expression for the IRBP and opsin genes was determined during the last proliferative cycle of the rod precursor cells before their differentiation. Mice at P2 or P3 were injected with bromodeoxyuridine (BrdU) and their retinal cells were dissociated and then double-labeled with antibodies against BrdU and either IRBP or opsin. Cells positive for both IRBP and BrdU were always observed as soon as 2 hr after injection but it took at least 40 hr before they became positive for both opsin and BrdU. Taken together, these results indicate that IRBP gene activation is associated with hypomethylation during the last mitosis before photoreceptor cell differentiation.

Identification of a retina-specific footprint within the retina-specific regulatory region of the human interphotoreceptor retinoid-binding protein gene.

Regulation of the retina-specific expression of the interphotoreceptor retinoid-binding protein (IRBP) gene in transgenic mice requires sequences between nucleotide (nt) -156 and +19. DNAse I footprinting and electrophoretic mobility shift assays identified a region between nt -69 and -40 that interacts with some nuclear proteins from bovine and mouse retinas but not from a variety of other neural and non-neural tissues. The selectivity of this sequence to elicit IRBP promoter activity is indicated by their failure to bind some nuclear proteins following mutagenesis. Therefore, IRBP promoter activity is linked to nuclear protein binding at a regulatory motif between nt-69 and -40.

Retina-specific expression from the IRBP promoter in transgenic mice is conferred by 212 bp of the 5'-flanking region.

IRBP is a photoreceptor-specific glycoprotein that has been suggested as a retinoid carrier in the visual process. Previous research has shown that 1.3 kb of 5'-flanking sequence from the human IRBP gene is sufficient to promote photoreceptor-specific expression of reporter genes in transgenic mice. To define more narrowly the sequences that promote tissue-specific expression, chimeric constructs with shorter promoters were used to generate transgenic mice. The bacterial CAT gene was fused to fragments of 706 bp or 212 bp from the 5' end of the human IRBP gene. Analysis of the three transgenic families bearing the 706 bp IRBP promoter revealed that CAT expression was confined to the neuro-retina and the pineal gland. Analysis of the four transgenic families bearing the 212 bp IRBP promoter revealed the same tissue-specific CAT expression in three families. These results establish that tissue-specific expression of IRBP can be regulated by a short 212 bp promoter which has been conserved between humans and mice.

Tissue-specific expression in transgenic mice directed by the 5'-flanking sequences of the human gene encoding interphotoreceptor retinoid-binding protein.

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular protein that has been suggested to participate in the visual process as a carrier for visual retinoids. A chimeric gene composed of the human IRBP promoter fused to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) was used to generate transgenic mice. Analysis of six transgenic families revealed that the CAT gene, concomitant with the endogenous IRBP gene, was expressed primarily in the retina and, to a lesser extent, in the pineal gland. These results establish that a 1.3-kilobase fragment from the 5' end of the human IRBP gene is sufficient to direct transgene expression to a visual subdivision of the central nervous system.

Visual sensitivity and interphotoreceptor retinoid binding protein in the mouse: regulation by vitamin A.

Interphotoreceptor retinoid binding protein (IRBP) is a retinoid and fatty acid binding glycoprotein secreted by rod and cone photoreceptors in all vertebrates. IRBP is believed to serve as a carrier for retinoids in the bleaching and regeneration cycle of rhodopsin. IRBP protein has been found to be decreased in vitamin A-deprived rats; it is rapidly recovered after retinol repletion. To understand the mechanism for this recovery, we determined whether vitamin A affects transcription and translation of the IRBP gene. Wild-type and transgenic mice harboring the IRBP promoter-CAT reporter fusion gene were maintained on a retinol-deficient diet supplemented with retinoic acid (-A) or on a control diet (+A) for up to 60 wk postweaning. Some of the -A mice were given retinol repletion for 7 days (-A+A). Electroretinography analysis revealed alterations in waveform and a 2 log unit decrease in b-wave sensitivity in the -A mice over a broad range of stimulus wavelengths. Retinol repletion effected a full recovery. Immunochemistry showed a significant decrease in the immunogold-labeled IRBP between the retinal pigment epithelium and the outer segments of the -A mice compared with +A and -A+A mice. Northern blots showed no differences in the amounts of IRBP or CAT mRNA between these three treatment groups. These results suggest that the regulation of IRBP by retinol is not transcriptional.

Early onset photoreceptor abnormalities induced by targeted disruption of the interphotoreceptor retinoid-binding protein gene.

Vision in all vertebrates is dependent on an exchange of retinoids between the retinal pigment epithelium and the visual photoreceptors. It has been proposed that the interphotoreceptor retinoid-binding protein (IRBP) is essential for this intercellular exchange, and that it serves to prevent the potentially cytotoxic effects of retinoids. Although its precise function in vivo has yet to be defined, the early expression of IRBP suggests that it may also be required for normal photoreceptor development. To further assess the biological role of IRBP, we generated transgenic mice with targeted disruption of the IRBP gene (IRBP-/- mice). Specifically, homologous recombination was used to replace the first exon and promoter region of the IRBP gene with a phosphoglycerate kinase-promoted neomycin-resistant gene. Immunocytochemical and Western blot analyses demonstrated the absence of IRBP expression in the IRBP-/- mice. As early as postnatal day 11, histological examination of the retinas of IRBP-/- mice revealed a loss of photoreceptor nuclei and changes in the structural integrity of the receptor outer segments. At 30 d of age, the photoreceptor abnormalities in IRBP-/- mice were more severe, and electroretinographic recordings revealed a marked loss in photic sensitivity. In contrast, no morphological or electrophysiological changes were detected in age-matched heterozygotes. These observations indicate that normal photoreceptor development and function are highly dependent on the early expression of IRBP, and that in the absence of IRBP there is a slowly progressive degeneration of retinal photoreceptors.